Natural photosynthetic system for restoring homeostasis of animal organelle interaction network

Domain: biology
Published: 2026-03-31
Editorial outcome: major_revision

Abstract
Disruption of organelle interactions due to metabolic stress is a crucial factor in the pathological processes of many degenerative diseases. Compared with animal cells, the participation of chloroplasts enables plant cells to show defensive adaptation under stress. Therefore, delivering plant-derived photosynthetic systems into animal cells may help to establish a more stable organelle interaction network. Here, we show that plant-derived photosynthetic systems can effectively restore homeostasis of the interaction network of animal organelles. Specifically, plant-derived nanothylakoid units provide energy to animal cells, regulate intracellular Ca2+ homeostasis, increase endoplasmic reticulum (ER) lipid unsaturation and global membrane fluidity, reduce abnormal contact between mitochondria and ER, and alleviate mitochondrial dysfunction. By combining implantable light-emitting diodes with wireless charging, we expand photosynthesis therapy, enabling treatments for deeper tissues. This study provides a proof-of-concept for disease treatment based on the regulation of organelle interaction networks by natural photosynthetic systems and establishes a therapeutic approach for treating deep tissues.

Introduction
The early evolution of life underwent a process of formation from simple organic molecules to pre-cellular and primitive lives1,2. For the evolution of complex life, the acquisition of mitochondria in eukaryotes is a prerequisite3,4. The endosymbiotic event that gave rise to mitochondria enabled a substantial expansion in gene expression and protein synthesis in eukaryotes relative to prokaryotes. This increase was driven by the emergence of specialized bioenergetic membranes and enhanced ATP-producing capacity, which together provided the energetic foundation for the biological diversity and complexity4,5. However, different types of diseases take a huge toll on life by placing stress on cells. Plants possess mitochondria and chloroplasts and exhibit unique defensive adaptation to stress6,7. Malate/oxaloacetate translocators and malate dehydrogenases form the malate valve, which metabolically links chloroplasts and mitochondria by transferring excess reducing equivalents from the chloroplast to maintain stromal redox balance. Under stresses such as high light, elevated CO2, cold, drought, or salinity, malate valve activity adjusts to maintain ATP/NAD(P)H homeostasis, limit ROS formation, and support stress resilience8–10. Additionally, plants also enhance the unsaturation of fatty acids in membrane lipids that preserves membrane fluidity and sustains the activity of essential membrane-associated proteins under cold stress11–18. Together, these redox-balancing and membrane-stabilizing mechanisms enable plants to maintain metabolic function and tolerate abiotic stress. However, compared with plant cells, animal cells lack the ability to tolerate different stressful environments. Therefore, transplanting plant-derived photosynthetic systems into animal cells may enhance their stress tolerance.

A previous study has used cell membrane camouflage encapsulation to deliver plant-derived nanothylakoid units (NTUs) into animal cells to control the deficient anabolism of pathological animal cells under light19. Healthy cells usually have a highly sophisticated spatiotemporal architecture, enabling precise organization of dense organelles and macromolecular complexes, accurate spatial intersections, and timely metabolite exchange20. However, as miniature organelles that restore the functions of pathological animal cells, whether NTUs can transplant the defensive adaptation capabilities of plants into animal cells remains unclear.

The premise of organelle interactions is the proximity between the organelles21. As the largest organelle in eukaryotic cells, the endoplasmic reticulum (ER) forms a network throughout the cell, maintains many membrane contact sites, and acts as a hub for intracellular protein and lipid synthesis22. The ER dedicates a special domain for metabolic interaction with mitochondria and is called the mitochondria-associated ER membrane (MAM)23. MAMs play important roles in various cellular functions, including acting as a tight platform for lipid synthesis and lipid transfer between the ER and mitochondria23,24. Mitochondria, the energy factories of eukaryotic cells, support the cascade of organelle interactions by generating energy25. The contact sites between the outer mitochondrial membrane and ER (MERCS) ensures spatiotemporal communication between mitochondria and the ER26,27. Animal cells exhibit ER–mitochondria contact alterations under stress28,29. Targeting individual organelles may not systematically correct dysregulated organelle interactions, and it is urgent to identify methods to restore these dysregulated interaction networks. Intervertebral disc degeneration (IVDD) is a degenerative disease of the deep retroperitoneal tissues that disrupts ER and mitochondrial functions in nucleus pulposus cells (NPCs), which are crucial pathological factors in its pathogenesis30,31. Given the central position of the ER-mitochondrial network in cell metabolism and because NTUs participate in cellular metabolism by producing energy and reducing equivalents19, NTUs may have the potential to protect dysregulated organelle interaction networks under pathological conditions.

Selecting the appropriate light to initiate the photosynthetic reaction is essential to achieve the in vivo therapeutic applications of NTUs. However, commonly used external light sources (lasers, light-emitting diodes, and lamps) are limited by poor penetration of light into tissues. Previous studies have attempted to introduce optical fibers into the body through endoscopy to achieve light transmission32,33; however, the wires required for optical fibers are rigid, fragile, bulky, and difficult to stabilize. Wireless power transfer involves transmitting electrical energy using a cable-free method, and this method is used to deliver controlled doses of light into deep tissues for therapeutic purposes34,35.

In this work, we aim to explore whether plant-derived NTUs can restore animal cell homeostasis under pathological conditions by modulating the ER–mitochondria interaction network. The IVDD animal model is used for conceptual verification. Similarly, we use an NPC membrane to nanoencapsulate NTUs (NM-coated NTUs, NM-NTUs), achieving light-controlled regulation of the organelle interaction network in rat IVDD models. Moreover, to simulate deep-tissue treatment in clinical practice, we design a wirelessly powered light-emitting diode (wp-LED) as an implantable light source and combine it with a wireless controller connected to a smartphone to remotely control the photosynthetic response of NTUs in deep tissues. The efficacy of combining wp-LED with NTU injection is validated in rabbit IVDD models (Fig. 1). In summary, we find that implanting plant-derived photosynthetic systems into mammalian cells can effectively restore homeostasis of the ER–mitochondria interaction network. By combining implantable wp-LED, we enable more effective treatments for deeper tissues.

Results
Disruption of the ER-mitochondria interaction network in human IVDD
ER stress and mitochondrial metabolic imbalance have been proposed to be involved in the pathological process of IVDD36,37. Transcriptomic analyses were performed on normal and degenerative human nucleus pulposus (NP) (Fig. 2a). The Pfirrmann degeneration grade indicates the degree of disc degeneration between the two groups (Supplementary Fig. 1a). Principal component analysis (PCA) showed that the genes in the normal group were distinct from those in the degeneration group (Fig. 2b). Compared with the normal group, 799 genes were differentially expressed in the degeneration group, with 369 upregulated and 430 downregulated (Fig. 2c). Gene expression patterns in the degeneration group showed a significant increase in the expression of oxidative stress-related (CYBB, RAC2, and EDN1) and ER stress-related (IGFBP1, STC2, and PTX3) genes (Fig. 2d). To further determine the levels of ER- and mitochondrial-related pathway enrichment, we performed gene set enrichment analysis (GSEA). The degeneration group exhibited an upregulated expression of genes involved in the reactive oxygen species (ROS) and reactive nitrogen species (RNS) production, ER stress, calcium import into the mitochondrion, and mitochondria permeability (Fig. 2e; Supplementary Fig. 1b, c).

To validate the findings of transcriptomic analyses and study cellular changes of human NPCs, we established a primary culture of normal and degenerated human NPCs. On the basis of previous reports, the phenotypic gene expression of human NPCs changes significantly after approximately 11–13 days of in vitro culture38,39. To evaluate the impact of in vitro culture on the changes in the phenotype of degenerated NPCs in this study, we monitored the phenotypic profiles of degenerated human NPCs cultured for 1–9 days. Referring to previous studies38,39, we examined the following NPC phenotypic markers: genes (CD24, KRT18, KRT19, SOX9, MIA, FN1, COL1A1, COL2A1, and COL3A1) and proteins (CD24 and KRT19). The results indicated that the phenotype of degenerated NPCs remained stable during the first 5 days of culture, whereas changes in the expression of certain markers began to emerge on day 7 (Supplementary Fig. 2a, b). Therefore, a 5-day culture time was selected in this study to reduce the impact of phenotypic changes on subsequent experiments. Next, we measured cell viability to evaluate the cellular status. In the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, the cell viability of degenerated human NPCs decreased to 68.2% compared with that of normal human NPCs (Supplementary Fig. 3a). However, in the live/dead staining assay, we detected no significant difference in the percentage of live cells between the degenerated NPCs and normal NPCs (Supplementary Fig. 3b, c). These findings suggested that although degenerated NPCs exhibited reduced metabolic activity and viability, substantial cell death did not occur. These results also suggested that the in vitro model matched IVDD, where a large percentage of NPCs were alive and that the findings in the following series of experiments were not due to a change in cell number but rather cellular metabolic changes.

The ER, the primary storage organelle for intracellular Ca2+, pumps cytoplasmic Ca2+ through sarcoplasmic/ER Ca2+-ATPase. Fluo-4/AM (intracellular Ca2+ indicator) and Rhod-2/AM (mitochondrial Ca2+ indicator) staining results showed that cytosolic Ca2+ ([Ca2+]c) and mitochondrial Ca2+ ([Ca2+]m) levels were significantly increased in degenerative NPCs (Fig. 2f). Since the ER is the primary intracellular Ca2+ store, the rise in [Ca2+]c and [Ca2+]m may reflect a decrease in the Ca2+ concentration within the ER ([Ca2+]ER). Ca2+, an ER signaling molecule, participates in protein folding inside the ER. A decrease in the [Ca2+]ER may cause ER stress through an unfolded protein response40. Our results showed that degenerative NPCs had increased levels of ER stress-related proteins (CHOP and ATF4) (Fig. 2g). ER stress causes increased MERCS and increased mitochondrial Ca2+ uptake from the ER41. To further clarify the ER–mitochondrial interaction network, we assessed the MERCS structure of normal and degenerative NPCs by immunostaining the ER and mitochondria with calnexin and translocase of the outer mitochondrial membrane 20 (TOMM20), respectively. The results revealed increased colocalization of the ER and mitochondria in degenerative NPCs (Fig. 2h, i). This was confirmed using transmission electron microscopy (TEM). ER–mitochondria contacts can be characterized by gap distances, which are usually in the range from 10–80 nm42,43. In accordance with a previous study44, we defined contact sites as those with tethered proximity between membrane-bound organelles. The contacts were categorized and quantified into three distinct ranges: ≤25 nm, 25 < distance ≤ 50 nm, and 50 < distance ≤ 100 nm. Compared with normal NPCs, degenerated NPCs presented significantly increased ER tubules closely apposed to mitochondria, indicating increased formation of MERCS (Fig. 2j, k). There was no significant difference in the number of mitochondria or the average length of ER tubules between normal and degenerated NPCs (Fig. 2l, m). The MERCS is crucial in regulating Ca2+ homeostasis from the ER to the mitochondria. Aberrant MERCS formation promotes [Ca2+]m overload45,46 and triggers the permeability of the inner mitochondrial membrane by opening the mitochondrial permeability transition pore (mPTP)47. Direct assessment of mPTP opening through the calcein release48 assay confirmed increased mPTP opening in degenerative NPCs (Fig. 2n). mPTP opening caused the depolarization of the mitochondrial membrane potential and oxidative stress49. Degenerated NPCs showed higher ROS production (Fig. 2o) and lower mitochondrial membrane potential than normal NPCs (Fig. 2p). Taken together, the disrupted ER-mitochondrial interaction network plays a crucial role in the process of IVDD, and restoring the dysfunctional organelle interaction network may ameliorate IVDD.

Preparation and characterization of NM-NTUs
The above results reveal that under pathological conditions, the ER-mitochondrial interaction network is disrupted. Given that plants possess chloroplasts and mitochondria and show defensive adaptations to stressful environments6,7, we used our previously constructed cell membrane-encapsulated NTUs, which can be transplanted across species19, to investigate their potential to regulate the interaction network of animal organelles. We coextruded the purified NPC membranes and NTUs to obtain NM-NTUs. To evaluate the encapsulation efficiency of the NTUs by NM, we used nanoscale flow cytometry (nanoFCM). The nanoFCM data indicated that the encapsulation efficiency was dependent on the initial ratio of NTUs to NMs. At the optimal formulation ratio (NTU to NM protein ratio of 1:150), 77.83 ± 4.01% of the NTUs could be encapsulated into NMs (Supplementary Fig. 4a), with approximately 51.37 ± 2.08% of the NMs successfully loaded with NTUs (Supplementary Fig. 4b). This ratio was used in subsequent experiments. The NM-NTU particle size was approximately 229 nm (Fig. 3a). The surface zeta potential results showed that the NTUs were coated with the cell membrane (Fig. 3b). The result of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) further confirmed the retention of the photosynthetic system proteins in NM-NTUs (Fig. 3c). The NM-NTU core-shell structure was observed using cryogenic transmission electron microscopy (cryo-TEM) (Fig. 3d). The cell counting kit-8 (CCK-8) assay revealed that the NM-NTU treatment had no evident cell toxicity (Fig. 3e).

Cell membrane-coated nanoparticles have unique characteristics such as immune escape, molecular recognition, and cell targeting50,51. We evaluated the selective targeting ability of the NM-NTUs. NTUs were first labeled with DiI. In the intervertebral disc environment, the primary cells present are NPCs, annulus fibrosus cells (AFCs), and endplate chondrocytes (EPCs). Therefore, rat NPCs, AFCs, and EPCs were treated with NM-NTUs (NTUs labeled with DiI). NM-NTUs were more highly taken up by NPCs than by AFCs or EPCs (Fig. 3f, g), indicating that NM-NTUs were preferentially taken up by NPCs. To confirm that membrane fusion had occurred, rat NPCsDiI (NPC membrane labeled with DiI) were treated with NMDiO-NTUs (NM labeled with DiO; NTU unlabeled). Owing to the characteristics of cell membrane fusion, the fluorescence signal of NMDiO colocalized with the fluorescence signal of NPCDiI, confirming the occurrence of membrane fusion (Fig. 3h). Lysosomes are organelles that degrade macromolecules via various membrane-trafficking pathways52. Many pathogens avoid lysosomal degradation by escaping the endocytic pathway52. In our study, rat NPCs were coincubated with either NTUDiI (NTU labeled with DiI) or NM-NTUDiI (NM unlabeled; NTU labeled with DiI). The results revealed that in NPCs treated with NTU alone, the NTUDiI fluorescence signal colocalized with that of the lysosomes (labeled with LysoTracker Green). In contrast, in NPCs treated with NM-NTUDiI, only a few signals of NTUDiI colocalized with lysosomes (Fig. 3i, j). These results indicated that the NM coating enabled the NTUs to bypass lysosomal degradation. Furthermore, the NTU’s post-internalization behavior after NM-NTU uptake was highly significant; the colocalization experiments revealed that the NTU signals colocalized with actin and tubulin to a high degree, and only a few NTU signals colocalized with the ER, mitochondria, and Golgi apparatus (Supplementary Fig. 5a, b). These results indicated that the NTU might achieve motility after entering the cell through the cytoskeleton. This phenomenon has similarities to previous reports of cytoskeleton-driven chloroplast movement in plants53,54. Moreover, to visualize MERCS and NTU colocalization in NPCs, we transfected immortalized human primary NPCs (IH-NPCs) with a previously validated split-GFP-based ER–mitochondria contact site sensor (SPLICS)55,56. Following the transfection of IH-NPCs with SPLICS, we treated them with NM-NTUs and performed confocal microscopy observations. Analysis of the colocalization between the MERCS signal (reconstituted GFP, green) and the NM-NTU signal (DiI labeled NTUs, red) revealed that the Mander’s coefficient for the percentage of NTUs that colocalized with MERCS was 5.29 ± 1.55% (Supplementary Fig. 6a–c). Taken together, the above results indicated that in the intervertebral disc microenvironment, NM-NTUs were preferentially taken up by NPCs. After entering NPCs, NTUs could evade lysosomal digestion and probably did not physically interact with MERCS.

NM-NTUs enhance NPC anabolism
Similar to that in other degenerative diseases, degenerated NPCs show an imbalance between anabolic and catabolic processes57. The ATP generation ability of NTUs has been confirmed in our previous study19. Studies have used interleukin (IL)-1β to induce NPC degeneration, which may be related to its ability to increase the expression of matrix degradation-related genes (MMP3, MMP13, and ADAMTS-4) and decrease the expression of matrix synthesis-related genes (ACAN and COL2A1), resulting in an imbalance between catabolic and anabolic events in NPCs58–60. Here, we measure the ability of NM-NTU to increase ATP levels in IL-1β-induced degenerated NPCs. We treated IL-1β-stimulated NPCs with a range of NM-NTU concentrations (0.125, 0.25, 0.5, 1.0, and 2.0 μg mL−1 for chlorophyll). The results revealed that as the concentration of NM-NTU increased from 0.125 to 0.5 μg mL−1 for chlorophyll, its ability to increase ATP in degenerative NPCs gradually increased, demonstrating the dose-dependent effects of NM-NTUs (Supplementary Fig. 7). A plateau was observed at NM-NTU concentrations higher than 0.5 μg mL−1 for chlorophyll. On the basis of these results, we selected a concentration of 0.5 μg mL−1 for chlorophyll in the subsequent in vitro experiments. Our further experiments also suggested that, compared with IL-1β group, the IL-1β + NM-NTU + dark group could not increase the intracellular ATP level (Fig. 4a). These results indicated that nonfunctional NM-NTUs (without light illumination) did not change the bioenergetics of cells and that light exposure was necessary for NM-NTUs to generate ATP in mammalian cells. To specifically quantify the ATP contribution from the NM-NTUs, we employed a pharmacological inhibition strategy to suppress endogenous cellular ATP production. We used 2-DG and rotenone to inhibit ATP production through glycolysis and oxidative phosphorylation in nucleus pulposus cells (NPCΔATP)61,62. Moreover, we used NM-NTUs + light to increase the ATP content in NPCΔATP, hypothesizing that the increased ATP in these cells was derived mainly from NM-NTUs. The results revealed that the rate of ATP production by NM-NTUs in NPCΔATP was 8.50 ± 1.42 pmol min−1 per 105 cells (Supplementary Fig. 8). Moreover, results of quantitative real-time polymerase chain reaction revealed that NM-NTUs under light illumination significantly upregulated Col2a1, Acan, and Sox9 and decreased Mmp3, Mmp13, and Adamts5 mRNA levels in degenerated NPCs (Fig. 4b). Immunofluorescence showed results consistent with the upregulation of Col II and aggrecan and the downregulation of MMP13 and ADAMTS-5 protein levels in NPCs treated with NM-NTUs + light, indicating that NTUs could improve the anabolism of degenerated NPCs (Fig. 4c).

Persistent ATP deficiency in cells is crucial in the dysregulation of cell metabolism. We used RNA-Seq to determine the possible mechanisms and molecular pathways underlying the protective effects of NM-NTUs on NPCs. Compared with the control group, the IL-1β group and the IL-1β + NM-NTU + dark group presented a greater number of differentially expressed genes, whereas compared with the control group, the IL-1β + NM-NTU + light group presented a lower number of differentially expressed genes (Fig. 4d; Supplementary Fig. 9a–d). In addition, we compared differentially expressed genes from the transcriptomes of normal and degenerative human NPCs, as well as from NPCs with and without IL-1β treatment. The results suggested that IL-1β treatment upregulated the Adamts7 (matrix degradation-related gene63) and Il1b genes and downregulated the Col2a1 (matrix synthesis-related gene) and Sesn3 (protection against oxidative stress64) genes. These differentially expressed genes were identical in the human transcriptome and exhibited consistent trends in the degenerative group, strengthening the validity of the IL-1β-induced NPC degeneration model. We further evaluated the effects of NM-NTUs on degenerated NPCs. PCA revealed distinct clustering among samples within groups, with similar gene expression profiles in the control and IL-1β + NM-NTU + light groups, while both groups clustered separately from the IL-1β and IL-1β + NM-NTU + dark groups (Fig. 4e). Furthermore, in contrast to the IL-1β + NM-NTU + dark group, the IL-1β + NM-NTU + light group presented lower levels of ROS-related gene (Prdx6) and mitochondrial apoptosis-related genes (Nptx1 and Bid) (Fig. 4f). GSEA results indicated that, compared to the control group, the IL-1β group and the IL-1β + NM-NTU + dark group presented upregulated expression of genes related to ROS signaling, negative regulation of [Ca2+]ER, and mitochondrial apoptosis and downregulated expression of genes related to ECM assembly (Supplementary Fig. 9e, f). In contrast to the IL-1β group and the IL-1β + NM-NTU + dark group, the IL-1β + NM-NTU + light group exhibited downregulation of genes associated with ROS signaling, negative regulation of [Ca2+]ER, and mitochondrial apoptosis and upregulation of genes associated with ECM assembly (Fig. 4g; Supplementary Fig. 9g). Taken together, these results demonstrated that NM-NTUs could restore metabolic homeostasis in degenerated NPCs in a light-dependent manner and might have positive effects on the ER and mitochondria within NPCs.

NM-NTUs correct dysfunctional organelle interaction network
We further evaluated the effect of NM-NTUs on the interaction network between the ER and mitochondria. The ER is the primary store of cytosolic Ca2+, and the ATP produced by NTUs may enhance the ER Ca2+ storage function through energy supplementation. To validate this finding, we utilized the genetically encoded ER Ca2+ indicator (G-CEPIA1er)65 to specifically monitor the ER intraluminal Ca2+ concentration ([Ca2+]ER) and used 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)66 to disrupt light-dependent ATP synthesis by NM-NTUs. The results showed that the NM-NTUs + light treatment significantly increased [Ca2+]ER compared with the NM-NTUs + dark treatment in 30 min (Supplementary Fig. 10a, b). Moreover, the inhibition of ATP production in NM-NTUs + light by the DCMU significantly attenuated the NM-NTU-induced increase in [Ca2+]ER (Supplementary Fig. 10a, b). Next, we evaluated [Ca2+]c using Fluo-4/AM dye. The NM-NTUs + light treatment decreased [Ca2+]c compared with that in the NM-NTUs + dark group (Fig. 5a, b). In summary, these results suggested that NM-NTUs might restore intracellular and ER Ca2+ homeostasis. Because ER Ca2+ perturbation may cause ER stress, we examined the effect of NM-NTUs on ER stress and found that NM-NTU effectively reduced the abundance of ER stress-related proteins (IRE1, ATF6, ATF4, and CHOP) in degenerated cells (Fig. 5c). We subsequently assessed the formation of MERCS by immunostaining the ER and mitochondria and observed that the NM-NTU + light treatment resulted in a decrease in MERCS (Fig. 5d, e). We further evaluated the effect of NM-NTUs on the ER stress-related MERCS structure by TEM. Compared with the IL-1β or IL-1β + NM-NTU + dark group, the IL-1β + NM-NTU + light group had significantly fewer ER closely apposed to mitochondria (Fig. 5f, g). ER length and mitochondrial counts were calculated for each field, and the results revealed no significant difference in the number of mitochondria or the average length of ER tubules among the four groups (Fig. 5h, i). An aberrant increase in MERCS causes mitochondrial Ca2+ overload and impairs cellular metabolic homeostasis67. As Ca2+ influx in the mitochondria can trigger mPTP opening, we measured [Ca2+]m and found that NM-NTU + light treatment caused an evident decrease in [Ca2+]m compared with that in the IL-1β group (Fig. 5j, k). Moreover, NM-NTU + light treatment significantly decreased the mPTP opening in degenerated NPCs (Fig. 5j, l). The continued opening of the mPTP may cause dissipation of the mitochondrial membrane potential and bioenergetic failure of the cell68. Next, we examined the effect of NM-NTUs on the restoration of mitochondrial function and found that NM-NTU + light treatment increased the mitochondrial membrane potential in degenerated NPCs (Fig. 5m, n). To investigate whether NM-NTU treatment led to increased ATP production from endogenous mitochondria in degenerated NPCs, we transfected IH-NPCs with a genetically encoded mitochondrial ATP indicator (mito-AT1.03) to measure changes in mitochondrial ATP concentrations69–71. The ratio of yellow fluorescence protein (YFP) to cyan fluorescence protein (CFP) was measured by flow cytometry to determine the mitochondrial ATP level. The results showed that following NM-NTUs + light treatment, the mitochondria within degenerated cells produced more ATP compared with the IL-1β group (Supplementary Fig. 11a, b). These results indicated that NM-NTUs might stimulate endogenous mitochondria in degenerated NPCs to produce ATP. In conclusion, while the NTUs themselves produced a quantifiable amount of ATP, a key therapeutic mechanism involved the NTU-generated ATP acting as a signal to restore the homeostasis of organelle interaction networks and promote the energy production of endogenous mitochondria. NTUs may also contribute to cellular metabolism through the generation of NADPH, which plays a critical role in redox homeostasis19,72. We further evaluated the effects of NM-NTUs + light on the regulation of intracellular NADPH levels. The results revealed that compared with that in the control NPCs, the NADPH content in the IL-1β-induced degenerated NPCs was decreased (Supplementary Fig. 12a). The IL-1β + NM-NTUs + light treatment significantly restored cellular NADPH levels compared with the IL-1β + NM-NTUs + dark group (Supplementary Fig. 12a). Furthermore, we found that IL-1β + NM-NTUs + light treatment resulted in reduced intracellular ROS levels compared with the IL-1β + NM-NTUs + dark group (Supplementary Fig. 12b, c). These findings supported the conclusion that, in addition to producing energy, NTUs might also help to mitigate cellular oxidative stress through the generation of NADPH. Studies have shown that the ER-mitochondrial interface contains a nanodomain of H2O2 originating from mitochondrial cristae73 and that high mitochondrial ROS levels promote MAM formation74. We hypothesized that NADPH produced by NTUs might indirectly affect the contact between mitochondria and the ER through the regulation of redox homeostasis. Our results indicate that NM-NTUs can restore intracellular Ca2+ homeostasis, inhibit abnormal MERCS formation, and reestablish a stable ER-mitochondrial interaction network (Fig. 5o).

NM-NTUs remodel ER lipid composition
ER-localized enzymes synthesize most cellular lipids, and the ER has a major influence on cellular lipid biomass and membrane biogenesis75. ER membrane lipid composition is altered in response to stressful conditions. For example, under prolonged nutrient and oxidative stress, the ER lipid composition is remodeled to involve an increased proportion of saturated phospholipids76. Moreover, acyl chain saturation of membrane lipids is crucial for membrane fluidity, with unsaturated lipid acyl chains enabling better fluidity and saturated lipid acyl chains forming nonfluid and packed structures77,78. Study has shown that increased membrane saturation/order leads to a loss of ER fluidity, which may affect the dynamic association and disassociation between organelles, further leading to an increase in MERCS44. Therefore, we investigated the lipidome of the ER to assess the relationships among ER lipid composition, membrane fluidity, and ER–mitochondria contact, as well as the regulatory role of NM-NTUs in this process.

We identified 877 metabolites in the ER lipidome, with 392 significantly different metabolites between the IL-1β + NM-NTU + light group and the IL-1β + NM-NTU + dark group (Fig. 6a; Supplementary Fig. 13a). PCA revealed distinct clustering among samples within groups, with similar metabolite profiles in the control and IL-1β + NM-NTU + light groups, while both groups clustered separately from the IL-1β and IL-1β + NM-NTU + dark groups (Fig. 6b). Compared with the IL-1β + NM-NTU + dark group, the IL-1β + NM-NTU + light group significantly affected Ca2+-dependent events and lipid metabolism (Fig. 6c; Supplementary Fig. 13b). Kyoto Encyclopaedia of Genomes enrichment analysis revealed that NM-NTU + light treatment affected pathways related to the biosynthesis of unsaturated fatty acids (Supplementary Fig. 13c).

We subsequently analyzed the distribution and changes in ER lipid categories among the groups. Compared with those in the control group, the triglyceride (TG) levels were significantly reduced in the IL-1β and IL-1β + NM-NTU + dark groups, whereas they were restored in the IL-1β + NM-NTU + light treatment group (Fig. 6d). The levels of other major ER lipid species (e.g., phosphatidylcholine, phosphatidylethanolamines, phosphatidylinositol, and sphingomyelin) did not change significantly among the groups (Fig. 6e–h).

We further used thin-layer chromatography (TLC) to determine the TG content in the ER of each group. Compared with the IL-1β + NM-NTU + dark group, the IL-1β + NM-NTU + light group contained more TGs (Fig. 6i). To address whether ER–mitochondria contact was related to TG levels, we modulated TG levels in the ER by applying inhibitors of TG synthesis. The final and committed step of TG synthesis is catalyzed by two diacylglycerol acyltransferases, DGAT1 and DGAT2, which are ER-resident transmembrane enzymes79. We treated NPCs with a DGAT1 inhibitor (A-922500) and a DGAT2 inhibitor (PF-06424439) or a combination of both. TLC analysis of ER lipids confirmed that inhibiting DGAT1 and DGAT2, either individually or in combination, reduced the TG levels in the ER (Fig. 6j). We then performed immunofluorescence experiments using calnexin and TOMM20 in cells treated with DGAT1 and DGAT2 inhibitors. Compared with the control group, the inhibition of TG synthesis significantly increased the number of MERCS (Fig. 6k, l). These results suggested a link between TG levels and the phenotype of MERCS. We further discuss the reasons for this link. Since inhibition of TG synthesis disrupts the channeling of fatty acids into neutral lipid storage80 and disruption of lipid homeostasis elicits ER stress81, the formation of MERCS may increase as an adaptive response to the unfolded protein response82,83. In general, our findings indicated that an altered ER lipidome (reduced TG levels) might contribute to increased ER–mitochondria contact.

We subsequently analyzed the acyl chain compositions of TG. Compared with those in the IL-1β + NM-NTU + dark group, the proportions of TG (54:3) [NL-18:1], TG (50:2) [NL-18:1], TG (54:2) [NL-18:0], TG (56:3) [NL-18:1], and TG (52:3) [NL-16:1] significantly increased in the IL-1β + NM-NTU + light group (Fig. 6m). The TG volcano map further confirmed these results (Fig. 6n). The proportion of unsaturated TG in the IL-1β + NM-NTU + light group was significantly greater than that in the IL-1β + NM-NTU + dark group (Fig. 6o). In summary, NM-NTU + light treatment increased the unsaturation levels of TGs. Given that lipid unsaturation significantly increased membrane fluidity77 and affected the contact between intracellular organelles44, we speculated that the NM-NTU reduced the formation of MERCS in degenerative cells by enhancing membrane fluidity. Next, we measured global membrane fluidity in NPCs using a membrane fluidity kit. Membrane fluidity was quantified by measuring the ratio of excimer to monomer fluorescence of lipophilic pyrene probes84. With increasing membrane fluidity, the probe underwent excimer formation. We found that NPCs in the IL-1β + NM-NTU + light group exhibited greater membrane fluidity than did those in the IL-1β + NM-NTU + dark group (Fig. 6p). Overall, NM-NTUs with light illumination might affect ER–mitochondria contact by remodeling the lipid composition of the ER.

NM-NTUs ameliorate IVDD in rats
To further evaluate the in vivo efficacy of the NM-NTUs, we constructed a puncture-induced IVDD model in rats85,86. Co7/8 intervertebral discs were selected for surgery (Fig. 7a). At 4 and 8 weeks post-surgery, micro-computed tomography (CT) radiographs showed that the intervertebral discs that underwent needle puncture without subsequent NM-NTU + light treatment had a low disc height index (DHI). The DHI of the NM-NTU + light treatment group was higher than that of the NM-NTU + dark group (Fig. 7b, c; Supplementary Fig. 14), indicating that the NM-NTU + light treatment maintained the DHI. DHI restoration is usually accompanied by increased disc water content. Magnetic resonance imaging (MRI) was used to assess disc water content in the different groups. The results showed that NM-NTU + light treatment significantly delayed progressive water loss (T2 signal intensity) in IVDD (Fig. 7d, e). We evaluated the histological changes in the intervertebral disc after surgery. The discs in the sham group showed intact and well-defined microstructures. Compared with that in the vehicle and NM-NTU + dark groups, NM-NTU + light treatment attenuated NP destruction at 4 and 8 weeks post-surgery (Fig. 7f, g). Alcian blue, safranin O-fast green, and Picrosirius red staining were used to further evaluate NP histological morphology (Fig. 7h–m). NP was replaced by fibrous tissues in the vehicle and NM-NTU + dark groups. In contrast, the NM-NTUs in the light groups showed a normal NP appearance and a clear boundary between the NP and the annulus fibrosus. Fluorescence staining was performed to investigate the NM-NTU in vivo mechanism of action. Compared with the vehicle and NM-NTU + dark groups, the NM-NTU + light group showed higher Col II and aggrecan protein levels in the intervertebral discs (Fig. 8a, b). Similarly, ER stress-related proteins (AFT4 and CHOP) were detected. The results showed that NM-NTU + light treatment reduced AFT4 and CHOP levels compared with those in the vehicle and NM-NTU + dark groups (Fig. 8a, b). We subsequently conducted immunofluorescence costaining of calnexin and TOMM20 in rat IVD tissue sections. The results indicated that compared with the sham group, MERCS increased in the puncture + vehicle and puncture + vehicle + NM-NTU + dark group. Moreover, MERCS significantly decreased in the NM-NTU + light treatment group compared with the NM-NTU + dark treatment group (Fig. 8c, d), which revealed that the abnormal contact between the ER and mitochondria in rat IVDD was attenuated. We further extracted cells from the NP tissue and performed TEM to further evaluate the MERCS in the tissue samples. For each field of view, we systematically quantified the ER tubules that were closely apposed to the mitochondria, the number of mitochondria, and the average length of the ER tubules. Interestingly, the NM-NTU + light group showed a significant reduction of ER closely apposed to mitochondria compared with the puncture + vehicle and puncture + NM-NTU + dark group (Fig. 8e, f). ER length and mitochondrial counts were calculated for each field, and the results revealed no significant difference in the number of mitochondria or the average length of ER tubules among the four groups (Fig. 8g, h). These results provided partial evidence that the in vivo therapeutic effects of NM-NTUs were functionally linked to their ability to restore the organelle interaction network. In addition, histological staining of multiple organs indicated NM-NTU biosafety in vivo (Supplementary Fig. 15). Taken together, these results reveal that NM-NTUs have therapeutic effects in rat IVDD models.

Remote wireless controllable device for deep illumination
Although NM-NTU has shown therapeutic potential in rats, its application to the intervertebral discs of larger animals may be limited by poor light penetration. Therefore, developing a safe and implantable light device for NTU treatment is crucial. The development of wireless power technology facilitates light delivery to deep tissues through wireless devices87. First, we assembled the wp-LED by connecting the LED parallel to the inductor and capacitor (Fig. 9a; Supplementary Fig. 16). Various color lighting was achieved using wireless charging coils (Fig. 9b). To enable remote control of the wp-LED, we connected the wireless coil to a smart socket, which served as the receiver. Hence, we controlled the receiver using a mobile phone (via a wireless network) and remotely controlled the wp-LED switch (Supplementary Movie 1). In addition, the lighting duration was precisely controlled using a mobile phone (Supplementary Movie 2). As wp-LEDs need to be implanted in the body, an insulating, biocompatible, and nondegradable transparent packaging material is required. Polyurethane (PU) materials have been used for the insulation packaging of cardiac implantable electronic devices because of their good biocompatibility and non-degradability88. Therefore, PU was used to package the wp-LED (PU-wp-LED), minimizing the risk of inflammation and device rejection. The PU-wp-LED was immersed in a phosphate buffer solution to confirm whether it could be insulated in a wet environment. The results showed the effectiveness of PU encapsulation, as the wp-LED remained functional and emitted light (Fig. 9c). Subsequently, we assessed the wp-LED output efficiency. In the horizontal plane, the closer to the center of the coil, the lower the brightness of the wp-LED, according to the principle of electromagnetic induction. In the vertical plane, the wp-LED light intensity decreased with increasing distance from the coil center (Fig. 9d). The PU and PU-wp-LED leachates also exhibited no evident cytotoxicity (Fig. 9e). We subcutaneously implanted the PU-wp-LEDs to test their feasibility in animals. Upon activating wireless power via the mobile phone, the device effectively emitted red light from the rabbit subcutaneous tissue (Fig. 9f; Supplementary Movie 3). Similarly, the results of rabbit body temperature and blood tests showed that the PU-wp-LED exhibited good biosafety (the early postoperative temperature increase may have been caused by surgery) (Fig. 9g; Supplementary Table 1). Furthermore, we simulated deep intervertebral disc illumination by surgically implanting a PU-wp-LED ventral to the disc and injecting NM-NTUs into the disc using X-ray fluoroscopy (Fig. 9h, i). The CT and X-ray results confirmed fixation of the PU-wp-LED ventral to the intervertebral disc and the feasibility of intradiscal NM-NTU injection (Fig. 9i, j). Moreover, we simulated the use of PU-wp-LED on the human body, in which the device was fixed to the human forearm. When the mobile power supply was turned on, the red light was effectively emitted, confirming the potential application value of this device (Supplementary Fig. 17). Overall, we established a therapeutic approach for treating deep degenerative tissues using wireless-powered light combined with NTUs.

PU-wp-LED combined with NM-NTUs alleviate IVDD in rabbits
To determine whether the light from the PU-wp-LED had sufficient intensity to activate NM-NTUs, we first determined that NM-NTUs had significant therapeutic effects in cell experiments with light intensities above 10.3 μmol photons m−2 s−1 (Supplementary Fig. 18a). This light intensity was defined as the light threshold for the activation of NM-NTUs. Furthermore, in the in vivo rabbit photoirradiation experiment, we measured the distance from the back skin of the intervertebral disc to the anterior longitudinal ligament by MRI, which was 4.45 ± 0.36 cm (Supplementary Fig. 18b). We then estimated that the PU-wp-LED output power in the rabbit body (intervertebral disc area) within the coil was approximately 60–70 μmol photons m-2 s-1. Considering the approximately 60% red light transmission efficiency through the annulus fibrosus (Supplementary Fig. 18c), we estimated that the light intensity reaching the nucleus pulposus was above the activation threshold of NM-NTUs. We further verified the therapeutic effect of PU-wp-LED combined with NM-NTUs in the rabbit IVDD model (Fig. 10a). At 4 and 8 weeks post-surgery, MRI results showed the signal intensity of discs in the NM-NTU + PU-wp-LED + light group reduced less compared to the dark group (Fig. 10b, c). Then we obtained fresh disc tissues and observed their appearance. The discs in the vehicle group and NM-NTU + PU-wp-LED + dark group both showed severe degeneration, while the NM-NTU + PU-wp-LED + light group showed mild changes compared with sham group (Fig. 10d). Moreover, compared to the dark group, the NM-NTU + PU-wp-LED + light group attenuated NP degeneration in histological analysis (Fig. 10e–h). We also detect the protein levels of Col II and aggrecan in different groups. The NM-NTU + PU-wp-LED + light treatment elevated both Col II and aggrecan levels compared to the dark group (Fig. 10i). We also conducted experiments in the rabbit IVDD model, comparing PU-wp-LED and external light (Supplementary Fig. 19a). Compared with the NM-NTU + external light group, MRI results revealed that discs treated with the NM-NTU + PU-wp-LED + light maintained significantly greater signal intensity, indicating better water content and reduced disc dehydration (Supplementary Fig. 19b, c). Histological evaluation via H&E staining further supported these findings. In the NM-NTU + PU-wp-LED + light group, the overall disc morphology appeared well preserved compared with that in the NM-NTU + external light group (Supplementary Fig. 19d). Histological scoring based on an established grading system confirmed a significant improvement in the NM-NTU + PU-wp-LED + light group compared with the NM-NTU + external light group (Supplementary Fig. 19e). Overall, compared with direct external light, the PU-wp-LED system achieved favorable therapeutic outcomes in the rabbit IVDD model. The reason for the external light system failure might be its inability to sufficiently penetrate dorsal anatomical structures and reach deep tissues. Taken together, the remote controllable wireless irradiation device combined with NM-NTU effectively alleviated deep disc degeneration in rabbits.

In summary, organelle interaction networks can become dysfunctional under pathological conditions, such as degeneration. Implanting plant-derived independent photosynthetic systems into mammalian cells can effectively restore homeostasis of the interaction network of animal organelles, potentially owing to cellular energy supply by NTUs. Furthermore, NTUs can regulate intracellular Ca2+ homeostasis and membrane fluidity by increasing ER lipid unsaturation and reducing MERCS formation and mitochondrial dysfunction. By combining implantable LEDs with wireless charging technology, we expanded photosynthesis therapy, enabling smarter and more effective treatments for deeper tissues. This treatment model has broad application prospects for various diseases involving imbalanced organelle interaction networks.

Methods
Cell culture
Rat and human NPCs were obtained from NP tissues by collagenase digestion for 4 h89. Rat AFCs were obtained from annulus fibrosus tissues by collagenase and hyaluronidase digestion for 1.5 h90. Rat EPCs were obtained from endplate cartilage tissues by collagenase digestion for 6 h91. Rat NP tissues, annulus fibrosus tissues and endplate cartilage tissues were macroscopically isolated from 4-week-old male Sprague–Dawley rats. Rat NPCs, AFCs, and EPCs were seeded with media changed every 72 h, passaged when the confluence reached 80%, and utilized before passage 2. Human NP tissues were obtained from human participants undergoing spinal surgery for vertebral fractures or IVDD. Cultures of primary human NPCs were maintained under standard conditions (5% CO2 and 37°C), and the medium was changed every 72 h. The human NPCs were passaged on the sixth day. To evaluate the impact of in vitro culture on the phenotypic changes in degenerated human NPCs, cell samples were collected every other day from day 1 to day 9 of culture. These samples were subsequently used for gene and protein analysis of NPC phenotypic markers. The use of patient samples was approved by the Ethics Committee of Sir Run Run Shaw Hospital. Informed consent was obtained from all the patients involved in the study. The cells were seeded in a humidified incubator and cultured in Dulbecco’s modified Eagle medium (DMEM), supplemented with 10% fetal bovine serum (Gibco). For plasmid transfection, we used IH-NPCs (iCell Bioscience, Inc., iCell-0028a), which were cultured in specialized culture medium (iCell Bioscience, Inc.).

NTU and NM-NTU preparation
Chloroplasts were isolated from fresh spinach leaves using differential centrifugation and Percoll density gradient centrifugation, and then suspended in a hypotonic lysis buffer to obtain thylakoids19,92. The thylakoids were extruded sequentially through polycarbonate membranes with pore sizes of 800-, 400-, 200-, and 100-nm to obtain NTUs. The NPC membranes were isolated from disrupted NPCs by differential centrifugation19 and suspended in water at a concentration of 2 mg mL−1. Subsequently, the NPC membrane solution was mixed with an equal volume of the NTUs and allowed to stand for 30 min. Next, the mixed solution was filtered through 1000-, 400-, and 200-nm polycarbonate membranes using an Avanti mini extruder. NM-NTUs were obtained through centrifugation at 10,000 × g for 5 min and were resuspended for further use. The chlorophyll content was determined by spectrophotometric analysis93. To evaluate the encapsulation efficiency of the NTUs by NM, we used nanoFCM. NTUs were stained with Star Red and detected in the PC5 channel. The NMs were labeled with a FITC-conjugated marker, allowing their specific detection in the FITC channel. Different NTU and NM protein ratios (ranging from 1:10 to 1:200) were tested. Events that were positive for both FITC and PC5 (FITC+/PC5+) were classified as NMs successfully loaded with NTUs, whereas events that were positive for FITC but negative for PC5 (FITC+/PC5-) were categorized as empty NMs. Events that were positive for PC5 but negative for FITC (FITC-/PC5+) were categorized as free NTUs. NTU and NM-NTU morphologies were observed using cryo-TEM (200 kV, FEI Tecnai G2 F20). The size and surface zeta potential of the nanoparticles were determined via dynamic light scattering using a Malvern Zetasizer Nano ZS49. SDS-PAGE and Coomassie Blue (Beyotime) staining were used to detect proteins in the thylakoids, NTUs, NPC membranes, and NM-NTUs.

Antibodies
The following antibodies were used for western blotting: CHOP (Proteintech, 15204-1-AP, 1:1,500); IRE1 (Proteintech, 27528-1-AP, 1:1,500); ATF6 (Proteintech, 66563-1-Ig, 1:10,000); ATF4 (Proteintech, 60035-1-Ig, 1:2000)；β-actin (Proteintech, 20536-1-AP, 1:5000).

For immunofluorescence analysis, the following antibodies were used: KRT19 (Proteintech, 10712-1-AP, 1:200), CD24 (Proteintech, 10600-1-AP, 1:200), CHOP (Proteintech, 15204-1-AP, 1:100); ATF4 (Proteintech, 60035-1-Ig, 1:200); ColII (Proteintech, 28459-1-AP, 1:800); aggrecan (Proteintech, 13880-1-AP, 1:200); MMP13 (Proteintech, 18165-1-AP, 1:200); ADAMTS-5 (Affinity Biosciences, DF13268, 1:200); TOMM20 (Abcam, ab56783, 1:200); Calnexin (Proteintech, 10427-2-AP, 1:200).

Cell viability assay
Human NPC viability was evaluated using two independent assays: the MTT assay (Beyotime) and the live/dead staining assay (Beyotime). An MTT assay was performed to measure cellular metabolic activity. Briefly, yellow, water-soluble MTT tetrazolium salt was added to the cell culture. The absorbance at 570 nm was measured spectrophotometrically. A live/dead staining assay was used to simultaneously distinguish viable cells from nonviable cells on the basis of membrane integrity. Cells were stained with a combination of calcein AM and propidium iodide. Images were acquired using a laser confocal microscope (LCM, Nikon), and the percentages of live and dead cells were quantified using Fiji/ImageJ software. Rat NPCs were incubated with NTU or NM-NTUs (0.5 μg mL−1 for chlorophyll) for 24 h. A CCK-8 assay was performed to determine cell viability (Dojindo).

Cellular uptake of NM-NTUs
We evaluated the cellular uptake of the NM-NTUs by rat AFCs, EPCs and NPCs. Briefly, cells were incubated with a medium containing NM-NTUs (DiI labeled NTUs, 0.5 μg mL−1 for chlorophyll) for 6 h, followed by washing, fixation with 4% formalin, and DAPI staining. The cells were imaged and analyzed using an LCM (Nikon). NPCs were cultured, labeled with DiI, and treated with a medium containing NM-NTUs (DiO labeled NM, 0.5 μg mL−1 for chlorophyll) for 1 h to detect the membrane fusion ability of NM-NTUs. Samples were fixed with 4% formalin, stained with DAPI, and photographed using an LCM (Nikon).

Intracellular trafficking of NTUs in NPCs
NTUs or NM-NTUs (DiI labeled NTUs, 0.5 μg mL−1 for chlorophyll) were co-cultured with rat NPCs for 6 h, and lysosomes were labeled with lysoTracker Green (Beyotime) to investigate the co-localization of NTUs and lysosomes in cells. Subsequently, cells were fixed with 4% formalin and stained with DAPI; images were captured using an LCM (Nikon). Next, Mander’s overlap coefficients of lysosomes and NTUs were analyzed using Fiji/ImageJ (JACoP plugin). Colocalization studies were performed to determine the intracellular distribution of internalized NM-NTUs. After rat NPCs were incubated with NM-NTUsDiI (NTUs labeled with DiI) for 6 h, the samples were stained for specific cellular structures, including the endoplasmic reticulum (ER-Tracker Green, Beyotime), mitochondria (Mito-Tracker Green, Beyotime), Golgi apparatus (Golgi-Tracker Green, Beyotime), actin filaments (Actin-Tracker Green, Beyotime), and microtubules (Tubulin-Tracker Green, Beyotime). Confocal microscopy images were acquired, and colocalization was quantitatively analyzed using Mander’s overlap coefficients. The Mander’s coefficients (M1) quantify the fraction of the signal intensity of NTUs that colocalizes with lysosomes relative to the total signal intensity of NTUs.

To visualize MERCS, IH-NPCs were transfected with SPLICSMt-ER Short P2A (Addgene plasmid #164108) encoding the mitochondrial outer membrane-targeted OMM-GFP1-10 and the endoplasmic reticulum-targeted short protein GFPβ11 (1 μg plasmid of a 35 mm glass bottom dish) using Lipofectamine 3000, strictly following the manufacturer’s protocol. At 36–48 h post-transfection, the cells were treated with DiI-labeled NM-NTUs for 6 h. After treatment, the cells were imaged using confocal microscopy. The reconstituted GFP signal, which indicates close-range (~8–10 nm) MERCS, and the DiI signal (NTUs) were captured. Colocalization between NTUs and MERCS puncta was quantitatively analyzed using the JACoP plugin in Fiji/ImageJ software, with the results expressed as the Mander’s coefficient (M1) representing the fraction of the NTU signal overlapping with MERCS.

Effects of NM-NTUs on cell metabolism
Rat NPCs were treated with IL-1β (20 ng mL−1) for 24 h and incubated with NM-NTUs (0.5 μg mL−1 for chlorophyll) for 6 h, with or without red light irradiation (from light-emitting diodes, peaking at 630 nm at an intensity of 80 μmol photons m−2 s−1, 30 min). The NPCs were incubated with the corresponding antibodies for immunofluorescence staining. Images were captured using an LCM (Nikon, Tokyo, Japan). Images were analyzed and fluorescence intensity was measured using the Fiji/ImageJ software. The intracellular ATP concentration was analyzed using an ATP assay kit (Beyotime). To evaluate the ATP production efficiency of the NM-NTU at different concentrations, we measured ATP levels under NM-NTU concentrations ranging from 0.125 to 2 μg mL−1 for chlorophyll. Similarly, ATP production under varying light conditions (10.3–108.1 μmol photons m−² s−¹) was also assessed. To specifically quantify ATP production from NM-NTUs, we inhibited mitochondrial oxidative phosphorylation and glycolysis using rotenone (500 nM) and 2-DG (10 mM). After the NM-NTUs were subjected to light treatment for 30 min, ATP levels were measured using an ATP assay kit. For intracellular NADPH quantification, cells from each group were lysed and analyzed using an NADPH assay kit (Beyotime) according to the manufacturer’s instructions.

Assessment of endogenous mitochondrial functional recovery
IH-NPCs were transfected with pCMV-Mito-AT1.03 (Beyotime, D2606). Cells were seeded in 6-well plates, and each well was transfected with 1 μg of plasmid using Lipofectamine 3000. At 24–36 h post-transfection, the cells were treated with IL-1β for an additional 24 h. Mitochondrial ATP levels were subsequently quantified by flow cytometry on the basis of the ratio of YFP to CFP fluorescence following NM-NTUs + light treatment.

Quantitative polymerase chain reaction (qPCR)
Total RNA was extracted from the lysed NPCs using the SteadyPure RNA Extraction Kit (Accurate Biology). RNA was reverse-transcribed into cDNA using Evo M-MLV RT Master Mix (Accurate Biology), and qPCR was performed using the Hieff UNICON qPCR SYBR Green Master Mix on a QuantStudioTM 6 Flex Real-Time PCR System (Thermo Fisher Scientific). Target gene sequences are listed in Supplementary Table 2.

Determination of cytoplasmic and mitochondrial Ca2+ concentration
NPCs were loaded with 2 μM Rhod-2/AM (50 min; Yeasen). The cells were then washed and reloaded with 5 μM Fluo-4/AM (30 min; Beyotime)94. Images were recorded using an LCM (Nikon). Image analysis and fluorescence intensity measurements were performed using the Fiji/ImageJ software.

Detection of ER Ca2+ concentration
To investigate the contribution of photosynthetic ATP to ER calcium storage, IH-NPCs were transfected with pCMV G-CEPIA1er (Addgene# 58215), which encodes an ER calcium indicator65. Photosynthetic ATP production in the NM-NTU was inhibited by treatment with 10 μM DCMU. The experimental design included eight treatment groups analyzed at 0, 15, and 30-min time points: control, IL-1β, IL-1β + NM-NTU + dark, and IL-1β + NM-NTU + light, with each condition further divided into subgroups either treated with or without DCMU. Cells were imaged using an LCM (Nikon), and quantitative analysis of fluorescence intensity was performed using Fiji/ImageJ software to assess [Ca2+]ER.

mPTP evaluation
The mPTP of the NPCs was evaluated based on the calcein release assay95 using an mPTP assay kit (Beyotime) and photographed using an LCM (Nikon).

MERCS observation
MERCS structures of the NPCs were observed using TEM (H-9500; Hitachi). MERCS in TEM images were manually analyzed in Fiji/ImageJ. Briefly, the image scale was first set with the Set Scale tool, and the freehand line tool was subsequently used to measure the length of the ER or membrane or the perimeter of the mitochondria. The contact length was defined as the ER membrane length (within 0–100 nm of the mitochondria) divided by the mitochondrial perimeter. Mitochondria and ER architecture in the TEM images were reconstructed graphically using Adobe Illustrator software. Similarly, the MERCS structure of the NPCs was observed by immunostaining the ER and mitochondria with calnexin and TOMM20. To quantify MERCS, we performed colocalization analysis using Fiji/ImageJ plugins (Coloc2 and JACoP) following established methods96. The Mander’s coefficients (M1) quantify the percentage of the signal intensity of mitochondria that colocalizes with the ER relative to the total signal intensity of the mitochondria.

Membrane fluidity detection
The global membrane fluidity of the NPCs was evaluated using a membrane fluidity kit (Abcam), which utilizes fluorescent lipophilic pyrene probes that are incorporated into the cell membrane. The monomeric pyrene probe undergoes excimer formation, shifting its emission from Em 400 nm to Em 470 nm (excitation at 360 nm). Membrane fluidity was quantified by determining the ratio of excimer (Em 470 nm) to monomer (Em 400 nm) fluorescence using a fluorescence microplate reader (BioTek).

Intracellular ROS and mitochondrial membrane potential detection
A ROS assay kit (Beyotime) was used to detect intracellular ROS. The DCFH-DA (Beyotime) fluorescence emission was acquired by flow cytometry or LCM. Furthermore, a JC-1 fluorescent probe (Solarbio) was used to detect mitochondrial membrane potential in NPCs. Image analysis and fluorescence intensity measurement were performed using the Fiji/ImageJ software.

Western blot analysis
NPCs were lysed using RIPA buffer (FDbio). SDS-PAGE gels (FDbio) were used to separate the proteins. Proteins were transferred onto polyvinylidene difluoride membranes (FDbio) after electrophoresis. After blocking with skim milk (FDbio) and incubation with the primary antibody, bands were developed using Horseradish peroxidase-labeled secondary antibodies (Beyotime). Following chemiluminescence detection, bands were visualized using the ChemiDoc Touch imaging system (Bio-Rad).

Transcriptomic and lipidomic study
Human NP tissues and rat NPCs were used for transcriptomic analysis. Briefly, total RNA was extracted using TRIzol reagent, following the manufacturer’s instructions. Sequencing was performed using an Illumina NovaSeq 6000 following the manufacturer’s protocol recommended by the supplier. Genes with a P value < 0.05 and an absolute log(fold changes) ≥ 1 were considered differentially expressed. GSEA was performed using GSEA software (https://www.broadinstitute.org/gsea/). Volcanomaps and other visual analyses were performed using the OmicStudio tools (https://www.omicstudio.cn/tool).

For the lipidomic experiment, the ER from rat NPCs was isolated using an ER isolation kit (Sigma), and sample pretreatment and analysis were conducted. Data analysis and acquisition were primarily performed using a Vanquish Flex UHPLC system (Thermo Fisher Scientific) and a TSQ Altis Plus mass spectrometer (Thermo Fisher Scientific). Quantification of lipid species was determined by comparison to the relevant internal standard using Skyline 22.2.0527. All detected lipids were annotated and classified based on the lipid databases (https://www.lipidmaps.org/; http://lipidontology.com/). Metabolites with P < 0.05 and fold change > 10% were considered significant.

TLC analysis
Thin-layer chromatography (TLC) was used for TG quantification97. Specifically, the total lipids in the ER extracts were obtained by suspending the ER in 1 mL of PBS supplemented with 0.1 mM phenylmethylsulphonyl fluoride (PMSF) and 2 mL of chloroform/methanol (2:1 vol/vol) supplemented with 0.01% butylated hydroxytoluene. After centrifugation at 1500 × g for 5 min, the organic phases were collected and dried with nitrogen and resuspended in chloroform. TLC was performed by spotting the total lipid extracts dissolved in chloroform onto 10 cm EMD TLC Silica Gel plates (Labshark) and developing them with hexane/diethyl ether/acetic acid (80:20:2, vol/vol). Lipids were visualized with iodine vapor prior to imaging.

PU-wp-LED assembly
The 220 pF multi-layer ceramic capacitor (221K, 0603) and surface-mount-device (SMD) LED (0805) were soldered on a 2.2 MH SMD power inductor (222, CD43) in parallel. Subsequently, wp-LED was encapsulated in PU and wrapped with poly-wires. The PU-wp-LEDs were detected immediately after assembly using a wireless charging coil (XINKETAI Electronic Co., Ltd.). The light intensity of the device was measured using a spectral illuminance meter (Linshang LS330). A laser power meter (Zhongxi Equipment) was used to measure the efficiency of red light penetrating the annulus fibrosus (3 mm thickness) from the sheep.

Biosafety of PU-wp-LED
To determine the biosafety of PU or PU-wp-LED in vitro, we soaked PU or PU-wp-LED in DMEM for 48 h to obtain the leachate. Subsequently, the NPCs were cultured with the leachate for 24 h. CCK-8 assay (Dojindo) was used to detect cell viability.

Animal models
The animal use protocol was reviewed and approved by the Institutional Animal Care and Use Committee of the Zhejiang Center of Laboratory Animals. Male Sprague–Dawley rats (12 weeks old) were used for in vivo experiments. The IVDD rat model was established using the needle puncture method98. First, rats were anesthetized with pentobarbital sodium. Subsequently, the IVDD models were established using a 26G needle puncture of the coccyx spaces Co7–8. The needle reached the NP center and was removed after 30 s. Rat were randomly divided into four groups, namely sham surgery (sham), puncture and 5 μL vehicle (Puncture + Vehicle) injection per week, puncture + 5 μL NM-NTUs (20 μg mL−1 for chlorophyll) injection per week without red light irradiation (Puncture + NM-NTUs + dark), and puncture + 5 μL NM-NTU injection (20 μg mL−1 for chlorophyll) per week with red light irradiation for 30 min daily (Puncture + NM-NTUs + light). At week 4 and 8, the rats were euthanized for further evaluation.

Male New Zealand rabbits (6–7 months old) were used for in vivo experiments. The rabbits were anesthetized with pentobarbital sodium and placed in a prone position on a platform. The PU-wp-LEDs were implanted subcutaneously to assess their safety and lighting performance. Briefly, a small incision was made in the dorsal skin of the rabbit, and the subcutaneous tissue was separated using a blunt probe; subsequently, the PU-wp-LED was inserted subcutaneously, and the wound was sutured (PU-wp-LED group). The illumination capability of the PU-wp-LED in vivo was tested immediately after surgery. The sham group underwent a surgical procedure similar to that of the implantation group; however, it did not involve PU-wp-LED implantation (sham group). The body temperature of the rabbits was measured daily for 1 week after implantation, and routine blood tests were performed 2 weeks after implantation. For the intervertebral disc illumination simulation, rabbit spines were exposed using a lateral retroperitoneal approach with aseptic techniques. Next, PU-wp-LED was implanted anterior to the L4/5 disc, fixed to the soft tissue structure with a silk thread, and the incision was closed. The intervertebral disc was punctured using a 20G needle under X-ray guidance to simulate deep NM-NTU injection.

For rabbit IVDD treatment, the rabbits were anesthetized and their spines were exposed. L4/5 IVD were punctured with an 18G needle at a depth of 5 mm, then PU-wp-LED was implanted anterior to the L4/5 disc and fixed to the soft tissue structure with silk thread, and the incision was closed. The following intra-disc injection was via X-ray fluoroscopy guide. Rabbits were randomly divided into 4 groups: sham surgery (sham), puncture and 10 μL vehicle (Puncture + Vehicle) injection per week, puncture + 10 μL NM-NTUs (20 μg mL−1 for chlorophyll) injection per week with PU-wp-LED turn off (Puncture + PU-wp-LED + NM-NTUs + dark), and puncture + 10 μL NM-NTU injection (20 μg mL−1 for chlorophyll) per week with PU-wp-LED turn on for 30 min daily (Puncture + PU-wp-LED + NM-NTUs + light). At week 4 and 8, the rabbits were euthanized for further evaluation. And PU-wp-LED were removed before imaging evaluation. To compare the effects of implantable versus non implantable optical stimulation, the rabbits were randomly assigned to one of four groups: sham surgery (sham), puncture and 10 μL vehicle (Puncture + Vehicle) injection per week, puncture + 10 μL NM-NTUs (20 μg mL−1 for chlorophyll) injection per week with external red light illumination for 30 min daily (Puncture + NM-NTUs + External illumination), and puncture + 10 μL NM-NTU injection (20 μg mL−1 for chlorophyll) per week with PU-wp-LED turn on for 30 min daily (Puncture + NM-NTUs + PU-wp-LED illumination). The PU-wp-LED implants were removed prior to imaging evaluation. All the animals were euthanized at week 8 for subsequent analysis.

In vivo radiological assessment
Magnetic resonance imaging was performed on vertebral samples 4 and 8 weeks postoperatively using a GE Sigma CV/I (1.5 T). The rat caudal vertebral samples were scanned using micro-CT (Skyscan 1275). We defined the regions of interest to cover the upper and lower normal intervertebral spaces. The calculation method for DHI is shown in Supplementary Fig. 14. DHI was calculated using the following equation:
DHI = 2 × (A + B + C) / (D + E + F + G + H + I) (1)
where A, B, and C represent the disc height between adjacent vertebrae; and D, E, F, G, H, and I represent the length of the vertebral bone immediately rostral and caudal to the intervertebral disc. Changes in the DHI were expressed as a percentage (%DHI = measured DHI / Sham group DHI × 100). Fiji/ImageJ software was used to measure the signal intensity on the T2-weighted central images. A CT scan was used to confirm the fixation of the PU-wp-LED anterior to the rabbit intervertebral disc.

Histological analysis
Disc samples were fixed, decalcified, and embedded to obtain the sections. These sections were stained with hematoxylin and eosin (H&E), Alcian blue, Safranin O-fast green, or Picrosirius red. The disc samples were scored according to the table shown in Supplementary Table 3. Subsequently, the sections were incubated with the corresponding antibodies for immunofluorescence staining. Rat major organs were collected at 8 weeks for systemic toxicity assessment (through H&E staining).

Statistics and reproducibility
Data were presented as mean ± standard deviations. The statistical analysis methods were described in the figure legends. Unpaired two-tailed Student’s or Welch’s t test (two independent groups) and one-way analysis of variance with Tukey’s post hoc test (three or more groups) were used. Ordinal grading system data were analyzed using the Kruskal–Wallis and post-hoc Dunn’s tests. Statistical significance was set at P < 0.05. Statistical analyses were performed using Excel and GraphPad Prism software version 10.0. Particle size analysis, zeta potential, SDS-PAGE, cryo-TEM, Western blot, and in vitro fluorescence imaging experiments were independently repeated three times. H&E staining of major organs in rats were replicated in two independent experiments.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study explores a novel approach to restoring organelle interaction network homeostasis in animal cells by introducing plant-derived photosynthetic systems. Under stress, animal cells often suffer from dysregulated organelle interactions, while plant cells exhibit robust adaptive mechanisms, partly due to chloroplast activity. The researchers delivered nanothylakoid units (NTUs) from plants into animal cells, demonstrating their ability to provide energy, regulate Ca²⁺ and lipid homeostasis, improve membrane fluidity, and reduce mitochondrial dysfunction. By integrating wireless-powered implantable LEDs, the therapy was extended to deeper tissues, offering a proof-of-concept for treating diseases through photosynthetic regulation of organelle networks. The work highlights the potential of cross-kingdom organelle-inspired therapeutics for enhancing cellular stress tolerance. This article already demonstrates a high level of academic quality, and through detailed refinement, its persuasiveness and clarity can be further enhanced.\n1.It is suggested that after mentioning \"animal cells lack stress resistance\" (lines 56–57), a couple of sentences should be added to explain the current technological bottlenecks in regulating organelle network homeostasis in the medical field (e.g., existing methods only target individual organelles), thereby more directly pointing to the innovation of this study.\n2.It is recommended to use the sentence structure \"This study aims to address...\" in the final paragraph of the introduction (or in a separate paragraph) to clearly state the research question, for example: \"This study aims to explore whether plant-derived NTUs can restore homeostasis in animal cells under pathological conditions by modulating the ER-mitochondria interaction network.\".\n3.The full name of \"MAM\" (mitochondria-associated ER membrane) was provided when it was first mentioned (line 74), and it appears again at line 124. It is recommended to check the abbreviation consistency throughout the text.\n4.Fig. 6 shows an increase in lipid unsaturation, but it does not directly prove the causal relationship between it and membrane fluidity or the reduction of MAM. Additional references could be cited to support the inference of \"unsaturation → fluidity → reduction of MAM.\".\n5.Fig. 9d mentions that the brightness of the LED varies with distance. What is the minimum light intensity threshold required for effective treatment?\n6.It is necessary to clearly describe the duration of cultivation after extracting NPCs from degenerated IVDD before staining and imaging, as the environment of cell culture in vitro differs from the microenvironment of degenerative changes in vivo. If the time is prolonged, the cells are likely to undergo alterations.\n7.Why does the Dil fluorescence appear in the nucleus in the NTU group image of Figure 3i?", "reviewer_2": "This is an interesting work demonstrating the therapeutic effect of NM-NTU in IVDD. The authors engineered NM-NTU to target nucleus pulposus cells within the intervertebral disc, exerting therapeutic effects by correcting their energy metabolism disorders and abnormal ER-mitochondria interactions. Moreover, the authors combined wp-LED to ensure that NM-NTU is applicable to deep tissues - which is the real environment where IVDD occurs in clinical. Although the authors have spent a lot of effort verifying the mechanism by which NM-NTU restores NPCs metabolism at the cellular level, there are some issues that need to be addressed through major revisions.\n1. Page 5, Line 129: From Fig 1h, the co-localization level of ER and mitochondria does not show a significant difference, although the authors claimed a significant difference in their co-localization level.\n2. Page 6, Line 163: If I understand correctly, the authors here want to show that the engineered NP-derived cell membrane NTU has the function of targeting NP cells, which is crucial for its role in IVDD. However, using RAW as the control cell to verify its targeting ability is confusing, as in the IVD environment, the main cells present are nucleus pulposus cells, annulus fibrosus cells, and endplate chondrocytes, which would be more suitable as controls to verify the targeting ability of NM-NTU.\n3. Page 6, Line 169: As shown in Fig 3h, NM-NTU is taken up by NP cells in a membrane fusion form, so its tracer dye almost perfectly aligns with the cell membrane. However, in Fig 3i, NM-NTU seems to change its intracellular distribution form and no longer presents a membrane distribution. The difference needs to be carefully explained by the authors. Additionally, based on the concept of membrane fusion, once the outer membrane structure fuses with the plasma membrane, its contents will be released into the cytoplasm. Therefore, detecting the subcellular localization of NTU after NM-NTU uptake is crucial for elucidating its mechanism of action.\n4. Page 7, Line 188: As the authors discussed in Fig 4a-c, NM-NTU can target and restore the intracellular metabolic processes of NP cells in a light-dependent manner. However, in the subsequent RNA-seq results, NM-NTU without light (if I understand correctly) also has the effect of altering the transcriptional process of NP cells, which is confusing. I think it is very important to compare the conditions with and without light.\n5. Page 9, Line 245: Similar to the previous issue, in the detection of metabolic changes in NPCs, the authors compared the blank, IL-1b, and IL-1b + NM-NTU groups but ignored the important factor of light. Therefore, the role of light in activating NM-NTU becomes ambiguous and confusing.\n6. Page 13, Line 356: Using a wirelessly controllable implantable LED to stimulate NM-NTU is an interesting idea because one of the major challenges in IVDD treatment is that the lesions are often located deep within the tissue, making light stimulation difficult to reach. However, the authors did not compare the therapeutic effects of NM-NTU under implantable LED and non-implantable light stimulation conditions, making it difficult to evaluate the activation effect of the wirelessly controllable implantable LED.", "reviewer_3": "Here the authors are reporting new methods to restore homeostasis of organelle interplay which is often disrupted in neurodegenerative diseases. They take advantage of plant-derived photosynthetic systems derived from spinach and transplant the nanothylakoid units (NTUs) into cells in vitro and in vivo. The rational is that upon exposure to light the NTUs produce ATP to support restoration of homeostasis of mitochondria-ER interactions. In vitro studies were performed i) in human nucleus pulposus cells (NPC) derived from controls or patients with intervertebral disc degeneration (IVDD); ii) in rat NPCs exposed to IL1-β+/- NPC-membrane-coated NTUs (NM-NTUs) and light. Two in vivo animal IVDD models, rat and rabbit, were used to evaluate the efficacy of NM-NTUs. In summary this is a well performed study, however some parts need to be strengthened and need further investigations.\nMajor comments\nFigure 2\nThere are large differences between normal NPCs and IVDD NPCs in parameters such as intracellular/mitochondrial [Ca2+], ROS, mitochondrial membrane potential, ER-stress, and mPTP opening indicate toxicity. Therefore, a viability test should be performed to show the status of the cells. The word ‘degeneration’ is misspelt throughout the figure (‘degenration’).\n2h) To quantify mitochondria-ER contacts (MERCS) authors used ICC of TOMM20 and PDI. PDI stain for ER lumen and not ER membrane and is therefore not a proper marker. The experiment should be repeated using antibodies directed against ER membrane proteins, eg calreticulin or calnexin. Pearson coefficient shows whether the intensity of one channel correlate with another channel. To show the spatial relationship between two channels, Manders’ colocalization analysis should be performed instead.\nThe resolution of the images is low, and the emission in the green channel is too strong. This also need to be corrected.\n2i) Authors need to define the cut-off distance between OMM and ER which was set as the upper limit to consider distance between the two membranes as MERCS (<30 nm? <50nm?). The number of cells analysed per sample should also be included.\nFigure 3\n3d) Quantification of cryo-EM data is missing. How many % of NM actually contain NTU?\n3i) Results show a clear co-localization of NTU and lysosomes, while NM-NTU do not co-localize with lysosomes. Co-localization with MERCS should also be investigated.\n3j) Pearson coefficient shows whether the intensity of one channel correlate with another channel. To show the spatial relationship between two channels, Manders’ colocalization analysis should be performed.\nFigure 4\nIL-1β exposure is a model of inflammation. Data or citation showing that IL-1β induces NPC degeneration should be presented. If not, it’s better to clarify that this model simulate only inflammation in IVDD.\nIs the therapeutic effect of NTU injection dosage-dependent? This is an important aspect since the study is about therapy.\n4a) NM-NTUs produce ATP under light exposure. How much ATP from NM-NTUs is produced per cell or protein amount?\nDo NTUs also generate a signal to stimulate ATP production by endogeneous mitochondria? Can ATP generated from NTUs alone produce the therapeutic effect?\n4d) The IL-1β+NM-NTU +light condition is missing in the heatmap. If IL-1β + NM-NTU already changed RNA level and lipid metabolism shown in figure 6, why is light required? That is inconsistent with 4b)\nFigure 5\n5e) The figure shows percentage, not number of mitochondria, please clarify number or percentage in text (Line 214). If number was quantified, the total number of mitochondria should also be quantified. Authors need to define the cut-off distance between OMM and ER which was set as the upper limit to consider distance between the two membranes as MERCS (<30 nm? <50nm?). The number of cells analysed per sample should also be included.\n5f) same comments as for Figure 2 regarding ICC.\nFigure 6\nThe authors tested the global membrane fluidity after NM-NTU treatment. Is it possible to show whether the formation of MAM is dependent on the level of TG to confirm that altered lipidomics change MAM formation?\n6a) The IL-1β+NM-NTU +light condition is missing in the heatmap.\nFigure 7 and 10\nThe in vivo experiments showing positive effects of NM-NTUs on the disc height index (DHI) in rats and the use of a remote wireless control system for illumination in rabbit are very promising and interestingly. However, the authors do neither show nor discuss any connection to the mechanism of restoration of organelle interaction network reported in vitro in NM-NTU treated cells. This point needs to be addressed, since the connection is missing now.\n7b, d, f, g and 10d) Use arrows to indicate points of interest and increase resolution of images\n7g and10e) Quantifications of the images are missing\nMinor comments\n-Line 29: What does ‘ignoring entire cell damage’ mean? Do authors mean ‘ignoring the role of organelle interactions in cell damage’?\n-Line 35: What does ‘independent’ mean in the phrase ‘plant-derived independent photosynthetic systems’. Is the word ‘independent’ redundant?\n-How are Line 47 & 48 related to mitochondria acquisition from Line 49?\n-How are malic acid cycle and energy homeostasis from Line 53 & 54 related to defensive adaptation mentioned in Line 52?\n-Line 55 & 56: Is changing the unsaturation of the membrane lipids related to mitochondrial or chloroplastic functions? If yes, add a few words to explain their role in this process to make connection with Line 52 ‘defensive adaptation by mitochondria and chloroplast’\n-Line 70: Better mention the role of MAM in lipid metabolism in introduction, instead of just mention ER.\n-Line 72: ‘support’ could be a better word than ‘amplify’\n-Line 73: The term MAM defines the part of the ER membrane which is in contact with outer mitochondrial membranes. The physical contacts between OMM and ER are referred to as MERCS (mitochondria-ER contact sites).\n-Line 76 & 77: Add reference for the statement ‘NTU primarily participate in cellular metabolism by producing energy’. Does NTU have additional roles in metabolism?\n-Line 195: should be ‘IL1β’ group instead of ‘IL1β+NM-NTU’ group\n-Line 204-206: The authors claim that ATP from NTU may enhance ER Ca2+ storage. Please provide either citation or experimental data showing the amount of ATP generated by NTU can affect ER Ca2+ storage\n-Lines 231-241: Make a better transition to justify the investigation of ER lipid composition. Add references regarding the connection between membrane fluidity to MAM.\n-Line 255: add ‘and’ before the last lipid category which is membrane lipid PC.\n-Line 284: mention the name of the method used to measure membrane fluidity\n-Line 347: ‘in front of’ is not a clear way to indicate position. Do authors mean ‘ventral to’?\n-Line 437: Add wavelength of red light.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Point-to-Point Response:\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nGeneral comment: “This study explores a novel approach to restoring organelle\ninteraction network homeostasis in animal cells by introducing plant-derived\nphotosynthetic systems. Under stress, animal cells often suffer from dysregulated\norganelle interactions, while plant cells exhibit robust adaptive mechanisms, partly due\nto chloroplast activity. The researchers delivered nanothylakoid units (NTUs) from\nplants into animal cells, demonstrating their ability to provide energy, regulate Ca2+ and\nlipid homeostasis, improve membrane fluidity, and reduce mitochondrial dysfunction.\nBy integrating wireless-powered implantable LEDs, the therapy was extended to deeper\ntissues, offering a proof-of-concept for treating diseases through photosynthetic\nregulation of organelle networks. The work highlights the potential of cross-kingdom\norganelle-inspired therapeutics for enhancing cellular stress tolerance. This article\nalready demonstrates a high level of academic quality, and through detailed refinement,\nits persuasiveness and clarity can be further enhanced.”\nResponse: We sincerely appreciate your thorough and constructive feedback on our\nmanuscript. Your insightful suggestions have significantly strengthened our study. We\nhave tried our best to revise the manuscript according to these valuable comments.\nComment-1: “1.It is suggested that after mentioning \"animal cells lack stress\nresistance\" (lines 56–57), a couple of sentences should be added to explain the current\ntechnological bottlenecks in regulating organelle network homeostasis in the medical\nfield (e.g., existing methods only target individual organelles), thereby more directly\npointing to the innovation of this study.”\nResponse: We thank the reviewer for this suggestion. Animal cells exhibit organelle\ninteraction network disruption under stress [PMID: 40745061, 40250411]. Traditional\ntherapies have focused primarily on the individual actions of individual organelles, such\nas mitochondria and the endoplasmic reticulum (ER) [PMID: 28792006, 23850759].\nHowever, few current treatments can systematically correct dysregulated organelle\ninteractions, and methods to systematically restore these dysregulated interaction\nnetworks are urgently needed. We have added the current technological bottlenecks to\nthe revised manuscript.\nAction: The technical bottlenecks have been added to the revised manuscript (Page 4).\n“Animal cells exhibit ER–mitochondria contact disruption under stress28,29. Targeting\nindividual organelles may not systematically correct dysregulated organelle\ninteractions, and it is urgent to identify methods to restore these dysregulated interaction\nnetworks.”\nComment-2: “2.It is recommended to use the sentence structure \"This study aims to\naddress...\" in the final paragraph of the introduction (or in a separate paragraph) to\nclearly state the research question, for example: \"This study aims to explore whether\nplant-derived NTUs can restore homeostasis in animal cells under pathological\nconditions by modulating the ER-mitochondria interaction network.\".”\nResponse: We thank the reviewer for this insightful input, which strengthens the\npaper’s narrative flow. The corresponding statements have been added to the final\nparagraph of the introduction.\nAction: The change has been highlighted (Page 4).\n“This study aimed to explore whether plant-derived NTUs could restore animal cell\nhomeostasis under pathological conditions by modulating the ER–mitochondria\ninteraction network.”\nComment-3: “3.The full name of \"MAM\" (mitochondria-associated ER membrane)\nwas provided when it was first mentioned (line 74), and it appears again at line 124. It\nis recommended to check the abbreviation consistency throughout the text.”\nResponse: We thank the reviewer for their careful attention to abbreviation consistency.\nAs suggested, we have thoroughly checked the entire manuscript to ensure that all\nabbreviations follow this consistent pattern of being fully defined at first use and\nsubsequently abbreviated.\nAction: We have removed the second full name, “mitochondria-associated ER\nmembrane (MAM)”, and we have maintained only the abbreviation after its initial\ndefinition.\nComment-4: “4.Fig. 6 shows an increase in lipid unsaturation, but it does not directly\nprove the causal relationship between it and membrane fluidity or the reduction of\nMAM. Additional references could be cited to support the inference of \"unsaturation\n→ fluidity → reduction of MAM.”.”\nResponse: We sincerely appreciate the reviewer’s insightful comment regarding the\ncausal relationship between lipid unsaturation, membrane fluidity, and MAM formation.\nWe agree that additional references would strengthen our interpretation. Biomembranes\nare materials composed of proteins embedded in a bilayer of lipids. One of the major\nfactors affecting membrane fluidity is the degree of unsaturation of fatty acid chains\n[PMID: 37652754, 28356571]. Saturated lipid acyl chains tend to form nonfluid, tightly\npacked gel phases at physiological temperatures, whereas unsaturated lipid acyl chains\nfluidize the bilayer [PMID: 29410529]. Membranes rich in unsaturated lipids can adopt\na fluid and disordered phase known as the liquid crystalline phase (Lα). Membranes\nthat mainly consist of saturated lipids can adopt an ordered and rigid gel phase (Lβ)\n[PMID: 37652754]. In summary, tightly packed lipids (saturated lipids) increase\nmembrane lipid order and make them stiffer, whereas loosely packed lipids (unsaturated\nlipids) increase membrane fluidity.\nWith respect to the connection between membrane fluidity and MAMs, loss of the\np97-UBXD8 complex increases the number of mitochondrial–endoplasmic reticulum\ncontact sites (MERCS) by reducing membrane lipid unsaturation through the SREBP1-\nSCD1 pathway [PMID: 36746962]. UBXD8 KO cells exhibited greater abundances of\nPCs, PEs, DGs, and TG lipids with saturated fatty acyl tails, leading to decreased global\nmembrane fluidity, which in turn resulted in increased MERCS. This aberrant contact\nphenotype could be rescued by unsaturated fatty acid supplementation. Ganji et al.\nreported that increased membrane saturation/order leading to loss of fluidity might\naffect the dynamic association and disassociation between organelles, further leading\nto an increase in MERCS [PMID: 36746962].\nThese studies reveal the connection between lipid unsaturation, membrane fluidity,\nand MAM formation.\nAction: We have added these additional references to the revised manuscript. The\nchanges are highlighted (Page 13).\n“Moreover, acyl chain saturation of membrane lipids is crucial for membrane fluidity,\nwith unsaturated lipid acyl chains enabling better fluidity and saturated lipid acyl chains\nforming nonfluid and packed structures73,74. Study has shown that increased membrane\nsaturation/order leads to a loss of fluidity, which may affect the dynamic association\nand disassociation between organelles, further leading to an increase in MERCS42.\nTherefore, we investigated the lipidome of the ER to assess the relationships among ER\nlipid composition, membrane fluidity, and ER–mitochondria contact, as well as the\nregulatory role of NM-NTUs in this process.”\nComment-5: “5.Fig. 9d mentions that the brightness of the LED varies with distance.\nWhat is the minimum light intensity threshold required for effective treatment?”\nResponse: We sincerely thank the reviewer for raising this important technical point\nregarding therapeutic light intensity thresholds. In response, we have conducted\nadditional experiments to systematically address this question. Using a spectral\nilluminance metre (Linshang LS330), we determined that NM-NTUs had significant\ntherapeutic effects in cell experiments with light intensities above 10.3 μmol photons\nm-2 s-1 (Response Figure 1a). This light intensity was defined as the light threshold for\nthe activation of NM-NTUs. In the in vivo rabbit photoirradiation experiment, we first\nmeasured the distance from the back skin of the intervertebral disc to the anterior\nlongitudinal ligament by MRI, which was 4.45 ± 0.36 cm (Response Figure 1b). We\nthen estimated that the PU-wp-LED output power in the rabbit body (intervertebral disc\narea) within the coil was approximately 60–70 μmol photons m-2 s-1 (Response Figure\n1c, d). Considering the 60% red light transmission efficiency through the annulus\nfibrosus (Response Figure 1e), we estimated that the light intensity reaching the nucleus\npulposus was above the activation threshold (10.3 μmol photons m-2 s-1) of the NM-\nNTUs.\nAction: We have added the corresponding information to the Results and Discussion\nsection. This change is highlighted (Page 18; Page 23; Page 26; Fig. 9d; Fig. S17).\n“To determine whether the light from the PU-wp-LED had sufficient intensity to\nactivate NM-NTUs, we first determined that NM-NTUs had significant therapeutic\neffects in cell experiments with light intensities above 10.3 μmol photons m-2 s-1 by\nusing a spectral illuminance metre (Fig. S17a). This light intensity was defined as the\nlight threshold for the activation of NM-NTUs. Furthermore, in the in vivo rabbit\nphotoirradiation experiment, we measured the distance from the back skin of the\nintervertebral disc to the anterior longitudinal ligament by MRI, which was 4.45 ± 0.36\ncm (Fig. S17b). We then estimated that the PU-wp-LED output power in the rabbit body\n(intervertebral disc area) within the coil was approximately 60–70 μmol photons m-2 s-\n1. Considering the 60% red light transmission efficiency through the annulus fibrosus\n(Fig. S17c), we estimated that the light intensity reaching the nucleus pulposus was\nabove the activation threshold of NM-NTUs.”\n“Similarly, ATP production under varying light conditions (10.3–108.1 µmol photons\nm⁻² s⁻¹) was also assessed.”\n“A laser power metre (Zhongxi Equipment) was used to measure the efficiency of red\nlight penetrating the annulus fibrosus (3 mm thickness) from the sheep.”\nResponse Figure 1\n(a) ATP levels of NPCs treated with NM-NTUs and red light irradiation for 30 min\nunder different light intensities. (b) MRI image of the distance between the rabbit\nintervertebral disc and the back (n = 6). (c) Schematic diagram of the use of PU-wp-\nLED in rabbits. (d) wp-LED light intensity in horizontal (left) or vertical (right)\ndistances from the centre of the wireless charging coil (n = 3). (e) Determination of the\nlight penetration ability of the intervertebral disc annulus (n = 3). All quantitative data\nare the mean ± s.d.\nComment-6: “6.It is necessary to clearly describe the duration of cultivation after\nextracting NPCs from degenerated IVDD before staining and imaging, as the\nenvironment of cell culture in vitro differs from the microenvironment of degenerative\nchanges in vivo. If the time is prolonged, the cells are likely to undergo alterations.”\nResponse: We sincerely appreciate the reviewer’s insightful comment. NP tissue was\nobtained from patients who underwent spinal surgery according to the standard\noperating procedures described in previous reports [PMID: 40610464, 40779637]. The\nharvested tissue samples were aseptically minced into approximately 1 mm³ pieces and\nwashed twice with precooled PBS to remove impurities. The tissue fragments were then\ndigested in 0.4% collagenase solution for 4 hours to fully release the cells, and the\nresulting cell suspension was centrifuged at 800 rpm for 5 minutes to pellet the cells,\nfollowed by two PBS washes to eliminate residual enzymes. The isolated cells were\nsubsequently seeded in a humidified incubator for culture. Primary cultures were\nmaintained under standard conditions (5% CO and 37°C) with media changes every\n2\n72 hours for 5 days, limiting cell expansion to passage 2 to preserve the phenotype\nintegrity. We have added this information to the Materials and methods section in the\nrevised manuscript.\nAction: This change is highlighted (Page 20).\n“All cells were seeded in a humidified incubator. Human NPCs were cultured for 5 days,\nlimiting cell expansion to passage 2 to preserve the phenotype integrity88-90.”\nComment-7: “7.Why does the Dil fluorescence appear in the nucleus in the NTU group\nimage of Figure 3i?”\nResponse: We sincerely appreciate the reviewer’s careful observation of the DiI\nfluorescence signal in the nucleus shown in Figure 3i. After a thorough re-evaluation,\nwe determined that this was an artefact caused by fluorescence signal overlap during\nimaging rather than actual nuclear localization of the NTUs. To address this concern,\nwe repeated the experiment and replaced the original image with a clearer,\nrepresentative image (Response Figure 2a). The new image presents a clear picture of\nthe colocalization status between NTUs (labelled with DiI) and lysosomes (labelled\nwith LysoTracker Green) (Response Figure 2b).\nAction: The experiment has been repeated, and the figure has been replaced (Page 8;\nFig. 3i and j).\n“In our study, rat NPCs were coincubated with either NTUDiI (NTU labelled with DiI)\nor NM-NTUDiI (NM unlabelled; NTU labelled with DiI). The results revealed that in\nNPCs treated with NTU alone, the NTUDiI fluorescence signal colocalized with that of\nthe lysosomes (labelled with LysoTracker Green). In contrast, in NPCs treated with\nNM-NTUDiI, only a few signals of NTUDiI colocalized with lysosomes (Fig. 3i and j).\nThese results indicated that the NM coating enabled the NTUs to bypass lysosomal\ndegradation.”\nResponse Figure 2\n(a) Colocalization and intensity profiles of NTUs or NM-NTUs with lysosomes. (b)\nMander’s coefficients (M1) of (a). The M1 quantifies the fraction of the signal intensity\nof NTUs that colocalizes with lysosomes relative to the total signal intensity of NTUs.\nn = 3 biological replicates, the same cells (n = 20) were acquired and analysed. All\nquantitative data are the mean ± s.d. P value was calculated using a two-tailed Student’s\nt-test (b). Scale bars: 5 μm (a).\nReviewer #2 (Remarks to the Author):\nGeneral comment: “This is an interesting work demonstrating the therapeutic effect\nof NM-NTU in IVDD. The authors engineered NM-NTU to target nucleus pulposus\ncells within the intervertebral disc, exerting therapeutic effects by correcting their\nenergy metabolism disorders and abnormal ER-mitochondria interactions. Moreover,\nthe authors combined wp-LED to ensure that NM-NTU is applicable to deep tissues -\nwhich is the real environment where IVDD occurs in clinical. Although the authors\nhave spent a lot of effort verifying the mechanism by which NM-NTU restores NPCs\nmetabolism at the cellular level, there are some issues that need to be addressed through\nmajor revisions.”\nResponse: We appreciate your interest regarding our research. We also thank you for\nyour helpful suggestion. We have tried our best to revise the manuscript according to\nthese valuable comments.\nComment-1: “1. Page 5, Line 129: From Fig 1h, the co-localization level of ER and\nmitochondria does not show a significant difference, although the authors claimed a\nsignificant difference in their co-localization level.”\nResponse: We sincerely appreciate the reviewer’s careful observation of the ER–\nmitochondria colocalization analysis. We apologize for the initial oversight. In response,\nwe have optimized our immunofluorescence protocol using alternative markers to more\nprecisely visualize ER–mitochondria contacts. Specifically, the ER membrane protein\ncalnexin was chosen as the marker for the ER [PMID: 11726513, 22045338], and\nmitochondrial outer membrane protein (TOMM20) was selected as the marker for\nmitochondria [PMID: 37328454, 35346689]. All merged images were reanalysed using\nthe Fiji/ImageJ process (white pixels to highlight contacts between the ER and\nmitochondria) [PMID: 28314612, 25870285], and enlarged views of the colocalization\nregions with clearer demarcations of organelle interfaces are also displayed to update\nthe visualization (Response Figure 3a). In addition, Mander’s coefficient analysis was\nconducted to evaluate the colocalization of ER and mitochondria (Response Figure 3b).\nThe revised figures and additional data collectively support our original conclusion\nwhile providing more comprehensive evidence of increased ER–mitochondria\ncolocalization in degenerative NPCs.\nAction: We have repeated the experiments and modified the image analysis process as\nmentioned above. The revised data, images and methodology have been added to\nthe revised manuscript. The changes are highlighted (Page 6; Page 25; Fig. 2h and i).\n“To further clarify the ER–mitochondrial interaction network, we assessed the MERCS\nstructure of normal and degenerative NPCs by immunostaining the ER and\nmitochondria with calnexin and translocase of the outer mitochondrial membrane 20\n(TOMM20), respectively. The results revealed increased colocalization of the ER and\nmitochondria in degenerative NPCs (Fig. 2h and i).”\n“Similarly, the MERCS structure of the NPCs was observed by immunostaining the ER\nand mitochondria with calnexin and TOMM20. To quantify MERCS, we performed\ncolocalization analysis using ImageJ plugins (Coloc2 and JACoP) following\nestablished methods95.”\nResponse Figure 3\n(a) Representative confocal image of human NPCs stained for calnexin (green, ER) and\nTOMM20 (red, mitochondria). Merged panel images were processed using an ImageJ-\nbased pipeline for quantifying MERCS (white dots indicate colocalized pixels). (b)\nMander’s coefficients (M1) of (a). The M1 quantifies the percentage of the signal\nintensity of mitochondria that colocalizes with the ER relative to the total signal\nintensity of the mitochondria. n = 3 biological replicates, the same cells (n = 20) were\nacquired and analysed. All quantitative data are the mean ± s.d. P value was calculated\nusing a two-tailed Student’s t-test (b). Scale bars: 50 μm (a).\nComment-2: “2. Page 6, Line 163: If I understand correctly, the authors here want to\nshow that the engineered NP-derived cell membrane NTU has the function of targeting\nNP cells, which is crucial for its role in IVDD. However, using RAW as the control cell\nto verify its targeting ability is confusing, as in the IVD environment, the main cells\npresent are nucleus pulposus cells, annulus fibrosus cells, and endplate chondrocytes,\nwhich would be more suitable as controls to verify the targeting ability of NM-NTU.”\nResponse: We sincerely appreciate the reviewer’s insightful suggestion regarding the\nvalidation of the targeting ability of the NM-NTU. The reviewer accurately points out\nthat RAW cells, while useful for assessing general cellular uptake, may not fully\nrepresent the intervertebral disc (IVD) microenvironment. To better demonstrate the\nNPC-targeting capability of NM-NTUs, we agree that comparing their uptake across\nIVD-relevant cell types—including NPCs, annulus fibrosus cells (AFCs), and endplate\nchondrocytes (EPCs)—would provide more biologically meaningful data. Therefore,\nwe performed additional uptake experiments in which AFCs and EPCs were combined\nwith NPCs to evaluate NM-NTU selectivity. In Response Figure 4, compared with\nAFCs or EPCs, NM-NTUs were more highly taken up by NPCs. These data indicated\nthat NM-NTUs were preferentially taken up by NPCs.\nAction: We have reported the abovementioned additional experiments in the revised\nmanuscript. The changes have been highlighted (Pages 7-8; Pages 19-20; Fig. 3f and\ng).\n“In the IVD environment, the primary cells present are NPCs, annulus fibrosus cells\n(AFCs), and endplate chondrocytes (EPCs). Therefore, rat NPCs, AFCs, and EPCs were\ntreated with NM-NTUs (NTUs labelled with DiI). NM-NTUs were more highly taken\nup by NPCs than by AFCs or EPCs (Fig. 3f and g), indicating that NM-NTUs were\npreferentially taken up by NPCs.”\n“The preparation of rat AFCs and EPCs was based on previously described methods86,87.\nRat NP tissues, annulus fibrosus tissues and endplate tissues were macroscopically\nisolated from 4-week-old male Sprague–Dawley rats.”\n“Rat NPCs, AFCs, and EPCs were seeded with media changed every 72 hours, passaged\nwhen the confluence reached 80%, and utilized before passage 2.”\nResponse Figure 4\n(a) Uptake of NM-NTUs (DiI-labelled NTUs) by AFCs, EPCs or NPCs. (b) The\nfluorescence intensity of the NTU per cell in cellular uptake experiments (n = 4).\nP values were calculated using one-way analysis of variance (ANOVA) (b). All\nquantitative data are the mean ± s.d. Scale bars: 10 μm.\nComment-3: “3. Page 6, Line 169: As shown in Fig 3h, NM-NTU is taken up by NP\ncells in a membrane fusion form, so its tracer dye almost perfectly aligns with the cell\nmembrane. However, in Fig 3i, NM-NTU seems to change its intracellular distribution\nform and no longer presents a membrane distribution. The difference needs to be\ncarefully explained by the authors. Additionally, based on the concept of membrane\nfusion, once the outer membrane structure fuses with the plasma membrane, its contents\nwill be released into the cytoplasm.”\nResponse: We sincerely appreciate the reviewer’s insightful observations regarding the\nintracellular distribution of NM-NTU. We acknowledge that our original description of\nthese two experiments (Figures 3h and 3i) was not sufficiently clear. The following\nprovides a detailed description.\nFor Figure 3h, as illustrated in the schematic diagram below (Response Figure 5a),\nthe experiment was conducted as follows: First, we labelled the NMs with DiO.\nUnlabelled NTUs were subsequently coated with NMDiO to form NMDiO-NTUs (NM\nlabelled with DiO; NTU unlabelled). The recipient NPCs were stained with the\nmembrane dye DiI (NPCDiI). A coincubation study revealed that the DiO signal from\nthe NMDiO-NTU colocalized with the DiI signal from the NPC membrane, confirming\nsuccessful membrane fusion (Response Figure 5b). This membrane alignment verified\nthe fusion event.\nFor Figure 3i, we provided schematic diagrams to illustrate the respective\nexperimental procedures for clarity (Response Figure 5c). We treated NPCs with either\nNTUDiI (NTU labelled with DiI) or NM-NTUDiI (NM unlabelled; NTU labelled with\nDiI). The results revealed that in NPCs treated with NTU alone, the NTUDiI\nfluorescence signal colocalized with that of the lysosomes (labelled with LysoTracker\nGreen). In contrast, in NPCs treated with NM-NTUDiI, only a few signals of NTUDiI\ncolocalized with lysosomes (Response Figure 5d, e). These results indicated that the\nNM coating enabled the NTUs to bypass lysosomal degradation.\nAction: We have revised the manuscript to explicitly distinguish the labelling strategies\nand objectives of Figures 3h and 3i. The changes are highlighted as follows (Page 8;\nFig. 3h-j).\n“To confirm that membrane fusion had occurred, rat NPCsDiI (NPC membrane labelled\nwith DiI) were treated with NMDiO-NTUs (NM labelled with DiO; NTU unlabelled).\nOwing to the characteristics of cell membrane fusion, the fluorescence signal of NMDiO\ncolocalized with the fluorescence signal of NPCDiI, confirming the occurrence of\nmembrane fusion (Fig. 3h).”\n“In our study, rat NPCs were coincubated with either NTUDiI (NTU labelled with DiI)\nor NM-NTUDiI (NM unlabelled; NTU labelled with DiI). The results revealed that in\nNPCs treated with NTU alone, the NTUDiI fluorescence signal colocalized with that of\nthe lysosomes (labelled with LysoTracker Green). In contrast, in NPCs treated with\nNM-NTUDiI, only a few signals of NTUDiI colocalized with lysosomes (Fig. 3i and j).\nThese results indicated that the NM coating enabled the NTUs to bypass lysosomal\ndegradation.”\nResponse Figure 5\n(a) Schematic diagram of the membrane fusion experiment. (b) Colocalization of\nNMDiO-NTUs (NM labelled with DiO; NTU unlabelled) and NPCDiI (NPC membrane\nlabelled with DiI). (c) Schematic diagram of the lysosomal colocalization experiments.\n(d) Colocalization and intensity profiles of NTUsDiI or NM-NTUsDiI with lysosomes\n(NTUs labelled with DiI; lysosomes labelled with LysoTracker Green). (e) Mander’s\ncoefficients (M1) of (d). The M1 quantifies the fraction of the signal intensity of NTUs\nthat colocalizes with lysosomes relative to the total signal intensity of NTUs. n = 3\nbiological replicates, the same cells (n = 20) were acquired and analysed. All\nquantitative data are the mean ± s.d. P value was calculated using a two-tailed Student’s\nt-test (e). Scale bars: 20 µm (b); 5 µm (d).\nComment-4: “Therefore, detecting the subcellular localization of NTU after NM-NTU\nuptake is crucial for elucidating its mechanism of action.”\nResponse: As the reviewer mentioned, the NTU’s post-internalization behaviour after\nNM-NTU uptake is highly important. We performed additional colocalization\nexperiments on NM-NTUsDiI (NTUs labelled with DiI) with organelles (endoplasmic\nreticulum, mitochondria, and the Golgi apparatus) and cytoskeletal components (actin\nfilaments and microtubules). Post-fusion NTU localization data revealed that the NTU\nsignals colocalized with actin and tubulin to a high degree, and only a few NTU signals\ncolocalized with the endoplasmic reticulum, mitochondria, and Golgi apparatus\n(Response Figure 6). The cytoskeleton is key to controlling cell polarity in cells, and\nits dynamic network controls the distribution of organelles within cells [PMID:\n18946476, 18648373]. Therefore, NTUs may achieve their motility after entering the\ncell through the cytoskeleton; this phenomenon has similarities to previous reports of\ncytoskeleton-driven chloroplast movement in plants [PMID: 19620714, 21772030].\nAction: We have reported the additional experimental results in the revised manuscript.\nThe changes are highlighted as follows (Page 8; Page 22; Fig. S4).\n“Furthermore, the NTU’s post-internalization behaviour after NM-NTU uptake was\nhighly significant; the colocalization experiments revealed that the NTU signals\ncolocalized with actin and tubulin to a high degree, and only a few NTU signals\ncolocalized with the endoplasmic reticulum, mitochondria, and Golgi apparatus (Fig.\nS4a and b). These results indicated that the NTU might achieve motility after entering\nthe cell through the cytoskeleton. This phenomenon has similarities to previous reports\nof cytoskeleton-driven chloroplast movement in plants51,52.”\n“Colocalization studies were performed to determine the intracellular distribution of\ninternalized NM-NTUs. After rat NPCs were incubated with NM-NTUsDiI (NTUs\nlabelled with DiI) for 6 h, the samples were stained for specific cellular structures,\nincluding the endoplasmic reticulum (ER-Tracker Green, Beyotime), mitochondria\n(Mito-Tracker Green, Beyotime), Golgi apparatus (Golgi-Tracker Green, Beyotime),\nactin filaments (Actin-Tracker Green, Beyotime), and microtubules (Tubulin-Tracker\nGreen, Beyotime). Confocal microscopy images were acquired, and colocalization was\nquantitatively analysed using Mander's overlap coefficients.”\nResponse Figure 6\n(a) Colocalization and intensity profiles of NM-NTUsDiI (NTUs labelled with DiI) with\norganelles (endoplasmic reticulum, mitochondria, Golgi apparatus) and cytoskeletal\ncomponents (actin filaments and microtubules) in rat NPCs. (b) Mander’s coefficients\n(M1) of (a). The M1 quantifies the fraction of the signal intensity of NTUs that\ncolocalizes with organelles/cytoskeletons relative to the total signal intensity of NTUs.\nn = 3 biological replicates, the same cells (n = 20) were acquired and analysed. All\nquantitative data are the mean ± s.d. Scale bars:10 μm (a).\nComment-5: “4. Page 7, Line 188: As the authors discussed in Fig 4a-c, NM-NTU can\ntarget and restore the intracellular metabolic processes of NP cells in a light-dependent\nmanner. However, in the subsequent RNA-seq results, NM-NTU without light (if I\nunderstand correctly) also has the effect of altering the transcriptional process of NP\ncells, which is confusing. I think it is very important to compare the conditions with\nand without light.”\nResponse: We sincerely thank the reviewer for this critical comment regarding the\nexperimental design of the RNA-seq analysis. The reviewer rightly emphasizes the\nimportance of comparing light and dark conditions to fully understand the light-\ndependent effects of NM-NTUs. In our initial submission, we inadvertently caused\nconfusion by not clearly specifying that the group labelled “IL-1β + NM-NTU” in the\nRNA-seq experiment actually referred to “IL-1β + NM-NTU + light”. We deeply\napologize for this oversight in labelling. Earlier in our study, we verified that NM-NTU\n+ dark treatment did not increase intracellular ATP levels (Fig. 4a). Furthermore, the\nqRCR and immunofluorescence results (Fig. 3b, c) indicated that without light\nirradiation, NM-NTUs could not enhance the anabolism of degenerated NPCs.\nTherefore, we did not include a dark condition in the initial RNA-seq experiment.\nHowever, we fully agree that comparing both conditions is essential for clarifying\nthe photodependency of the effects of NM-NTUs. To address this point, we repeated\nthe RNA-seq experiment and added an “IL-1β + NM-NTU + dark” group. Compared\nwith the control group, the IL-1β group and the IL-1β + NM-NTU + dark group\npresented a greater number of differentially expressed genes (DEGs), whereas\ncompared with the control group, the IL-1β + NM-NTU + light group presented a lower\nnumber of DEGs (Response Figures 7a, 8a-d). PCA revealed distinct clustering among\nsamples within groups, with the gene expression profiles of the control and IL-1β +\nNM-NTU + light groups being similar, while both groups clustered separately from the\nIL-1β and IL-1β + NM-NTU + dark groups (Response Figure 7b). Furthermore, in\ncontrast to the IL-1β + NM-NTU + dark group, the IL-1β + NM-NTU + light group\npresented lower levels of ROS-related genes (Sod2 and Prdx6) and mitochondrial\napoptosis-related genes (Nptx1 and Bid) (Response Figure 7c). Gene set enrichment\nanalysis (GSEA) results indicated that, compared to the control group, the IL-1β group\nand the IL-1β + NM-NTU + dark group presented upregulated expression of genes\nrelated to ROS signalling, ER calcium homeostasis, and mitochondrial apoptosis and\ndownregulated expression of genes related to ECM assembly (Response Figure 8e, f).\nIn contrast to the IL-1β group and the IL-1β + NM-NTU + dark group, the IL-1β + NM-\nNTU + light group exhibited downregulation of genes associated with ROS signalling,\nER calcium homeostasis, and mitochondrial apoptosis and upregulation of genes\nassociated with ECM assembly (Response Figures 7d, 8g). These results were\nconsistent with the conclusions from our initial RNA-seq data. Taken together, these\nresults revealed that NM-NTUs could restore metabolic homeostasis in degenerated\nNPCs in a light-dependent manner and might have positive effects on the ER and\nmitochondria within NPCs.\nAction: We performed a new RNA-seq experiment that included a dark condition group.\nThe updated RNA-seq results have now been incorporated into the revised manuscript\n(Page 10; Fig. 4d-g; Fig. S8).\n“Compared with the control group, the IL-1β group and the IL-1β + NM-NTU + dark\ngroup presented a greater number of differentially expressed genes, whereas compared\nwith the control group, the IL-1β + NM-NTU + light group presented a lower number\nof differentially expressed genes (Fig. 4d; Fig. S8a-d). PCA revealed distinct clustering\namong samples within groups, with similar gene expression profiles in the control and\nIL-1β + NM-NTU + light groups, while both groups clustered separately from the IL-\n1β and IL-1β + NM-NTU + dark groups (Fig. 4e). Furthermore, in contrast to the IL-\n1β + NM-NTU + dark group, the IL-1β + NM-NTU + light group presented lower levels\nof ROS-related genes (Sod2 and Prdx6) and mitochondrial apoptosis-related genes\n(Nptx1 and Bid) (Fig. 4f). GSEA results indicated that, compared to the control group,\nthe IL-1β group and the IL-1β + NM-NTU + dark group presented upregulated\nexpression of genes related to ROS signalling, ER calcium homeostasis, and\nmitochondrial apoptosis and downregulated expression of genes related to ECM\nassembly (Fig. S8e and f). In contrast to the IL-1β group and the IL-1β + NM-NTU +\ndark group, the IL-1β + NM-NTU + light group exhibited downregulation of genes\nassociated with ROS signalling, ER calcium homeostasis, and mitochondrial apoptosis\nand upregulation of genes associated with ECM assembly (Fig. 4g; Fig. S8g). Taken\ntogether, these results demonstrated that NM-NTUs could restore metabolic\nhomeostasis in degenerated NPCs in a light-dependent manner and might have positive\neffects on the ER and mitochondria within NPCs.”\nResponse Figure 7\n(a) Heatmap of differentially expressed genes in NPCs; six biological replicates are\nshown. (b) PCA of genes in NPCs with different treatments. (c) Volcano plots of genes\nbetween the IL-1β + NM-NTU + light group and the IL-1β + NM-NTU + dark group.\n(d) GSEA results of pathways in the IL-1β + NM-NTU + light group compared with\nthose in the IL-1β + NM-NTU + dark group.\nResponse Figure 8\n(a) Heatmap showing genes that are differentially expressed between different groups.\n(b-d) Volcano plots of differentially expressed genes between the following\nconditions: (b) control group versus the IL-1β group. (c) control group versus the IL-\n1β + NM-NTU + dark group. (d) control group versus the IL-1β + NM-NTU + light\ngroup. (e) GSEA results of the IL-1β group compared with the control group. (f) GSEA\nresults of the IL-1β + NM-NTU + dark group compared with the control group. (g)\nGSEA results of the IL-1β + NM-NTU + light group compared with the IL-1β group.\nComment-6: “5. Page 9, Line 245: Similar to the previous issue, in the detection of\nmetabolic changes in NPCs, the authors compared the blank, IL-1b, and IL-1b + NM-\nNTU groups but ignored the important factor of light. Therefore, the role of light in\nactivating NM-NTU becomes ambiguous and confusing.”\nResponse: We sincerely thank the reviewer for this comment regarding the\nexperimental design of the lipidome. Like in Comment-5, in our initial submission, we\ninadvertently raised confusion by not clearly specifying that the group labelled “IL-1β\n+ NM-NTU” in the ER lipidome experiment actually referred to “IL-1β + NM-NTU +\nlight”. We deeply apologize for this labelling oversight.\nWe fully agree that comparing both the IL-1β + NM-NTU + dark and the IL-1β +\nNM-NTU + light conditions is essential for clarifying the photodependency of the\neffects of the NM-NTU. To address this point, we repeated the lipidomic experiment\nand added an “IL-1β + NM-NTU + dark” group. In the new lipidomic results, we\nidentified 876 metabolites in the ER lipidome, with 392 significantly different\nmetabolites between the IL-1β + NM-NTU + light group and the IL-1β + NM-NTU +\ndark group (Response Figures 9a, 10a). PCA revealed distinct clustering among\nsamples within groups, with similar metabolite profiles in the control and IL-1β + NM-\nNTU + light groups, while both groups clustered separately from the IL-1β and IL-1β\n+ NM-NTU + dark groups (Response Figure 9b). Compared with the IL-1β + NM-NTU\n+ dark group, the IL-1β + NM-NTU + light group significantly affected Ca2+-dependent\nevents and lipid metabolism (Response Figures 9c, 10b). Kyoto Encyclopaedia of\nGenes and Genomes enrichment analysis revealed that NM-NTU + light treatment\naffected pathways related to the biosynthesis of unsaturated fatty acids (Response\nFigure 10c).\nWe subsequently analysed the distribution and changes in ER lipid categories\namong the groups. Compared with those in the control group, the triglyceride (TG)\nlevels were significantly reduced in the IL-1β and IL-1β + NM-NTU + dark groups,\nwhereas they were restored in the IL-1β + NM-NTU + light treatment group (Response\nFigure 9d). The levels of other major ER lipid species (e.g., phosphatidylcholine,\nphosphatidylethanolamines, phosphatidylinositol, and sphingomyelin) did not change\nsignificantly among the groups (Response Figures 9e-h).\nWe further used thin-layer chromatography (TLC) to determine the TG content in\nthe ER of each group. Compared with the IL-1β + NM-NTU + dark group, the IL-1β +\nNM-NTU + light group contained more TGs (Response Figure 9i). To address whether\nER–mitochondria contact was related to TG levels, we modulated TG levels in the ER\nby applying inhibitors of TG synthesis. The final and committed step of TG synthesis\nis catalysed by two diacylglycerol acyltransferases, DGAT1 and DGAT2, which are\nER-resident transmembrane enzymes [PMID: 32559414]. We treated NPCs with a\nDGAT1 inhibitor (A-922500) and a DGAT2 inhibitor (PF-06424439) or a combination\nof both. TLC analysis of ER lipids confirmed that inhibiting DGAT1 and DGAT2,\neither individually or in combination, reduced the TG levels in the ER (Response Figure 9j).\nWe then performed immunofluorescence experiments using calnexin and TOMM20 in\ncells treated with DGAT1 and DGAT2 inhibitors. Compared with the control group, the\ninhibition of TG synthesis significantly increased the number of mitochondrial–\nendoplasmic reticulum contact sites (MERCS) (Response Figures 9k, l). These results\nsuggested a link between TG levels and the phenotype of MERCS. We further discuss\nthe reasons for this link. Since inhibition of TG synthesis disrupts the channelling of\nfatty acids into neutral lipid storage [PMID: 28768178] and disruption of lipid\nhomeostasis elicits ER stress [PMID: 19966778], the formation of MERCS may\nincrease as an adaptive response to the unfolded protein response [PMID: 36586409,\n33335290]. In general, our findings indicated that an altered ER lipidome (reduced TG\nlevels) might contribute to increased ER–mitochondria contact.\nWe subsequently analysed the acyl chain compositions of TG. Compared with\nthose in the IL-1β + NM-NTU + light group, the proportions of TG (50:2)[NL-18:1],\nTG (54:2)[NL-18:0], TG (52:3)[NL-16:1], TG (56:3)[NL-18:1], and TG (54:3)[NL-\n18:1] significantly increased (Response Figure 9m). The TG volcano map further\nconfirmed these results (Response Figure 9n). The proportion of unsaturated TG in the\nIL-1β + NM-NTU + light group was significantly greater than that in the IL-1β + NM-\nNTU + dark group (Response Figure 9o). In summary, NM-NTU + light treatment\nincreased the unsaturation levels of TGs. Given that lipid unsaturation significantly\nincreased membrane fluidity [PMID: 29410529] and affected the contact between\nintracellular organelles [PMID: 36746962], we speculated that the NM-NTU reduced\nthe formation of MERCS in degenerative cells by enhancing membrane fluidity. Next,\nwe measured global membrane fluidity in NPCs using a membrane fluidity kit.\nMembrane fluidity was quantified by measuring the ratio of excimer to monomer\nfluorescence of lipophilic pyrene probes [PMID: 36103824]. With increasing\nmembrane fluidity, the probe underwent excimer formation. We found that NPCs in the\nIL-1β + NM-NTU + light group exhibited greater membrane fluidity than did those in\nthe IL-1β + NM-NTU + dark group (Response Figure 9p). Overall, NM-NTUs with\nlight illumination might affect ER–mitochondria contact by remodelling the lipid\ncomposition of the ER.\nAction: We performed a new lipidomic experiment that included a dark condition group.\nThe updated results have now been incorporated into the revised manuscript (Pages 13-\n15; Fig. 6; Fig. S12).\n“We identified 876 metabolites in the ER lipidome, with 392 significantly different\nmetabolites between the IL-1β + NM-NTU + light group and the IL-1β + NM-NTU +\ndark group (Fig. 6a; Fig. S12a). PCA revealed distinct clustering among samples within\ngroups, with similar metabolite profiles in the control and IL-1β + NM-NTU + light\ngroups, while both groups clustered separately from the IL-1β and IL-1β + NM-NTU\n+ dark groups (Fig. 6b). Compared with the IL-1β + NM-NTU + dark group, the IL-1β\n+ NM-NTU + light group significantly affected Ca2+-dependent events and lipid\nmetabolism (Fig. 6c; Fig. S12b). Kyoto Encyclopaedia of Genes and Genomes\nenrichment analysis revealed that NM-NTU + light treatment affected pathways related\nto the biosynthesis of unsaturated fatty acids (Fig. S12c).\nWe subsequently analysed the distribution and changes in ER lipid categories\namong the groups. Compared with those in the control group, the triglyceride (TG)\nlevels were significantly reduced in the IL-1β and IL-1β + NM-NTU + dark groups,\nwhereas they were restored in the IL-1β + NM-NTU + light treatment group (Fig. 6d).\nThe levels of other major ER lipid species (e.g., phosphatidylcholine,\nphosphatidylethanolamines, phosphatidylinositol, and sphingomyelin) did not change\nsignificantly among the groups (Fig. 6e-h).\nWe further used thin-layer chromatography (TLC) to determine the TG content in\nthe ER of each group. Compared with the IL-1β + NM-NTU + dark group, the IL-1β +\nNM-NTU + light group contained more TGs (Fig. 6i). To address whether ER–\nmitochondria contact was related to TG levels, we modulated TG levels in the ER by\napplying inhibitors of TG synthesis. The final and committed step of TG synthesis is\ncatalysed by two diacylglycerol acyltransferases, DGAT1 and DGAT2, which are ER-\nresident transmembrane enzymes75. We treated NPCs with a DGAT1 inhibitor (A-\n922500) and a DGAT2 inhibitor (PF-06424439) or a combination of both. TLC analysis\nof ER lipids confirmed that inhibiting DGAT1 and DGAT2, either individually or in\ncombination, reduced the TG levels in the ER (Fig. 6j). We then performed\nimmunofluorescence experiments using calnexin and TOMM20 in cells treated with\nDGAT1 and DGAT2 inhibitors. Compared with the control group, the inhibition of TG\nsynthesis significantly increased the number of MERCS (Fig. 6k and l). These results\nsuggested a link between TG levels and the phenotype of MERCS. We further discuss\nthe reasons for this link. Since inhibition of TG synthesis disrupts the channelling of\nfatty acids into neutral lipid storage76 and disruption of lipid homeostasis elicits ER\nstress77, the formation of MERCS may increase as an adaptive response to the unfolded\nprotein response78,79. In general, our findings indicated that an altered ER lipidome\n(reduced TG levels) might contribute to increased ER–mitochondria contact.\nWe subsequently analysed the acyl chain compositions of TG. Compared with\nthose in the IL-1β + NM-NTU + light group, the proportions of TG (50:2)[NL-18:1],\nTG (54:2)[NL-18:0], TG (52:3)[NL-16:1], TG (56:3)[NL-18:1], and TG (54:3)[NL-\n18:1] significantly increased (Fig. 6m). The TG volcano map further confirmed these\nresults (Fig. 6n). The proportion of unsaturated TG in the IL-1β + NM-NTU + light\ngroup was significantly greater than that in the IL-1β + NM-NTU + dark group (Fig.\n6o). In summary, NM-NTU + light treatment increased the unsaturation levels of TGs.\nGiven that lipid unsaturation significantly increased membrane fluidity73 and affected\nthe contact between intracellular organelles42, we speculated that the NM-NTU reduced\nthe formation of MERCS in degenerative cells by enhancing membrane fluidity. Next,\nwe measured global membrane fluidity in NPCs using a membrane fluidity kit.\nMembrane fluidity was quantified by measuring the ratio of excimer to monomer\nfluorescence of lipophilic pyrene probes80. With increasing membrane fluidity, the\nprobe underwent excimer formation. We found that NPCs in the IL-1β + NM-NTU +\nlight group exhibited greater membrane fluidity than did those in the IL-1β + NM-NTU\n+ dark group (Fig. 6p). Overall, NM-NTUs with light illumination might affect ER–\nmitochondria contact by remodelling the lipid composition of the ER.”\nResponse Figure 9\n(a) Heatmap showing differentially abundant lipids in the ER of NPCs; three biological\nreplicates are shown. (b) PCA of lipids in the ER of NPCs with different treatments. (c)\nReactome database analysis of the metabolomic data revealed that the NM-NTU + light\ntreatment group was associated with Ca2+-dependent events and pathways related to\nlipid metabolism. The topology of the associated pathways is shown. (d-h) The bar chart\nshows the proportions of lipid classes in the ER of the NPCs in different groups. The\nlipid species included triglycerides (TG) (d), phosphatidylcholine (PC) (e),\nphosphatidylethanolamines (PE) (f), phosphatidylinositol (PI) (g), and sphingomyelin\n(SM) (h). The proportion of each lipid species was normalized to that of total lipids (n\n= 3). (i) Thin layer chromatography (TLC) analysis imaging (left) and quantification\n(right) of TG levels in the ER of NPCs with different treatments (n = 3). (j) TLC analysis\nimaging (left) and quantification (right) of TG levels in the ER of NPCs treated\nwith/without a DGAT1 inhibitor (A-922500, 20 μg mL-1) or a DGAT2 inhibitor (PF-\n06424439, 20 μg mL-1) (n = 3). (k) Representative confocal image of NPCs stained for\ncalnexin (green, ER) and TOMM20 (red, mitochondria). Merged panel images were\nprocessed via an ImageJ-based pipeline for quantifying MERCS (white dots indicate\ncolocalized pixels). (l) Mander’s coefficients (M1) of (k). The M1 quantifies the\npercentage of the signal intensity of mitochondria that colocalizes with the ER relative\nto the total signal intensity of the mitochondria. n = 3 biological replicates, the same\ncells (n = 20) were acquired and analysed. (m) Proportion of lipid types in TGs in the\nER of NPCs with different treatments. The proportion of each lipid was normalized to\nthat of total lipids (n = 3). (n) Volcano plots of TG with different treatments (IL-1β +\nNM-NTU + light group versus IL-1β + NM-NTU + dark group). (o) The proportion of\nunsaturated TG in the ER of NPCs with different treatments (n = 3). (p) Global\nmembrane fluidity of NPCs (n = 3). All quantitative data are the mean ± s.d. P value\nwere calculated using one-way ANOVA (d–j, l, m, o, and p). Scale bars: 5 μm (k).\nResponse Figure 10\n(a) Heatmap and volcano plots showing differentially abundant lipids between different\ngroups. (b) Differentially abundant lipid metabolites were analysed using the Reactome\ndatabase. The major metabolic processes are annotated. (c) KEGG analysis between the\ndifferent groups.\nComment-7: “6. Page 13, Line 356: Using a wirelessly controllable implantable LED\nto stimulate NM-NTU is an interesting idea because one of the major challenges in\nIVDD treatment is that the lesions are often located deep within the tissue, making light\nstimulation difficult to reach. However, the authors did not compare the therapeutic\neffects of NM-NTU under implantable LED and non-implantable light stimulation\nconditions, making it difficult to evaluate the activation effect of the wirelessly\ncontrollable implantable LED.”\nResponse: We sincerely appreciate the reviewer’s insightful suggestion regarding the\nvalidation of our PU-wp-LED system. We fully agree that a direct comparison between\nimplantable and nonimplantable light stimulation is critical for evaluating the activation\neffect of wirelessly controllable implantable LEDs. To address this issue, we\nconducted new experiments in the rabbit IVDD model, comparing PU-wp-LED and\nexternal light (Response Figure 11a). Magnetic resonance imaging (MRI) assessment\nand histological analysis (H&E staining) were performed to evaluate the results.\nCompared with the NM-NTU + external light group, MRI results revealed that discs\ntreated with the NM-NTU + PU-wp-LED + light maintained significantly higher signal\nintensity, indicating better water content and reduced disc dehydration (Response\nFigure 11b, c). Histological evaluation via H&E staining further supported these\nfindings. In the NM-NTU + PU-wp-LED + light group, the overall disc morphology\nappeared well preserved compared with that in the NM-NTU + external light group\n(Response Figure 11d). Histological scoring based on an established grading system\nconfirmed a significant improvement in the NM-NTU + PU-wp-LED + light group\ncompared with the NM-NTU + external light group (Response Figure 11e). Overall,\ncompared with direct external light, the PU-wp-LED system achieved favourable\ntherapeutic outcomes in the rabbit IVDD model. The external light system might fail\nbecause of its inability to sufficiently penetrate dorsal anatomical structures and reach\ndeep tissues. These data confirmed that the wirelessly controllable implantable LED\nsystem was essential for achieving therapeutic effects of NM-NTUs in deep tissues.\nAction: We have reported additional experimental results and methods in the revised\nmanuscript. The changes are highlighted (Pages 18-19; Page 28; Fig. S18).\n“We also conducted experiments in the rabbit IVDD model, comparing PU-wp-LED\nand external light (Fig. S18a). Compared with the NM-NTU + external light group,\nMRI results revealed that discs treated with the NM-NTU + PU-wp-LED + light\nmaintained significantly greater signal intensity, indicating better water content and\nreduced disc dehydration (Fig. S18b and c). Histological evaluation via H&E staining\nfurther supported these findings. In the NM-NTU + PU-wp-LED + light group, the\noverall disc morphology appeared well preserved compared with that in the NM-NTU\n+ external light group (Fig. S18d). Histological scoring based on an established grading\nsystem confirmed a significant improvement in the NM-NTU + PU-wp-LED + light\ngroup compared with the NM-NTU + external light group (Fig. S18e). Overall,\ncompared with direct external light, the PU-wp-LED system achieved favourable\ntherapeutic outcomes in the rabbit IVDD model. The reason for the external light\nsystem failure might be its inability to sufficiently penetrate dorsal anatomical\nstructures and reach deep tissues.”\n“To compare the effects of implantable versus nonimplantable optical stimulation, the\nrabbits were randomly assigned to one of four groups: sham surgery (sham), puncture\nand 10 µL vehicle (Vehicle) injection per week, puncture + 10 µL NM-NTUs (20 μg\nmL-1 for chlorophyll) injection per week with external red light illumination for 30 min\ndaily (Puncture + NM-NTUs + External illumination), and puncture + 10 μL NM-NTU\ninjection (20 μg mL-1 for chlorophyll) per week with PU-wp-LED turn on for 30 min\ndaily (Puncture + NM-NTUs + PU-wp-LED illumination). The PU-wp-LED implants\nwere removed prior to imaging evaluation. All the animals were euthanized at week 8\nfor subsequent analysis.”\nResponse Figure 11\n(a) Schematic diagram of the experimental design for comparison of the therapeutic\neffects of the NM-NTUs under implantable LED and external light stimulation\nconditions. (b) Representative MR images of a rabbit IVD. The observed segments are\nindicated by red arrows. (c) Quantitative analysis of the T2-weighted signal intensity\nof the NPs in each group (n = 4). (d) H&E staining images of IVD samples. The nucleus\npulposus areas are outlined with a red dashed line. (e) Histological grade evaluation in\neach group (n = 4). All quantitative data are the mean ± s.d. P values were calculated\nusing one-way ANOVA (c) or nonparametric Kruskal–Wallis test (e). Scale bar: 625\nµm (d).\nReviewer #3 (Remarks to the Author):\nGeneral comment: “Here the authors are reporting new methods to restore\nhomeostasis of organelle interplay which is often disrupted in neurodegenerative\ndiseases. They take advantage of plant-derived photosynthetic systems derived from\nspinach and transplant the nanothylakoid units (NTUs) into cells in vitro and in vivo.\nThe rational is that upon exposure to light the NTUs produce ATP to support restoration\nof homeostasis of mitochondria-ER interactions. In vitro studies were performed i) in\nhuman nucleus pulposus cells (NPC) derived from controls or patients with\nintervertebral disc degeneration (IVDD); ii) in rat NPCs exposed to IL1-β+/- NPC-\nmembrane-coated NTUs (NM-NTUs) and light. Two in vivo animal IVDD models, rat\nand rabbit, were used to evaluate the efficacy of NM-NTUs. In summary this is a well\nperformed study, however some parts need to be strengthened and need further\ninvestigations.”\nResponse: We sincerely appreciate the reviewer’s thoughtful evaluation of our work.\nWe also thank you for your helpful suggestion. We have tried our best to revise the\nmanuscript according to these valuable comments.\nComment-1:\n“Major comments\nFigure 2\nThere are large differences between normal NPCs and IVDD NPCs in parameters such\nas intracellular/mitochondrial [Ca2+], ROS, mitochondrial membrane potential, ER-\nstress, and mPTP opening indicate toxicity. Therefore, a viability test should be\nperformed to show the status of the cells. The word ‘degeneration’ is misspelt\nthroughout the figure (‘degenration’).”\nResponse: We sincerely thank the reviewer for this valuable suggestion. We fully agree\nthat direct measurement of cell viability is crucial for evaluating cellular status.\nTherefore, we tested cell viability using 3-(4,5-dimethylthiazol-2-yl)-2,5-\ndiphenyltetrazolium bromide (MTT) and live/dead staining assays. In the MTT assay,\ncompared with that of normal NPCs, the viability of degenerated nucleus pulposus cells\n(NPCs) decreased to 68.2% (Response Figure 12a). However, in the live/dead staining\nassay, after the cells were incubated with calcein-AM/propidium iodide (PI), we\ndetected no significant difference in the percentage of live cells between the\ndegenerated NPCs and normal NPCs (Response Figure 12b, c).\nWe further attempted to determine the reasons for this difference. In the MTT assay,\nthe water-soluble yellow MTT dye is taken up by cells and reduced to purple formazan\nby enzymes such as NAD(P)H-dependent oxidoreductase enzyme and mitochondrial\nsuccinate dehydrogenase [PMID: 38593323]. The reduction in MTT occurs only in\nmetabolically active cells, and alterations in metabolic activity may lead to variations\nin outcomes. Therefore, the lower absorbance (cell viability) shown in the MTT assay\nof degenerated NPCs might be due to a decrease in cellular metabolic activity. In the\nlive/dead staining assay, although the cell viability of the degenerated NPCs decreased,\ncell death did not occur (PI staining negative).\nThese findings suggested that although degenerated NPCs exhibited reduced\nmetabolic activity and viability, substantial cell death did not occur. These results were\nalso consistent with pathological changes in degenerated NPCs (such as altered Ca2+\nlevels, oxidative stress, and decreased mitochondrial membrane potential). We also\napologize for the spelling error and have corrected the misspelling of “degeneration” in\nFigure 1. The labels are now accurate.\nAction: We have reported the abovementioned additional experiments in the revised\nmanuscript. The misspelling of “degeneration” in Figure 1 has been corrected. The\nchanges have been highlighted (Page 5; Page 21; Fig. S2).\n“Next, we measured cell viability to evaluate the cellular status. In the 3-(4,5-\ndimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, the cell viability\nof degenerated human NPCs decreased to 68.2% compared with that of normal human\nNPCs (Fig. S2a). However, in the live/dead staining assay, we detected no significant\ndifference in the percentage of live cells between the degenerated NPCs and normal\nNPCs (Fig. S2b and c). These findings suggested that although degenerated NPCs\nexhibited reduced metabolic activity and viability, substantial cell death did not occur.”\n“Human NPC viability was evaluated using two independent assays: the MTT assay\n(Beyotime) and the live/dead staining assay (Beyotime). An MTT assay was performed\nto measure cellular metabolic activity. Briefly, yellow, water-soluble MTT tetrazolium\nsalt was added to the cell culture. The absorbance at 570 nm was measured\nspectrophotometrically. A live/dead staining assay was used to simultaneously\ndistinguish viable cells from nonviable cells on the basis of membrane integrity. Cells\nwere stained with a combination of calcein AM and propidium iodide. Images were\nacquired using a laser confocal microscope (LCM, Nikon), and the percentages of live\nand dead cells were quantified using ImageJ software.”\nResponse Figure 12\n(a) MTT assays were used to evaluate the viability of normal and degenerated NPCs (n\n= 4). (b) Representative images of the live/dead staining assay. Live cells (green) and\ndead cells (red) were detected by calcein-AM and propidium iodide staining,\nrespectively. (c) Percentage of live normal and degenerative NPCs (n = 4). P values\nwere calculated using a two-tailed Student’s t-test (a, c). All quantitative data are the\nmean ± s.d. Scale bars: 100 μm (b).\nComment-2: “2h) To quantify mitochondria-ER contacts (MERCS) authors used ICC\nof TOMM20 and PDI. PDI stain for ER lumen and not ER membrane and is therefore\nnot a proper marker. The experiment should be repeated using antibodies directed\nagainst ER membrane proteins, eg calreticulin or calnexin. Pearson coefficient shows\nwhether the intensity of one channel correlate with another channel. To show the spatial\nrelationship between two channels, Manders’ colocalization analysis should be\nperformed instead.\nThe resolution of the images is low, and the emission in the green channel is too strong.\nThis also need to be corrected.”\nResponse: We sincerely thank the reviewer for these critical and insightful comments\nregarding the MERCS analysis. We appreciate the expert guidance on the appropriate\nmarkers and analytical methods, which has significantly improved the rigor of our study.\nWe have therefore repeated the immunofluorescence experiments using calnexin (ER\nmembrane marker) together with TOMM20. We have also improved the imaging\nconditions to address the issues of resolution and channel oversaturation. The new\nimages were acquired using higher resolution settings and with laser/power settings\ncarefully adjusted to prevent oversaturation in any channel, ensuring that the\nquantitative data extracted were reliable. All merged images were reanalysed using\nthe Fiji/ImageJ process (white pixels to highlight contacts between the ER and\nmitochondria) [PMID: 28314612, 25870285], and higher-magnification insets are also\ndisplayed to update the visualization (Response Figure 13a). As correctly suggested by\nthe reviewer, Mander’s overlap coefficients are superior for quantifying the spatial\noverlap and fraction of colocalizing structures. We have now analysed all the new\nimages using Mander’s colocalization analysis (M1: percentage of the signal intensity\nof mitochondria that colocalizes with the ER relative to the total signal intensity of\nmitochondria) to accurately quantify the extent of MERCS. The revised data clearly\nshow a significant increase in MERCS (Response Figure 13b) in the degeneration group\ncompared with the normal group. These new results, obtained with the revised\nmethodology, support our original conclusion.\nAction: We have repeated the experiments and modified the image analysis process as\nmentioned above. The revised data, images and methodology have been added to\nthe revised manuscript. The changes are highlighted (Page 6; Page 25; Fig. 2h and i).\n“To further clarify the ER–mitochondrial interaction network, we assessed the MERCS\nstructure of normal and degenerative NPCs by immunostaining the ER and\nmitochondria with calnexin and translocase of the outer mitochondrial membrane 20\n(TOMM20), respectively. The results revealed increased colocalization of the ER and\nmitochondria in degenerative NPCs (Fig. 2h and i).”\n“Similarly, the MERCS structure of the NPCs was observed by immunostaining the ER\nand mitochondria with calnexin and TOMM20. To quantify MERCS, we performed\ncolocalization analysis using ImageJ plugins (Coloc2 and JACoP) following\nestablished methods95.”\nResponse Figure 13\n(a) Representative confocal image of human NPCs stained for calnexin (green, ER) and\nTOMM20 (red, mitochondria). Merged panel images were processed using an ImageJ-\nbased pipeline for quantifying MERCS (white dots indicate colocalized pixels). (b)\nMander’s coefficients (M1) of (a). The M1 quantifies the percentage of the signal\nintensity of mitochondria that colocalizes with the ER relative to the total signal\nintensity of the mitochondria. n = 3 biological replicates, the same cells (n = 20) were\nacquired and analysed. All quantitative data are the mean ± s.d. P value was calculated\nusing a two-tailed Student’s t-test (b). Scale bars: 50 μm (a).\nComment-3: “2i) Authors need to define the cut-off distance between OMM and ER\nwhich was set as the upper limit to consider distance between the two membranes as\nMERCS (<30 nm? <50nm?). The number of cells analysed per sample should also be\nincluded.”\nResponse: We sincerely appreciate the reviewer’s insightful suggestions regarding the\nMERCS quantification criteria. We agree that a precise, standardized definition and\nrobust sampling are essential for accurate interpretation. In response to this comment,\nwe have thoroughly revised our MERCS analysis protocol on the basis of established\nmethodologies [PMID: 36746962, 40514428]. ER–mitochondria contacts can be\ncharacterized by gap distances, which are usually in the range from 10–80 nm,\nwith many focusing on 30 nm [PMID: 16982799, 30894536]. In accordance with a previous\nstudy [PMID: 36746962], we defined contact sites as those with a tethered proximity\nbetween membrane-bound organelles. The contacts were categorized and quantified\ninto three distinct ranges: ≤25 nm, 25<distance≤50 nm, and 50<distance≤100 nm. This\nstratified approach allowed for a more nuanced and biologically relevant assessment of\nchanges in MERCS under different conditions. Quantitative analysis was performed on\na rigorously increased number of samples, with n = 3 independent biological replicates\nper condition, encompassing a total of 50 cells and 63–65 randomly sampled fields of\nview. For each field of view, we systematically quantified the ER tubules closely\napposed to the mitochondria, the number of mitochondria, and the average length of\nthe ER tubules. The details of the quantitative method are as follows. Briefly, the scale\nof the image was set using the Set Scale tool in ImageJ. The length of the ER and the\nperimeter of the mitochondria were subsequently measured using a freehand line tool.\nThe percent contact length was determined by taking the ratio of the length of the ER\n(within contact distances of 0–100 nm) to the perimeter of the mitochondria.\nThe results revealed that compared with normal NPCs, degenerated NPCs\npresented significant increased ER tubules closely apposed to mitochondria, indicating\nincreased formation of MERCS (Response Figure 14a, b). There was no significant\ndifference in the number of mitochondria or the average length of ER tubules between\nnormal NPCs and degenerated NPCs (Response Figure 14c, d). We believe these\nmethodological improvements, prompted by the reviewer's insightful comment,\nprovide a more robust and definitive quantification of MERCS and strengthen the\nconclusions of our study.\nAction: We have now explicitly defined MERCS on the basis of the precise\nintermembrane distance. The analysis included 50 cells (from n = 3 biological replicates)\nand 63–65 randomly sampled fields of view per condition. The changes are highlighted\n(Page 6; Pages 24-25; Fig. 2j-m).\n“ER–mitochondria contacts can be characterized by gap distances, which are usually in\nthe range from 10–80 nm40,41. In accordance with a previous study42, we defined contact\nsites as those with tethered proximity between membrane-bound organelles. The\ncontacts were categorized and quantified into three distinct ranges: ≤25 nm,\n25<distance≤50 nm, and 50<distance≤100 nm. Compared with normal NPCs,\ndegenerated NPCs presented significant increased ER tubules closely apposed to\nmitochondria, indicating increased formation of MERCS (Fig. 2j and k). There was no\nsignificant difference in the number of mitochondria or the average length of ER tubules\nbetween normal and degenerated NPCs (Fig. 2l and m).”\n“MERCS in TEM images were manually analysed in ImageJ. Briefly, the image scale\nwas first set with the Set Scale tool, and the freehand line tool was subsequently used\nto measure the length of the ER or membrane or the perimeter of the mitochondria. The\ncontact length was defined as the ER membrane length (within 0–100 nm of the\nmitochondria) divided by the mitochondrial perimeter.”\nResponse Figure 14\n(a) Transmission electron microscopy (TEM) images of mitochondrial and ER\nmorphology. The ER and mitochondria were graphically reconstructed: mitochondria\n(red); ER (green). (b) Quantification of the mitochondrial–ER contact length in (a). The\ncontact length was quantified by taking the ratio of the length of the ER (within different\ncontact distances) to the perimeter of the mitochondria. (c) Mitochondrial count per\nfield from TEM in (a). (d) Quantification of ER lengths per field from the TEM image\nin (a). Measurements in b–d are from n = 3 biological replicates, with the normal\ngroup = 50 cells from 65 fields and the degeneration group = 50 cells from 63 fields.\nOMM: Outer mitochondrial membrane. All quantitative data are the mean ± s.d.\nP values were calculated using one-way ANOVA (b) or two-tailed Student’s t-test (c,\nd). Scale bars: 1 μm (a).\nComment-4:\n“Figure 3\n3d) Quantification of cryo-EM data is missing. How many % of NM actually contain\nNTU?”\nResponse: We thank the reviewer for this insightful comment regarding the\nquantification of the cryo-EM data. Through preliminary experiments, we identified an\noptimal NM-to-NTU protein ratio for NTU encapsulation. While cryo-EM provides\nunparalleled structural detail, its field of view is restricted, and the number of individual\nparticles captured in any given micrograph is relatively low. Consequently, gathering a\nstatistically robust dataset across multiple preparation batches can be challenging and\ncan lead to significant variance in manual counting. Therefore, we have presented the\ndata obtained using nanoscale flow cytometry (nanoFCM). NTUs were stained with\nStarRed and detected in the PC5 channel. The NMs were labelled with a FITC-\nconjugated marker, allowing their specific detection in the FITC channel.\nDifferent NTU and NM protein ratios (ranging from 1:10 to 1:200) were tested.\nEvents that were positive for both FITC and PC5 (FITC+/PC5+) were classified as NMs\nsuccessfully loaded with NTUs, whereas events that were positive for FITC but\nnegative for PC5 (FITC+/PC5-) were categorized as empty NMs. Events that were\npositive for PC5 but negative for FITC (FITC-/PC5+) were categorized as free NTUs.\nThe nanoFCM data indicated that the incorporation efficiency was dependent on the\ninitial ratio of NTUs to NMs. At the optimal formulation ratio (NTU to NM protein\nratio of 1:150), 77.83 ± 4.01% of the NTUs could be encapsulated into NMs (Response\nFigure 15a), with approximately 51.37 ± 2.08% of the NMs successfully loaded with\nNTUs (Response Figure 15b). We believe these additional data address the reviewer's\nconcern and enhance the quantitative rigor of our study.\nAction: We have reported the abovementioned experiments in the revised manuscript.\nThe changes are highlighted (Page 7; Pages 20-21; Fig. S3).\n“To evaluate the encapsulation efficiency of the NTUs by NM, we used nanoscale flow\ncytometry (nanoFCM). The nanoFCM data indicated that the incorporation efficiency\nwas dependent on the initial ratio of NTUs to NMs. At the optimal formulation ratio\n(NTU to NM protein ratio of 1:150), 77.83 ± 4.01% of the NTUs could be encapsulated\ninto NMs (Fig. S3a), with approximately 51.37 ± 2.08% of the NMs successfully loaded\nwith NTUs (Fig. S3b). This ratio was used in subsequent experiments.”\n“To evaluate the encapsulation efficiency of the NTUs by NM, we used nanoFCM.\nNTUs were stained with StarRed and detected in the PC5 channel. The NMs were\nlabelled with a FITC-conjugated marker, allowing their specific detection in the FITC\nchannel. Different NTU and NM protein ratios (ranging from 1:10 to 1:200) were tested.\nEvents that were positive for both FITC and PC5 (FITC+/PC5+) were classified as NMs\nsuccessfully loaded with NTUs, whereas events that were positive for FITC but\nnegative for PC5 (FITC+/PC5-) were categorized as empty NMs. Events that were\npositive for PC5 but negative for FITC (FITC-/PC5+) were categorized as free NTUs.”\nResponse Figure 15\n(a) The percentage of NTUs encapsulated in NMs (FITC+ % in PC5+) was assessed by\nnanoscale flow cytometry (n = 3). The gating strategy was as follows. NTUs were\nstained with StarRed (PC5 channel), and NMs were labelled with a FITC-conjugated\nmarker (FITC channel). Colocalization of signals (FITC+/PC5+) revealed successful\nloading of NTUs by NMs. Events that were positive for FITC but negative for PC5\n(FITC+/PC5-) were categorized as empty NMs. Events that were positive for PC5 but\nnegative for FITC (FITC-/PC5+) were categorized as free NTUs. Different formulation\nratios were tested (NTU to NM protein ratios ranging from 1:10 to 1:200). (b) The\npercentage of NMs successfully loaded with NTUs (PC5+ % in FITC+) was assessed\nby nanoscale flow cytometry (n = 3). All quantitative data are the mean ± s.d. P values\nwere calculated using one-way ANOVA (a, b).\nComment-5: “3i) Results show a clear co-localization of NTU and lysosomes, while\nNM-NTU do not co-localize with lysosomes. Co-localization with MERCS should also\nbe investigated.”\nResponse: We thank the reviewer for the valuable comments. To visualize MERCS in\nNPCs, we transfected immortalized human primary NPCs (IH-NPCs) with a previously\nvalidated split-GFP-based contact site sensor (SPLICS) consisting of two\nnonfluorescent fragments of GFP fused to targeting sequences to either the outer\nmitochondrial membrane (OMM-GFP1-10) or the ER (ER-β11) [PMID: 29229997,\n34686857]. A version of the sensor that detects close-range contacts between the ER\nand mitochondria (~8–10 nm) was used. The establishment of close-range contacts\nbetween the ER and mitochondria restored GFP fluorescence, resulting in the formation\nof puncta that could be imaged, thus providing a sensitive readout for MERCS.\nFollowing the transfection of IH-NPCs with SPLICS, we treated them with NM-NTUs\nand performed confocal microscopy observations. Analysis of the colocalization\nbetween the MERCS signal (reconstituted GFP, green) and the NM-NTU signal (DiI\nlabelled NTUs, red) revealed that the Mander’s coefficient for the percentage of NTUs\nthat colocalized with MERCS was 5.29±1.55% (Response Figure 16a-c).\nAction: We have reported the corresponding experimental results in the revised\nmanuscript. The changes are highlighted (Page 8; Page 20; Pages 22-23; Fig. S5).\n“Moreover, to visualize MERCS and NTU colocalization in NPCs, we transfected\nimmortalized human primary NPCs (IH-NPCs) with a previously validated split-GFP-\nbased ER–mitochondria contact site sensor (SPLICS)53,54. Following the transfection\nof IH-NPCs with SPLICS, we treated them with NM-NTUs and performed confocal\nmicroscopy observations. Analysis of the colocalization between the MERCS signal\n(reconstituted GFP, green) and the NM-NTU signal (DiI labelled NTUs, red) revealed\nthat the Mander’s coefficient for the percentage of NTUs that colocalized with MERCS\nwas 5.29±1.55% (Fig. S5).”\n“For plasmid transfection, we used IH-NPCs, which were purchased from iCell\nBioscience, Inc. and cultured in specialized culture medium (iCell Bioscience, Inc.).”\n“To visualize MERCS, IH-NPCs were transfected with SPLICS Mt-ER Short P2A\n(Addgene plasmid # 164108) encoding the mitochondrial outer membrane-targeted\nOMM-GFP1-10 and the endoplasmic reticulum-targeted short protein GFPβ11 (1 μg\nplasmid of a 35 mm glass bottom dish) using Lipofectamine 3000, strictly following\nthe manufacturer's protocol. At 36–48 hours post-transfection, the cells were treated\nwith DiI-labelled NM-NTUs for 6 h. After treatment, the cells were imaged using\nconfocal microscopy. The reconstituted GFP signal, which indicates close-range (~8–\n10 nm) MERCS, and the DiI signal (NTUs) were captured. Colocalization between\nNTUs and MERCS puncta was quantitatively analysed using the Coloc2 plugin in\nImageJ software, with the results expressed as the Mander's coefficient (M1)\nrepresenting the fraction of the NTU signal overlapping with MERCS.”\nResponse Figure 16\n(a, b) Representative microscopy image (a) of the colocalization and intensity profiles\n(b) of NM-NTUs (DiI-labelled NTUs, red) and MERCS (GFP-based contact site sensor,\ngreen). (c) Mander’s coefficients (M1) of (a). The M1 quantifies the fraction of the\nsignal intensity of NTUs that colocalizes with MERCS relative to the total signal\nintensity of NTUs. n = 3 biological replicates, the same cells (n = 20) were acquired and\nanalysed. All quantitative data are the mean ± s.d. Scale bars: 10 μm (a).\nComment-6: “3j) Pearson coefficient shows whether the intensity of one channel\ncorrelate with another channel. To show the spatial relationship between two channels,\nManders’ colocalization analysis should be performed.”\nResponse: We sincerely appreciate the reviewer’s expert guidance on the\ncolocalization analysis methodology. In response to this comment, we have replaced all\nPearson’s correlation coefficients with Mander’s overlap coefficients as the primary\nmetric for determining organelle/NTU spatial relationships. As shown in Response\nFigure 17a and b, the NTUs were directly labelled with DiI, and the lysosomes of NPCs\nwere labelled with LysoTracker Green. When these NTUDiI were coincubated with\nNPCs, they exhibited extensive colocalization with lysosomes. In contrast, when the\nNTUs were first labelled with DiI and then encapsulated in unlabelled NMs (forming\nNM-NTUDiI), the result showed significantly reduced colocalization with lysosomes\nafter co-incubation. These results collectively indicated that the NM coating enabled\nthe NTUs to bypass lysosomal degradation.\nAction: The experiment has been repeated, and the figure has been replaced. The\nchanges are highlighted (Fig. 3i and j).\nResponse Figure 17\n(a) Colocalization and intensity profiles of NTUs or NM-NTUs with lysosomes. (b)\nMander’s coefficients (M1) of (a). The M1 quantifies the fraction of the signal intensity\nof NTUs that colocalizes with lysosomes relative to the total signal intensity of NTUs.\nn = 3 biological replicates, the same cells (n = 20) were acquired and analysed. All\nquantitative data are the mean ± s.d. P value was calculated using a two-tailed Student’s\nt-test (b). Scale bars: 5 μm (a).\nComment-7:\n“Figure 4\nIL-1β exposure is a model of inflammation. Data or citation showing that IL-1β induces\nNPC degeneration should be presented. If not, it’s better to clarify that this model\nsimulate only inflammation in IVDD.”\nResponse: We sincerely appreciate the reviewer’s insightful comment regarding the\nrole of IL-1β in inducing NPC degeneration. As a key member of the IL-1 family, IL-\n1β is widely recognized for its critical role in inflammation-related diseases [PMID:\n30995499]. In the context of IVDD, IL-1β is considered a pivotal factor that amplifies\ninflammatory cascades. Study has shown that IL-1β expression is significantly elevated\nin degenerative intervertebral discs and that this cytokine is involved in multiple\npathological processes associated with IVDD [PMID: 17688691]. Moreover, increased\nIL-1β synthesis accelerated degenerative changes in IVDD [PMID: 37088216].\nFurthermore, studies have used IL-1β to induce NPC degeneration, which may be\nrelated to its ability to increase the expression of matrix degradation-related genes\n(MMP3, MMP13, and ADAMTS-4) and decrease the expression of matrix synthesis-\nrelated genes (ACAN and COL2A1), resulting in an imbalance between catabolic and\nanabolic events in NPCs [PMID: 35984761, 15987475, 32583517]. Therefore, the IL-\n1β exposure model serves not only as a simulation of inflammation but also as a well-\nestablished system for investigating pathological processes in NPC degeneration. We\nhave revised the manuscript and added these citations to clarify these points and ensure\na more precise description of the model’s relevance to IVDD.\nAction: We have revised the manuscript and added relevant citations to the revised\nmanuscript. The changes are highlighted (Page 9).\n“Studies have used IL-1β to induce NPC degeneration, which may be related to its\nability to increase the expression of matrix degradation-related genes (MMP3, MMP13,\nand ADAMTS-4) and decrease the expression of matrix synthesis-related genes (ACAN\nand COL2A1), resulting in an imbalance between catabolic and anabolic events in\nNPCs56-58.”\nComment-8: “Is the therapeutic effect of NTU injection dosage-dependent? This is an\nimportant aspect since the study is about therapy.”\nResponse: We are deeply grateful to the reviewer for raising this critical point regarding\nthe dosage dependency of the therapeutic effect of the NTUs. To assess whether the\nprotective effect of NM-NTUs against IL-1β-induced NPC degeneration was dose\ndependent, we treated IL-1β-stimulated NPCs with a range of NM-NTU concentrations\n(0.125, 0.25, 0.5, 1.0, and 2.0 μg mL-1 for chlorophyll). The results showed that as the\nconcentration of NM-NTU increased from 0.125 to 0.5 μg mL-1 for chlorophyll, its\nability to increase ATP in degenerative NPCs gradually increased, demonstrating the\ndose-dependent effects of NM-NTUs (Response Figure 18). A plateau was observed at\nNM-NTU concentrations higher than 0.5 μg mL-1 for chlorophyll. On the basis of these\nresults, we selected a concentration of 0.5 μg mL-1 for chlorophyll in the subsequent in\nvitro experiments.\nAction: We have reported the corresponding experimental results in the revised\nmanuscript. The changes are highlighted (Page 9; Page 23; Fig. S6).\n“We treated IL-1β-stimulated NPCs with a range of NM-NTU concentrations (0.125,\n0.25, 0.5, 1.0, and 2.0 μg mL-1 for chlorophyll). The results revealed that as the\nconcentration of NM-NTU increased from 0.125 to 0.5 μg mL-1 for chlorophyll, its\nability to increase ATP in degenerative NPCs gradually increased, demonstrating the\ndose-dependent effects of NM-NTUs (Fig. S6). A plateau was observed at NM-NTU\nconcentrations higher than 0.5 μg mL-1 for chlorophyll. On the basis of these results,\nwe selected a concentration of 0.5 μg mL-1 for chlorophyll in the subsequent in vitro\nexperiments.”\n“To evaluate the ATP production efficiency of the NM-NTU at different concentrations,\nwe measured ATP levels under NM-NTU concentrations ranging from 0.125 to 2 μg\nmL-1 for chlorophyll.”\nResponse Figure 18\nATP levels of NPCs treated with IL-1β+NM-NTU with light (different NM-NTU\nconcentrations, μg ml-1 for chlorophyll) (n = 3). All quantitative data are the mean ± s.d.\nP value was calculated using one-way ANOVA.\nComment-9: “4a) NM-NTUs produce ATP under light exposure. How much ATP from\nNM-NTUs is produced per cell or protein amount? ”\nResponse: We sincerely thank the reviewer for raising these insightful questions\nregarding ATP production by NM-NTUs. To address these points directly, we designed\nand performed the following experiments. ATP is the universal energy currency of the\ncell. It is primarily produced through oxidative phosphorylation in the mitochondria,\nwith a secondary, less efficient pathway being glycolysis in the cytoplasm [PMID:\n28331208]. To specifically quantify the ATP contribution from the NM-NTUs, we\nemployed a pharmacological inhibition strategy to suppress endogenous cellular ATP\nproduction. Rotenone is a potent and specific inhibitor of mitochondrial respiratory\ncomplex I (CI). It binds to the ubiquinone binding site of CI, preventing the transfer of\nelectrons from iron–sulphur clusters to ubiquinone, thereby halting oxidative\nphosphorylation [PMID: 36952383]. The 2-deoxy-D-glucose (2DG) is a glucose\nanalogue that differs in the removal of an oxygen atom at the 2-position. Upon\nphosphorylation, it cannot isomerize to fructose-6-phosphate, effectively and reversibly\ninhibiting glycolysis [PMID: 25218591].\nOn the basis of these studies, we used 2DG and rotenone to inhibit ATP production\nthrough glycolysis and oxidative phosphorylation in nucleus pulposus cells (NPC ).\nΔATP\nMoreover, we used NM-NTUs + light to increase the ATP content in NPC ,\nΔATP\nhypothesizing that the increased ATP in these cells was derived mainly from NM-NTUs.\nThe results revealed that the rate of ATP production by NM-NTUs in NPC was 8.50\nΔATP\n± 1.42 pmol min-1 per 105 cells (Response Figure 19).\nAction: We have reported the abovementioned additional experiments in the revised\nmanuscript. The changes are highlighted (Page 9; Page 23; Fig. S7).\n“To specifically quantify the ATP contribution from the NM-NTUs, we employed a\npharmacological inhibition strategy to suppress endogenous cellular ATP production.\nWe used 2DG and rotenone to inhibit ATP production through glycolysis and oxidative\nphosphorylation in nucleus pulposus cells (NPC )59,60. Moreover, we used NM-\nΔATP\nNTUs + light to increase the ATP content in NPC , hypothesizing that the increased\nΔATP\nATP in these cells was derived mainly from NM-NTUs. The results revealed that the\nrate of ATP production by NM-NTUs in NPC was 8.50 ± 1.42 pmol min-1 per 105\nΔATP\ncells (Fig. S7).”\n“To specifically quantify ATP production from NM-NTUs, we inhibited mitochondrial\noxidative phosphorylation and glycolysis using rotenone (500 nM) and 2-DG (10 mM).\nAfter the NM-NTUs were subjected to light treatment, ATP levels were measured using\nan ATP assay kit.”\nResponse Figure 19\nMeasurement of ATP content in NPCs under different conditions (n = 3). All\nquantitative data are the mean ± s.d. P value was calculated using one-way ANOVA.\nComment-10: “Do NTUs also generate a signal to stimulate ATP production by\nendogeneous mitochondria? Can ATP generated from NTUs alone produce the\ntherapeutic effect?”\nResponse: Regarding the second part of the question, degenerative NPCs are\ncharacterized by a disruption in the mitochondria–endoplasmic reticulum interaction\nnetwork. We hypothesized that the ATP produced by NM-NTUs in this context played\na crucial signalling role, helping to remodel and restore the homeostasis of this\ndysfunctional interorganellar network. In turn, this process might restore endogenous\nmitochondria, enabling them to resume their normal ATP-generating function.\nTo investigate whether NM-NTU treatment led to increased ATP production from\nendogenous mitochondria in degenerated NPCs, we transfected cells with a genetically\nencoded mitochondrial ATP indicator (mit-AT1.03) to measure changes in\nmitochondrial ATP concentrations65-67. The ratio of yellow fluorescence protein (YFP)\nto cyan fluorescence protein (CFP) was measured by flow cytometry to determine the\nmitochondrial ATP level. The results showed that following NM-NTUs + light\ntreatment, the mitochondria within degenerated NPCs produced more ATP compared\nwith the IL-1β group (Response Figures 20a, b). These results indicated that NM-NTUs\nmight stimulate endogenous mitochondria in degenerated NPCs to produce ATP. In\nconclusion, while the NTUs themselves produced a quantifiable amount of ATP, a key\ntherapeutic mechanism was that the NTU-generated ATP acted as a signal to restore the\nhomeostasis of organelle interaction networks and promote the energy production of\nendogenous mitochondria.\nAction: We have reported the abovementioned additional experiments in the revised\nmanuscript. The changes are highlighted (Page 12; Page 23; Fig. S10).\n“To investigate whether NM-NTU treatment led to increased ATP production from\nendogenous mitochondria in degenerated NPCs, we transfected IH-NPCs with a\ngenetically encoded mitochondrial ATP indicator (mit-AT1.03) to measure changes in\nmitochondrial ATP concentrations65-67. The ratio of yellow fluorescence protein (YFP)\nto cyan fluorescence protein (CFP) was measured by flow cytometry to determine the\nmitochondrial ATP level. The results showed that following NM-NTUs + light\ntreatment, the mitochondria within degenerated cells produced more ATP compared\nwith the IL-1β group (Fig. S10a and b). These results indicated that NM-NTUs might\nstimulate endogenous mitochondria in degenerated NPCs to produce ATP. In\nconclusion, while the NTUs themselves produced a quantifiable amount of ATP, a key\ntherapeutic mechanism involved the NTU-generated ATP acting as a signal to restore the\nhomeostasis of organelle interaction networks and promote the energy production\nof endogenous mitochondria.”\n“Assessment of endogenous mitochondrial functional recovery\nIH-NPCs were transfected with pCMV-Mito-AT1.03 (Beyotime). Cells were seeded in\n6-well plates, and each well was transfected with 1 µg of plasmid using Lipofectamine\n3000. At 24–36 hours post-transfection, the cells were treated with IL-1β for an\nadditional 24 h. Mitochondrial ATP levels were subsequently quantified by flow\ncytometry on the basis of the ratio of YFP to CFP fluorescence following NM-NTUs +\nlight treatment.”\nResponse Figure 20\n(a) Representative flow cytometry histograms for each group. The mitochondrial ATP\nlevels in the different groups were measured by mit-AT1.03. (b) Quantitative analysis\nof the mitochondrial ATP level (YFP/CFP ratio) (n = 3). All quantitative data are the\nmean ± s.d. P value was calculated using one-way ANOVA.\nComment-11: “4d) The IL-1β+NM-NTU +light condition is missing in the heatmap.\nIf IL-1β + NM-NTU already changed RNA level and lipid metabolism shown in figure\n6, why is light required? That is inconsistent with 4b)”\nResponse: We sincerely thank the reviewer for this critical comment regarding the\nexperimental design of the RNA-seq analysis. In our initial submission, we\ninadvertently caused confusion by not clearly specifying that the group labelled “IL-1β\n+ NM-NTU” in the RNA-seq experiment (and for the ER lipidome in Figure 6)\nactually referred to “IL-1β+NM-NTU + light”. We deeply apologize for this oversight in\nlabelling. Earlier in our study, we verified that NM-NTUs could not increase\nintracellular ATP levels in the absence of light (Fig. 4a). Furthermore, the\nqRCR and immunofluorescence results (Fig. 3b, c) indicated that without light\nirradiation, NM-NTUs could not enhance the anabolism of degenerated NPCs.\nTherefore, we did not include a dark condition in the initial RNA-seq experiment.\nHowever, as comparing both dark and light conditions is essential for clarifying the\nphotodependency of the effects of NM-NTUs, we repeated the RNA-seq experiment\nand added an “IL-1β + NM-NTU + dark” group. Compared with the control group, the\nIL-1β group and the IL-1β + NM-NTU + dark group presented a greater number of\ndifferentially expressed genes (DEGs), whereas compared with the control group, the\nIL-1β + NM-NTU + light group presented a lower number of DEGs (Response Figure\n21a, Response Figure 22a-d). PCA revealed distinct clustering among samples within\ngroups, with the gene expression profiles of the control and IL-1β + NM-NTU + light\ngroups being similar, while both groups clustered separately from the IL-1β and IL-1β\n+ NM-NTU + dark groups (Response Figure 21b). Furthermore, in contrast to the IL-\n1β + NM-NTU + dark group, the IL-1β + NM-NTU + light group presented lower levels\nof ROS-related genes (Sod2 and Prdx6) and mitochondrial apoptosis-related genes\n(Nptx1 and Bid) (Response Figure 21c). Gene set enrichment analysis (GSEA) results\nindicated that, compared to the control group, the IL-1β group and the IL-1β + NM-\nNTU + dark group presented upregulated expression of genes related to ROS signalling,\nER calcium homeostasis, and mitochondrial apoptosis and downregulated expression\nof genes related to ECM assembly (Response Figure 22e, f). In contrast to the IL-1β\ngroup and the IL-1β + NM-NTU + dark group, the IL-1β + NM-NTU + light group\nexhibited downregulation of genes associated with ROS signalling, ER calcium\nhomeostasis, and mitochondrial apoptosis and upregulation of genes associated with\nECM assembly (Response Figures 21d, 22g). These results were consistent with the\nconclusions from our initial RNA-seq data. Taken together, these results revealed that\nNM-NTUs could restore metabolic homeostasis in degenerated NPCs in a light-\ndependent manner and might have positive effects on the ER and mitochondria within\nNPCs.\nAction: We performed a new RNA-seq experiment that included a dark condition group.\nThe updated RNA-seq results have now been incorporated into the revised manuscript\n(Page 10; Fig. 4d-g; Fig. S8).\n“Compared with the control group, the IL-1β group and the IL-1β + NM-NTU + dark\ngroup presented a greater number of differentially expressed genes, whereas compared\nwith the control group, the IL-1β + NM-NTU + light group presented a lower number\nof differentially expressed genes (Fig. 4d; Fig. S8a-d). PCA revealed distinct clustering\namong samples within groups, with similar gene expression profiles in the control and\nIL-1β + NM-NTU + light groups, while both groups clustered separately from the IL-\n1β and IL-1β + NM-NTU + dark groups (Fig. 4e). Furthermore, in contrast to the IL-\n1β + NM-NTU + dark group, the IL-1β + NM-NTU + light group presented lower levels\nof ROS-related genes (Sod2 and Prdx6) and mitochondrial apoptosis-related genes\n(Nptx1 and Bid) (Fig. 4f). GSEA results indicated that, compared to the control group,\nthe IL-1β group and the IL-1β + NM-NTU + dark group presented upregulated\nexpression of genes related to ROS signalling, ER calcium homeostasis, and\nmitochondrial apoptosis and downregulated expression of genes related to ECM\nassembly (Fig. S8e and f). In contrast to the IL-1β group and the IL-1β + NM-NTU +\ndark group, the IL-1β + NM-NTU + light group exhibited downregulation of genes\nassociated with ROS signalling, ER calcium homeostasis, and mitochondrial apoptosis\nand upregulation of genes associated with ECM assembly (Fig. 4g; Fig. S8g). Taken\ntogether, these results demonstrated that NM-NTUs could restore metabolic\nhomeostasis in degenerated NPCs in a light-dependent manner and might have positive\neffects on the ER and mitochondria within NPCs.”\nResponse Figure 21\n(a) Heatmap of differentially expressed genes in NPCs; six biological replicates are\nshown. (b) PCA of genes in NPCs with different treatments. (c) Volcano plots of genes\nbetween the IL-1β + NM-NTU + light group and the IL-1β + NM-NTU + dark group.\n(d) GSEA results of pathways in the IL-1β + NM-NTU + light group compared with\nthose in the IL-1β + NM-NTU + dark group.\nResponse Figure 22\n(a) Heatmap showing genes that are differentially expressed between different groups.\n(b-d) Volcano plots of differentially expressed genes between the following\nconditions: (b) control group versus the IL-1β group. (c) control group versus the IL-\n1β + NM-NTU + dark group. (d) control group versus the IL-1β + NM-NTU + light\ngroup. (e) GSEA results of the IL-1β group compared with the control group. (f) GSEA\nresults of the IL-1β + NM-NTU + dark group compared with the control group. (g)\nGSEA results of the IL-1β + NM-NTU + light group compared with the IL-1β group.\nComment-12:\n“Figure 5\n5e) The figure shows percentage, not number of mitochondria, please clarify number\nor percentage in text (Line 214). If number was quantified, the total number of\nmitochondria should also be quantified. Authors need to define the cut-off distance\nbetween OMM and ER which was set as the upper limit to consider distance between\nthe two membranes as MERCS (<30 nm? <50nm?). The number of cells analysed per\nsample should also be included.”\nResponse: We thank the reviewer very much for the comments. In fact, we wanted to\nexpress the mitochondrial percentage in the initial manuscript. In the revised manuscript,\nconsidering the similar comment raised by the reviewer in Fig. 2i (Comment-3), we\nhave modified our MERCS analysis protocol [PMID: 36746962, 40514428]. The\ndistances between the OMM and ER were categorized into three groups: ≤25 nm,\n25<distance≤50 nm, and 50<distance≤100 nm, after which we performed separate\nstatistical analyses. The measurements of each group are based on 3 biological\nreplicates with n = 50 cells from 59–65 fields. For each field of view, we systematically\nquantified the ER tubules closely apposed to the mitochondria, the number of\nmitochondria, and the average length of the ER tubules (details in Comment 3).\nInterestingly, compared with the IL-1β or IL-1β + NM-NTU + dark group, the IL-1β +\nNM-NTU + light group presented significantly reduced ER near mitochondria\n(Response Figure 23a, b). ER length and mitochondrial counts were calculated for each\nfield, and the results revealed that there was no significant difference in the number of\nmitochondria or the average length of ER tubules among the four groups (Response\nFigure 23c, d).\nAction: We have performed new analyses and clarified the methodology in the revised\nmanuscript. The changes are highlighted (Page 11; Pages 24-25; Fig. 5f-i).\n“Compared with the IL-1β or IL-1β + NM-NTU + dark group, the IL-1β + NM-NTU +\nlight group had significantly fewer ER closely apposed to mitochondria (Fig. 5f and g).\nER length and mitochondrial counts were calculated for each field, and the results\nrevealed no significant difference in the number of mitochondria or the average length\nof ER tubules among the four groups (Fig. 5h and i).”\n“MERCS in TEM images were manually analysed in ImageJ. Briefly, the image scale\nwas first set with the Set Scale tool, and the freehand line tool was subsequently used\nto measure the length of the ER or membrane or the perimeter of the mitochondria. The\ncontact length was defined as the ER membrane length (within 0–100 nm of the\nmitochondria) divided by the mitochondrial perimeter.”\nResponse Figure 23\n(a) TEM images of the mitochondrial and ER morphology. The ER and mitochondria\nwere graphically reconstructed: mitochondria (red); ER (green). (b) Quantification of\nthe mitochondrial–ER contact length in (a). The contact length was quantified by taking\nthe ratio of the length of the ER (within contact distances of 25–100 nm) to the\nperimeter of the mitochondria. (c) Mitochondrial count per field from TEM in (a). (d)\nQuantification of ER lengths per field from the TEM image in (a). Data are derived\nfrom 50 cells (sampled across 59 fields) for the control group, 50 cells (sampled across\n65 fields) for the IL-1β group, 50 cells (sampled across 62 fields) for the IL-1β + NM-\nNTU + dark group and 50 cells (sampled across 63 fields) for the IL-1β + NM-NTU +\nlight group; n = 3 biological replicates. P values were calculated using one-way\nANOVA (b - d).\nComment-13: “5f) same comments as for Figure 2 regarding ICC.”\nResponse: We thank the reviewer for this comment. We repeated the\nimmunofluorescence experiments using calnexin and TOMM20. We also implemented\nupdated image processing and statistical approaches as described in Comment-2.\nCompared with those in the control group, the MERCS in the IL-1β and IL-1β + NM-\nNTU + dark groups were increased (Response Figure 24a, b). In contrast, the IL-1β +\nNM-NTU + light treatment resulted in a decrease in MERCS (Response Figure 24a, b).\nAction: We have repeated the experiments and modified the image analysis process.\nThe changes are highlighted (Page 11; Page 25; Fig. 5d and e).\n“We subsequently assessed the formation of MERCS by immunostaining the ER and\nmitochondria and observed that the NM-NTU + light treatment resulted in a decrease\nin MERCS (Fig. 5d and e).”\n“Similarly, the MERCS structure of the NPCs was observed by immunostaining the ER\nand mitochondria with calnexin and TOMM20. To quantify MERCS, we performed\ncolocalization analysis using ImageJ plugins (Coloc2 and JACoP) following\nestablished methods95.”\nResponse Figure 24\n(a) Representative confocal image of NPCs stained for calnexin (green, ER) and\nTOMM20 (red, mitochondria). Merged panel images were processed using an ImageJ-\nbased pipeline for quantifying MERCS (white dots indicate colocalized pixels). (b)\nMander’s coefficients (M1) of (a). The M1 quantifies the percentage of the signal\nintensity of mitochondria that colocalizes with the ER relative to the total signal\nintensity of the mitochondria. n = 3 biological replicates, the same cells (n = 20) were\nacquired and analysed. P value was calculated using one-way ANOVA (b). Scale bars:\n5 μm (a).\nComment-14:\n“Figure 6\nThe authors tested the global membrane fluidity after NM-NTU treatment. Is it possible\nto show whether the formation of MAM is dependent on the level of TG to confirm that\naltered lipidomics change MAM formation?”\nResponse: We sincerely thank the reviewer for raising this important point. Our\nlipidomic data revealed that the triglyceride (TG) levels in the control group and the\nIL-1β + NM-NTU + light group were greater than those in the IL-1β group. To address\nwhether MAM formation was dependent on TG levels, we modulated TG levels in the\nER using inhibitors of TG synthesis. The final and committed step of TG synthesis is\ncatalysed by two diacylglycerol acyltransferases, DGAT1 and DGAT2, which are\nER-resident transmembrane enzymes [PMID: 32559414]. We treated NPCs with a\nDGAT1 inhibitor (A-922500), a DGAT2 inhibitor (PF-06424439), or a combination of both for\n24 hours. Thin-layer chromatography (TLC) analysis of ER lipids confirmed that\ninhibiting DGAT1 and DGAT2, either individually or in combination, reduced the TG\nlevels in the ER (Response Figures 25a, b).\nTo investigate the role of TG levels in ER–mitochondria contact, we performed\nimmunofluorescence experiments using calnexin and TOMM20 in cells treated with\nDGAT1 and DGAT2 inhibitors. Compared with the control group, the inhibition of TG\nsynthesis significantly increased the number of MERCS (Response Figure 25c, d).\nThese results suggested a link between TG levels and the phenotype of MERCS. We\nfurther discussed the reasons for this link. Since inhibition of TG synthesis disrupted\nthe channelling of fatty acids into neutral lipid storage [PMID: 28768178] and\ndisruption of lipid homeostasis elicited ER stress [PMID: 19966778], the formation of\nMERCS might increase as an adaptive response to the unfolded protein response\n[PMID: 36586409, 33335290]. In general, our findings indicated that an altered ER\nlipidome (reduced TG levels) might contribute to increased ER–mitochondria contact.\nAction: We have reported the abovementioned additional experiments in the revised\nmanuscript. The changes are highlighted (Page 14; Page 26; Fig. 6j-l).\n“To address whether ER–mitochondria contact was related to TG levels, we modulated\nTG levels in the ER by applying inhibitors of TG synthesis. The final and committed\nstep of TG synthesis is catalysed by two diacylglycerol acyltransferases, DGAT1 and\nDGAT2, which are ER-resident transmembrane enzymes75. We treated NPCs with a\nDGAT1 inhibitor (A-922500) and a DGAT2 inhibitor (PF-06424439) or a combination\nof both. TLC analysis of ER lipids confirmed that inhibiting DGAT1 and DGAT2, either\nindividually or in combination, reduced the TG levels in the ER (Fig. 6j). We then\nperformed immunofluorescence experiments using calnexin and TOMM20 in cells\ntreated with DGAT1 and DGAT2 inhibitors. Compared with the control group, the\ninhibition of TG synthesis significantly increased the number of MERCS (Fig. 6k and\nl). These results suggested a link between TG levels and the phenotype of MERCS. We\nfurther discuss the reasons for this link. Since inhibition of TG synthesis disrupts the\nchannelling of fatty acids into neutral lipid storage76 and disruption of lipid homeostasis\nelicits ER stress77, the formation of MERCS may increase as an adaptive response to\nthe unfolded protein response78,79. In general, our findings indicated that an altered ER\nlipidome (reduced TG levels) might contribute to increased ER–mitochondria contact.”\n“TLC analysis\nTLC was used for TG quantification96. Specifically, the total lipids in the ER extracts\nwere obtained by suspending the ER in 1 mL of PBS supplemented with 0.1 mM\nphenylmethylsulphonyl fluoride (PMSF) and 2 mL of chloroform/methanol (2:1 vol/vol)\nsupplemented with 0.01% butylated hydroxytoluene. After centrifugation at 1500 × g\nfor 5 min, the organic phases were collected and dried with nitrogen and resuspended\nin chloroform. TLC was performed by spotting the total lipid extract dissolved in\nchloroform onto 10 cm EMD TLC Silica Gel plates (Labshark) and developing them\nwith hexane/diethyl ether/acetic acid (80:20:2, vol/vol). Lipids were visualized with\niodine vapour prior to imaging.”\nResponse Figure 25\n(a) TLC analysis imaging of TG levels in the ER of NPCs treated with/without a\nDGAT1 inhibitor (A-922500, 20 μg mL-1) or a DGAT2 inhibitor (PF-06424439, 20 μg\nmL-1) (n = 3). (b) Quantitative analysis of TLC (n = 3). (c) Representative confocal\nimage of NPCs stained for calnexin (green, ER) and TOMM20 (red, mitochondria).\nMerged panel images were processed via an ImageJ-based pipeline for quantifying\nMERCS (white dots indicate colocalized pixels). (d) Mander’s coefficients (M1) of (c).\nThe M1 quantifies the percentage of the signal intensity of mitochondria that\ncolocalizes with the ER relative to the total signal intensity of the mitochondria. n = 3\nbiological replicates, the same cells (n = 20) were acquired and analysed. P values were\ncalculated using one-way ANOVA (b, d). Scale bars: 5 μm (c).\nComment-15: “6a) The IL-1β+NM-NTU +light condition is missing in the heatmap.”\nResponse: Thank you for your suggestion. We sincerely thank the reviewer for this\ncomment regarding the experimental design of the lipidome. Like in Comment-11, in\nour initial submission, we inadvertently introduced confusion by not clearly specifying\nthat the group labelled “IL-1β + NM-NTU” in the ER lipidome experiment actually\nreferred to “IL-1β + NM-NTU + light”. We deeply apologize for this oversight in\nlabelling.\nHowever, comparing both the IL-1β + NM-NTU + dark and the IL-1β + NM-NTU\n+ light conditions is essential for clarifying the photodependency of the effects of NM-\nNTUs. To address this point, we repeated the lipidomic experiment and added an “IL-\n1β + NM-NTU + dark” group. In the new lipidomic results, we identified 876\nmetabolites in the ER lipidome, with 392 significantly different metabolites between\nthe IL-1β + NM-NTU + light group and the IL-1β + NM-NTU + dark group (Response\nFigures 26a, 27a). PCA revealed distinct clustering among samples within groups, with\nsimilar metabolite profiles in the control and IL-1β + NM-NTU + light groups, while\nboth groups clustered separately from the IL-1β and IL-1β + NM-NTU + dark groups\n(Response Figure 26b). Compared with the IL-1β + NM-NTU + dark group, the IL-1β\n+ NM-NTU + light group significantly affected Ca2+-dependent events and lipid\nmetabolism (Response Figures 26c, 27b). Kyoto Encyclopaedia of Genes and Genomes\nenrichment analysis revealed that NM-NTU + light treatment affected pathways related\nto the biosynthesis of unsaturated fatty acids (Response Figure 27c).\nWe subsequently analysed the distribution and changes in ER lipid categories\namong the groups. Compared with those in the control group, the triglyceride (TG)\nlevels were significantly reduced in the IL-1β and IL-1β + NM-NTU + dark groups,\nwhereas they were restored in the IL-1β + NM-NTU + light treatment group (Response\nFigure 26d). The levels of other major ER lipid species (e.g., phosphatidylcholine,\nphosphatidylethanolamines, phosphatidylinositol, and sphingomyelin) did not change\nsignificantly among the groups (Response Figures 26e-h).\nWe further used thin-layer chromatography (TLC) to determine the TG content in\nthe ER of each group. Compared with the IL-1β + NM-NTU + dark group, the IL-1β +\nNM-NTU + light group contained more TGs (Response Figure 26i). To address whether\nER–mitochondria contact was related to TG levels, we modulated TG levels in the ER\nby applying inhibitors of TG synthesis. The final and committed step of TG synthesis\nis catalysed by two diacylglycerol acyltransferases, DGAT1 and DGAT2, which are\nER-resident transmembrane enzymes [PMID: 32559414]. We treated NPCs with a\nDGAT1 inhibitor (A-922500) and a DGAT2 inhibitor (PF-06424439) or a combination\nof both. TLC analysis of ER lipids confirmed that inhibiting DGAT1 and DGAT2,\neither individually or in combination, reduced the TG levels in the ER (Response Figure 26j).\nWe then performed immunofluorescence experiments using calnexin and TOMM20 in\ncells treated with DGAT1 and DGAT2 inhibitors. Compared with the control group, the\ninhibition of TG synthesis significantly increased the number of MERCS (Response\nFigures 26k, l). These results suggested a link between TG levels and the phenotype of\nMERCS. We further discuss the reasons for this link. Since inhibition of TG synthesis\ndisrupts the channelling of fatty acids into neutral lipid storage [PMID: 28768178] and\ndisruption of lipid homeostasis elicits ER stress [PMID: 19966778], the formation of\nMERCS may increase as an adaptive response to the unfolded protein response [PMID:\n36586409, 33335290]. In general, our findings indicated that an altered ER lipidome\n(reduced TG levels) might contribute to increased ER–mitochondria contact.\nWe subsequently analysed the acyl chain compositions of TG. Compared with\nthose in the IL-1β + NM-NTU + light group, the proportions of TG (50:2)[NL-18:1],\nTG (54:2)[NL-18:0], TG (52:3)[NL-16:1], TG (56:3)[NL-18:1], and TG (54:3)[NL-\n18:1] significantly increased (Response Figure 26m). The TG volcano map further\nconfirmed these results (Response Figure 26n). The proportion of unsaturated TG in\nthe IL-1β + NM-NTU + light group was significantly greater than that in the IL-1β +\nNM-NTU + dark group (Response Figure 26o). In summary, NM-NTU + light treatment\nincreased the unsaturation levels of TGs. Given that lipid unsaturation significantly\nincreased membrane fluidity [PMID: 29410529] and affected the contact between\nintracellular organelles [PMID: 36746962], we speculated that the NM-NTU reduced\nthe formation of MERCS in degenerative cells by enhancing membrane fluidity.\nNext, we measured global membrane fluidity in NPCs using a membrane fluidity kit.\nMembrane fluidity was quantified by measuring the ratio of excimer to monomer\nfluorescence of lipophilic pyrene probes [PMID: 36103824]. With increasing\nmembrane fluidity, the probe underwent excimer formation. We found that NPCs in the\nIL-1β + NM-NTU + light group exhibited greater membrane fluidity than did those in\nthe IL-1β + NM-NTU + dark group (Response Figure 26p). Overall, NM-NTUs with\nlight illumination might affect ER–mitochondria contact by remodelling the lipid\ncomposition of the ER.\nAction: We performed a new lipidomic experiment that included a dark condition group.\nThe updated results have now been incorporated into the revised manuscript (Pages 13-\n15; Fig. 6; Fig S12).\n“We identified 876 metabolites in the ER lipidome, with 392 significantly different\nmetabolites between the IL-1β + NM-NTU + light group and the IL-1β + NM-NTU +\ndark group (Fig. 6a; Fig. S12a). PCA revealed distinct clustering among samples within\ngroups, with similar metabolite profiles in the control and IL-1β + NM-NTU + light\ngroups, while both groups clustered separately from the IL-1β and IL-1β + NM-NTU\n+ dark groups (Fig. 6b). Compared with the IL-1β + NM-NTU + dark group, the IL-1β\n+ NM-NTU + light group significantly affected Ca2+-dependent events and lipid\nmetabolism (Fig. 6c; Fig. S12b). Kyoto Encyclopaedia of Genes and Genomes\nenrichment analysis revealed that NM-NTU + light treatment affected pathways related\nto the biosynthesis of unsaturated fatty acids (Fig. S12c).\nWe subsequently analysed the distribution and changes in ER lipid categories\namong the groups. Compared with those in the control group, the triglyceride (TG)\nlevels were significantly reduced in the IL-1β and IL-1β + NM-NTU + dark groups,\nwhereas they were restored in the IL-1β + NM-NTU + light treatment group (Fig. 6d).\nThe levels of other major ER lipid species (e.g., phosphatidylcholine,\nphosphatidylethanolamines, phosphatidylinositol, and sphingomyelin) did not change\nsignificantly among the groups (Fig. 6e-h).\nWe further used thin-layer chromatography (TLC) to determine the TG content in\nthe ER of each group. Compared with the IL-1β + NM-NTU + dark group, the IL-1β +\nNM-NTU + light group contained more TGs (Fig. 6i). To address whether ER–\nmitochondria contact was related to TG levels, we modulated TG levels in the ER by\napplying inhibitors of TG synthesis. The final and committed step of TG synthesis is\ncatalysed by two diacylglycerol acyltransferases, DGAT1 and DGAT2, which are\nER-resident transmembrane enzymes75. We treated NPCs with a DGAT1 inhibitor (A-\n922500) and a DGAT2 inhibitor (PF-06424439) or a combination of both. TLC analysis\nof ER lipids confirmed that inhibiting DGAT1 and DGAT2, either individually or in\ncombination, reduced the TG levels in the ER (Fig. 6j). We then performed\nimmunofluorescence experiments using calnexin and TOMM20 in cells treated with\nDGAT1 and DGAT2 inhibitors. Compared with the control group, the inhibition of TG\nsynthesis significantly increased the number of MERCS (Fig. 6k and l). These results\nsuggested a link between TG levels and the phenotype of MERCS. We further discuss\nthe reasons for this link. Since inhibition of TG synthesis disrupts the channelling of\nfatty acids into neutral lipid storage76 and disruption of lipid homeostasis elicits ER\nstress77, the formation of MERCS may increase as an adaptive response to the unfolded\nprotein response78-79. In general, our findings indicated that an altered ER lipidome\n(reduced TG levels) might contribute to increased ER–mitochondria contact.\nWe subsequently analysed the acyl chain compositions of TG. Compared with\nthose in the IL-1β + NM-NTU + light group, the proportions of TG (50:2)[NL-18:1],\nTG (54:2)[NL-18:0], TG (52:3)[NL-16:1], TG (56:3)[NL-18:1], and TG (54:3)[NL-\n18:1] significantly increased (Fig. 6m). The TG volcano map further confirmed these\nresults (Fig. 6n). The proportion of unsaturated TG in the IL-1β + NM-NTU + light\ngroup was significantly greater than that in the IL-1β + NM-NTU + dark group (Fig.\n6o). In summary, NM-NTU + light treatment increased the unsaturation levels of TGs.\nGiven that lipid unsaturation significantly increased membrane fluidity73 and affected\nthe contact between intracellular organelles42, we speculated that the NM-NTU reduced\nthe formation of MERCS in degenerative cells by enhancing membrane fluidity. Next,\nwe measured global membrane fluidity in NPCs using a membrane fluidity kit.\nMembrane fluidity was quantified by measuring the ratio of excimer to monomer\nfluorescence of lipophilic pyrene probes80. With increasing membrane fluidity, the\nprobe underwent excimer formation. We found that NPCs in the IL-1β + NM-NTU +\nlight group exhibited greater membrane fluidity than did those in the IL-1β + NM-NTU\n+ dark group (Fig. 6p). Overall, NM-NTUs with light illumination might affect ER–\nmitochondria contact by remodelling the lipid composition of the ER.”\nResponse Figure 26\n(a) Heatmap showing differentially abundant lipids in the ER of NPCs; three biological\nreplicates are shown. (b) PCA of lipids in the ER of NPCs with different treatments. (c)\nReactome database analysis of the metabolomic data revealed that the NM-NTU + light\ntreatment group was associated with Ca2+-dependent events and pathways related to\nlipid metabolism. The topology of the associated pathways is shown. (d-h) The bar chart\nshows the proportions of lipid classes in the ER of the NPCs in different groups. The\nlipid species included triglycerides (TG) (d), phosphatidylcholine (PC) (e),\nphosphatidylethanolamines (PE) (f), phosphatidylinositol (PI) (g), and sphingomyelin\n(SM) (h). The proportion of each lipid species was normalized to that of total lipids (n\n= 3). (i) Thin layer chromatography (TLC) analysis imaging (left) and quantification\n(right) of TG levels in the ER of NPCs with different treatments (n = 3). (j) TLC analysis\nimaging (left) and quantification (right) of TG levels in the ER of NPCs treated\nwith/without a DGAT1 inhibitor (A-922500, 20 μg mL-1) or a DGAT2 inhibitor (PF-\n06424439, 20 μg mL-1) (n = 3). (k) Representative confocal image of NPCs stained for\ncalnexin (green, ER) and TOMM20 (red, mitochondria). Merged panel images were\nprocessed via an ImageJ-based pipeline for quantifying MERCS (white dots indicate\ncolocalized pixels). (l) Mander’s coefficients (M1) of (k). The M1 quantifies the\npercentage of the signal intensity of mitochondria that colocalizes with the ER relative\nto the total signal intensity of the mitochondria. n = 3 biological replicates, the same\ncells (n = 20) were acquired and analysed. (m) Proportion of lipid types in TGs in the\nER of NPCs with different treatments. The proportion of each lipid was normalized to\nthat of total lipids (n = 3). (n) Volcano plots of TG with different treatments (IL-1β +\nNM-NTU + light group versus IL-1β + NM-NTU + dark group). (o) The proportion of\nunsaturated TG in the ER of NPCs with different treatments (n = 3). (p) Global\nmembrane fluidity of NPCs (n = 3). All quantitative data are the mean ± s.d. P value\nwere calculated using one-way ANOVA (d–j, l, m, o, and p). Scale bars: 5 μm (k).\nResponse Figure 27\n(a) Heatmap and volcano plots showing differentially abundant lipids between different\ngroups. (b) Differentially abundant lipid metabolites were analysed using the Reactome\ndatabase. The major metabolic processes are annotated. (c) KEGG analysis between the\ndifferent groups.\nComment-16:\n“Figure 7 and 10\nThe in vivo experiments showing positive effects of NM-NTUs on the disc height index\n(DHI) in rats and the use of a remote wireless control system for illumination in rabbit\nare very promising and interestingly. However, the authors do neither show nor discuss\nany connection to the mechanism of restoration of organelle interaction network\nreported in vitro in NM-NTU treated cells. This point needs to be addressed, since the\nconnection is missing now.”\nResponse: We sincerely appreciate the reviewer’s insightful observation regarding the\nmechanistic link in vitro and in vivo. To clarify the correlation between the in vitro and\nin vivo results, we conducted immunofluorescence costaining for calnexin and\nTOMM20 in rat IVD tissue sections. The results indicated that compared with the sham\ngroup, MERCS increased in the puncture + vehicle and puncture + vehicle + NM-NTU\n+ dark group. Moreover, MERCS significantly decreased in the NM-NTU + light\ntreatment group compared with the NM-NTU + dark treatment group (Response\nFigures 28a, b), which revealed that the abnormal contact between the ER and\nmitochondria in rat IVDD was attenuated. We further extracted cells from the NP tissue\nand performed TEM to further evaluate the MERCS in the tissue samples. For each\nfield of view, we systematically quantified the ER tubules that were closely apposed to\nthe mitochondria, the number of mitochondria, and the average length of the ER tubules.\nInterestingly, compared with the puncture + vehicle and puncture + vehicle + NM-NTU\n+ dark group, the NM-NTU + light group had significantly fewer ER closely apposed\nto mitochondria (Response Figures 28c, d). ER length and mitochondrial counts were\ncalculated for each field, and the results revealed that there was no significant difference\nin the number of mitochondria or the average length of ER tubules among the four\ngroups (Response Figures 28e, f). These results provided partial evidence that the in\nvivo therapeutic effects of NM-NTUs (evidenced by the improved DHI and disc water\ncontent) were functionally linked to their ability to restore the organelle interaction\nnetwork.\nAction: We have reported the abovementioned additional experiments in the revised\nmanuscript. The changes are highlighted (Page 16; Fig. 8c-h).\n“We subsequently conducted immunofluorescence costaining of calnexin and\nTOMM20 in rat IVD tissue sections. The results indicated that compared with the sham\ngroup, MERCS increased in the puncture + vehicle and puncture + vehicle + NM-NTU\n+ dark group. Moreover, MERCS significantly decreased in the NM-NTU + light\ntreatment group compared with the NM-NTU + dark treatment group (Fig. 8c and d),\nwhich revealed that the abnormal contact between the ER and mitochondria in rat IVDD\nwas attenuated. We further extracted cells from the NP tissue and performed TEM to\nfurther evaluate the MERCS in the tissue samples. For each field of view, we\nsystematically quantified the ER tubules that were closely apposed to the mitochondria,\nthe number of mitochondria, and the average length of the ER tubules. Interestingly,\nthe NM-NTU + light group showed a significant reduction of ER closely apposed to\nmitochondria compared with the puncture + vehicle and puncture + vehicle + NM-NTU\n+ dark group (Fig. 8e and f). ER length and mitochondrial counts were calculated for\neach field, and the results revealed no significant difference in the number of\nmitochondria or the average length of ER tubules among the four groups (Fig. 8g and\nh). These results provided partial evidence that the in vivo therapeutic effects of NM-\nNTUs were functionally linked to their ability to restore the organelle interaction\nnetwork.”\nResponse Figure 28\n(a) Representative fluorescence staining images of calnexin (green, ER) and TOMM20\n(red, mitochondria). Merged panel images were processed using an ImageJ-based\npipeline for quantifying MERCS (white dots indicate colocalized pixels). (b) Mander’s\ncoefficients (M1) of (a). The M1 quantifies the percentage of the signal intensity of\nmitochondria that colocalizes with the ER relative to the total signal intensity of the\nmitochondria. n = 4 biological replicates, the same fields of vision (n = 12) were\nacquired and analysed. (c) TEM images of mitochondrial and ER morphology. The ER\nand mitochondria were graphically reconstructed: mitochondria (red); ER (green). (d)\nQuantification of the mitochondrial–ER contact length in (c). The contact length was\nquantified by taking the ratio of the length of the ER (within contact distances of 0–\n100 nm) to the perimeter of the mitochondria. (e) Mitochondrial count per field from\nTEM in (c). (f) Quantification of ER lengths per field from the TEM image in (c). All\nquantitative data are the mean ± s.d. P value was calculated using one-way ANOVA (b,\nd, e, and f). Scale bars: 50 µm (a); 1 µm (c).\nComment-17: “7b, d, f, g and 10d) Use arrows to indicate points of interest and\nincrease resolution of images”\nResponse: We sincerely thank the reviewer for the valuable suggestions regarding the\nimprovement in the quality and interpretability of these figures. As recommended, we\nhave added arrows or frames to indicate points of interest in these figures (Response\nFigures 29, 30). These markers now highlight the specific intervertebral space that is\ncritical for the interpretation of our data. Moreover, we have re-exported these figures\nto improve their resolution (minimum of 600 dpi). The updated figures have been\nsubmitted with the revised manuscript.\nAction: These figures have been revised (Fig. 7b, d, f, h, j, and l; Fig. 10d).\nResponse Figure 29\n(a) Representative micro-CT images of rat caudal vertebrae at 4 and 8 weeks. The\nobserved segments are indicated by red arrows. (b) H&E staining images of rat IVDs\nat 4 and 8 weeks. The nucleus pulposus areas are outlined with a red dashed line. (c)\nAlcian blue, Safranin O-Fast Green, and Picrosirius red staining images of the rat IVD\nat 4 and 8 weeks. The nucleus pulposus areas are outlined with a red dashed line. (d)\nRepresentative magnetic resonance imaging (MRI) images of a rat IVD at 4 and 8\nweeks. The regions of interest are marked by red triangles.\nResponse Figure 30\nMacroscopic view of IVD samples. The region highlighted by the red square is shown\nat higher magnification. The nucleus pulposus is delineated by a red dashed circle.\nComment-18: “7g and10e) Quantifications of the images are missing”\nResponse: We sincerely thank the reviewer for pointing out the need for image\nquantification. As suggested, we have performed statistical analyses on the histological\nstaining results for both Figure 7g (rat IVDD model) and Figure 10e (rabbit IVDD\nmodel).\nAs shown in Figure 7g (Figures 7h, j, l in the revised manuscript), Alcian blue and\nSafranin O/Fast Green staining are commonly employed histological techniques for the\nassessment of proteoglycan content in tissue sections. The presence and relative\nabundance of proteoglycans within the tissue can be evaluated on the basis of the\nstaining intensities [PMID: 36152244]. Quantitative analysis revealed that compared\nwith the NM-NTU + dark group, the NM-NTU + light group exhibited significantly\ngreater staining intensity (higher proteoglycan content) at 4 and 8 weeks post-surgery\nin a rat IVDD model (Response Figures 31a, b). Picrosirius red staining serves as a tool\nfor analysing pathological nucleus pulposus fibrosis [PMID: 25023614]. Quantitative\nanalysis of Picrosirius red staining revealed a significantly lower intensity in the NM-\nNTU + light group than in the NM-NTU + dark group at 4 and 8 weeks post-surgery\n(Response Figure 31c), suggesting a reduced degree of fibrosis within the nucleus\npulposus tissue by NM-NTU + light treatment. As shown in Figure 10e, we performed\na similar quantitative analysis on the rabbit IVDD model. These results were consistent\nwith those observed in the rat model, which revealed that NM-NTU + PU-wp-LED +\nlight treatment attenuated nucleus pulposus degeneration (Response Figures 31d-f).\nAction: We have performed quantitative statistical analyses on the histological staining\nresults in both figures (Fig. 7h-m; Fig. 10e-h).\nResponse Figure 31\n(a) Alcian blue staining images and quantification of rat IVDs at 4 and 8 weeks (n = 4).\n(b) Safranin O-Fast Green staining images and quantification of rat IVDs at 4 and 8\nweeks (n = 4). (c) Picrosirius red staining images and quantification of rat IVDs at 4\nand 8 weeks (n = 4). (d) H&E staining images of rabbit IVDs at 4 and 8 weeks.\nHistological grade evaluation was performed at 4 and 8 weeks in each group (n = 4).\n(e) Alcian blue staining images and quantification of rabbit IVDs at 4 and 8 weeks (n =\n4). (f) Safranin O-Fast Green staining images and quantification of rabbit IVDs at 4 and\n8 weeks (n = 4). All quantitative data are the mean ± s.d. P values were calculated using\none-way ANOVA (a - f). Scale bar: 400 µm (a - f).\nComment-19:\n“Minor comments\n-Line 29: What does ‘ignoring entire cell damage’ mean? Do authors mean ‘ignoring\nthe role of organelle interactions in cell damage’?”\nResponse: We sincerely appreciate the reviewer’s careful reading and constructive\nsuggestion. Our intended meaning was indeed “ignoring the role of organelle\ninteractions in cell damage.” We have revised this sentence in the revised manuscript.\nAction: The changes are highlighted (Page 2).\n“Current treatments usually focus on individual organelles while ignoring the role of\norganelle interactions in cell damage caused by cascade reactions.”\nComment-20: “-Line 35: What does ‘independent’ mean in the phrase ‘plant-derived\nindependent photosynthetic systems’. Is the word ‘independent’ redundant?”\nResponse: We sincerely appreciate the reviewer’s keen attention to linguistic precision.\nWe agree that the term “independent” was indeed redundant in this context. We have\nremoved the word “independent” in the phrase.\nAction: The changes are highlighted (Page 2).\n“In this study, plant-derived photosynthetic systems effectively restored homeostasis of\nthe interaction network of animal organelles.”\nComment-21: “-How are Line 47 & 48 related to mitochondria acquisition from Line\n49?”\nResponse: We thank the reviewer for the valuable comment. We have strengthened the\ndescription of the relationship between life evolution and mitochondrial acquisition.\nAccording to the timeline of life evolution, approximately 3.5 billion years ago,\nDNA/protein-containing organisms evolved into cyanobacteria [PMID: 8681375,\n15073369]. According to molecular phylogeny and biogeochemistry studies,\neukaryotes appeared approximately 1.5 billion years ago [PMID: 11452306]. The origin\nand cellular complexity of eukaryotes represent a major enigma in biology. Current data\nsupport scenarios in which an archaeal host cell and an alphaproteobacterial\n(mitochondrial) endosymbiont merged together, resulting in the first eukaryotic cell\n[PMID: 28077874]. Research has shown that all complex life is composed of eukaryotic\ncells. Eukaryotic cells arising from prokaryotes and otherwise prokaryotes show no\ntendency to evolve greater complexity [PMID: 20962839]. The main reason for this\nphenomenon is the energetics of genome complexity. The prokaryotic genome size is\nconstrained by bioenergetics. The acquisition of mitochondria in eukaryotes was a\nrevolution in cell structure. The endosymbiosis that gave rise to mitochondria\nrestructured the distribution of DNA in relation to bioenergetic membranes, permitting\na remarkable expansion in the number of genes that were expressed [PMID: 32428484].\nIn reality, mitochondrial power probably enabled an expansion in eukaryotic genome\nsize (from a maximum of 13 Mb in bacteria up to ∼150,000 Mb in eukaryotes), an\nincrease in the number of protein-coding genes (4-fold on average) and an increase in\ngene expression of hundreds to thousands of times [PMID: 32428484]. This vast\nincrease in genomic capacity was strictly dependent on mitochondrial power and was a\nprerequisite for eukaryotic complexity, the key innovation en route to multicellular life.\nAction: To clarify this relationship, we have revised the relevant content in the\nmanuscript (Page 2).\n“The early evolution of life underwent a process of formation from simple organic\nmolecules to precellular and primitive lives1,2. For the evolution of complex life, the\nacquisition of mitochondria in eukaryotes is a prerequisite3,4. The endosymbiosis that\ngave rise to mitochondria restructured the distribution of DNA in relation to\nbioenergetic membranes, thereby allowing eukaryotes to significantly increase the\nnumber of gene expressed compared with prokaryotes, based on which the diversity\nand complexity of life have evolved4,5.”\nComment-22: “-How are malic acid cycle and energy homeostasis from Line 53 & 54\nrelated to defensive adaptation mentioned in Line 52?”\nResponse: We thank the reviewer for the insightful comment. The reviewer raises an\nimportant point regarding the connection between the malic acid cycle (malate valve)\nand energy homeostasis on the one hand, and defensive adaptation on the other.\nMalate valves are systems for balancing the ATP/NAD(P)H ratio required in\nvarious subcellular compartments in plant cells. For energy-consuming reactions, the\ndelivery of NAD(P)H and ATP must take place at the required rates and in specific\ncompartments, since the pool sizes of these energy carriers are rather limited and, in\ngeneral, they are not directly transported across biomembranes [PMID: 15032873].\nPlant cells possess specific translocators enabling the indirect exchange of reducing\nequivalents. These translocators enable the exchange of dicarboxylates such as malate\nand oxaloacetate (malate/OAA shuttles; Response Figure 32) [PMID: 29933514].\nMalate is a versatile compound involved in plant metabolism that can easily be\ntransported across subcellular membranes and utilised as a substrate for mitochondrial\nATP production or for the provision of NADH to the cytosol (Response Figure 32).\nMalate/OAA shuttles act in combination with malate dehydrogenases (MDHs) as key\nenzymes connecting compartments. They are powerful systems for balancing metabolic\nfluxes by enabling the indirect transfer of reducing equivalents and play a key role in\nplant metabolism.\nTherefore, the malate valve is a crucial regulatory mechanism that helps plants\nmaintain metabolic stability (energy homeostasis) under fluctuating or stressful\nenvironmental conditions—which is, in itself, a fundamental defensive adaptation\n[PMID: 15032873]. Plants are frequently exposed to rapid changes in environmental\nconditions. The imposed changes can occur within seconds, minutes, hours or days.\nAt all instants, metabolism should run smoothly without major disturbances, and\nmetabolite pools should be kept constant. Clearly, regulation at various sites and levels\nis required to achieve homeostasis of all metabolic fluxes and pools, particularly those\nconnected with the generation and consumption of reducing equivalents and ATP. The\nvarious forms of energy should consistently be available at certain rates, at defined\nratios, such as those of NADPH to ATP, and in the various compartments according to\nactual needs. The malate valve is a useful means to balance the ATP/NADPH ratio in\nchloroplasts. Export of excess NAD(P)H through the malate valves will allow for the\ncontinued production of ATP in photosynthesis and in oxidative phosphorylation. When\nplants are subjected to conditions such as high light, high CO , NH + nutrition, and cold\n2 4\nstress, which require altered activity of malate valve enzymes, altered expression levels\nof the MDH isoforms can be observed to balance the cellular reducing equivalents\n[PMID: 15032873, 29933514, 22140244].\nAction: To clarify this point, we have modified the manuscript to directly present both\nthe malate valve and the maintenance of energy homeostasis as integral components of\nthe plant’s defensive adaptation to stress (Pages 2-3).\n“Plants possess mitochondria and chloroplasts and exhibit unique defensive adaptations\nto stress6,7. Malate/oxaloacetate translocators together with malate dehydrogenases\n(malate valves) connect chloroplasts and mitochondria in plant cells. When plants are\nsubjected to conditions such as high light, high CO , and cold stress, the activities of\n2\nthe enzymes of the malate valves change to balance the ATP/NAD(P)H ratio required\nin subcellular compartments and therefore balance metabolic fluxes under stress\nconditions8-10.”\nResponse Figure 32\nLocalisation of malate dehydrogenase isoforms. Malate/OAA translocators together\nwith malate dehydrogenases (malate valves) enable the indirect transfer of reducing\nequivalents between different subcellular compartments in plant cells. Figure from J.\nSelinski, Plant Biol (Stuttg). 2019 Jan; 21 Suppl 1(Suppl Suppl 1):21-30.\nComment-23: “-Line 55 & 56: Is changing the unsaturation of the membrane lipids\nrelated to mitochondrial or chloroplastic functions? If yes, add a few words to explain\ntheir role in this process to make connection with Line 52 ‘defensive adaptation by\nmitochondria and chloroplast’”\nResponse: We thank the reviewer for the valuable suggestion. Changes in membrane\nlipid unsaturation are related to the functions of chloroplasts. Generally, membrane\nfluidity is affected by changes in temperature, with the most common response to\nchilling being an increase in fatty acid unsaturation in membrane lipids. This\nphenomenon enables the membranes to maintain their functional fluidity and is\nessential for plant survival during cold stress [PMID: 15519313, 27835850, 23585650,\n11607410]. The ω3 fatty acid desaturase FAD8 is localized in chloroplasts and is\nresponsible for trienoic fatty acid production in plants [PMID: 16930600, 12232435].\nThe FAD8 gene was identified as a cold-inducible temperature-sensitive ω3 desaturase.\nIn Arabidopsis and maize, the overall level of FAD8 mRNA is elevated at relatively low\ntemperatures [PMID: 7846164, 9484441], indicating the connection between\nchloroplast-localized FAD8 and fatty acid desaturation in cold environments.\nAction: We have revised the manuscript to clarify this mechanistic connection (Page\n3).\n“The increase in fatty acid unsaturation in membrane lipids enables the membranes to\nmaintain their functional fluidity and is essential for plants to survive during cold\nstress11-14. To adapt to chilling or freezing temperatures, plants can utilise chloroplast-\nlocalized fatty acid desaturase (FAD8) to alter the unsaturation of membrane lipids. The\ncold-inducible ω3 fatty acid desaturase FAD8 is responsible for trienoic fatty acid\nproduction in plants15,16. In Arabidopsis and maize, the overall level of FAD8 mRNA is\nelevated at relatively low temperatures17,18, indicating the connection between\nchloroplast-localized FAD8 and fatty acid desaturation in cold environments.”\nComment-24: “-Line 70: Better mention the role of MAM in lipid metabolism in\nintroduction, instead of just mention ER.”\nResponse: We thank the reviewer for this insightful suggestion. We agree that explicitly\nhighlighting the role of the mitochondria-associated ER membrane (MAM) in lipid\nmetabolism in the Introduction would strengthen the background of the manuscript.\nThe endoplasmic reticulum (ER) dedicates a special domain for metabolic\ninteractions with mitochondria and is referred to as the mitochondria-associated ER\nmembrane (MAM) [PMID: 22575682]. MAM plays important roles in various cellular\nfunctions, including acting as a tight platform for lipid synthesis and lipid transfer\nbetween the ER and mitochondria [PMID: 22575682, 19144519]. The lipid metabolism\nfunction of the MAM has been well characterized. MAM is enriched in lipid\nmetabolism-related enzymes, such as phosphatidylserine synthase,\nphosphatidylethanolamine methyltransferase-2, acyl-CoA:cholesterol acyltransferase,\nand diacylglycerol acyltransferase [PMID: 19144519].\nAction: We have revised the Introduction to mention the role of MAM in lipid\nmetabolism (Pages 3-4).\n“The ER dedicates a special domain for metabolic interaction with mitochondria and is\ncalled the mitochondria-associated ER membrane (MAM)23. MAMs play important\nroles in various cellular functions, including acting as a tight platform for lipid synthesis\nand lipid transfer between the ER and mitochondria23,24.”\nComment-25: “-Line 72: ‘support’ could be a better word than ‘amplify’”\nResponse: We sincerely appreciate the reviewer’s valuable suggestion regarding word\nchoice. We have changed the word “amplify” to “support” as recommended.\nAction: This change is highlighted (Page 4).\n“Mitochondria, the energy factories of eukaryotic cells, support the cascade of organelle\ninteractions by generating energy.”\nComment-26: “-Line 73: The term MAM defines the part of the ER membrane which\nis in contact with outer mitochondrial membranes. The physical contacts between\nOMM and ER are referred to as MERCS (mitochondria-ER contact sites).”\nResponse: We sincerely appreciate the reviewer’s insightful suggestion regarding the\nterminology of membrane contact sites. In accordance with this comment, we have\nrevised the manuscript to consistently use “MERCS” (instead of “MAM”) to describe\nthe contact sites between the outer mitochondrial membrane and ER.\nAction: The text has been revised accordingly (Page 4).\n“The contact sites between the outer mitochondrial membrane and ER (MERCS)\nensures spatiotemporal communication between mitochondria and the ER26,27.”\nComment-27: “-Line 76 & 77: Add reference for the statement ‘NTU primarily\nparticipate in cellular metabolism by producing energy’. Does NTU have additional\nroles in metabolism?”\nResponse: We thank the reviewer for this insightful comment. Linear electron transport\nin the thylakoid membrane drives photosynthetic energy (ATP) and reduces equivalent\n(NADPH) production [PMID: 31245694]. Therefore, in addition to their role in energy\nproduction, NTUs may also contribute to cellular metabolism through the generation of\nNADPH, which plays a critical role in redox homeostasis [PMID: 36477541,\n28514672]. NADPH is used as a cofactor by glutathione reductase to reduce oxidized\nglutathione (GSSG→2GSH) and, likewise, by thioredoxin reductase to reduce oxidized\nthioredoxin. Both of these molecules contribute to defence against oxidative stress\n[PMID: 36505922, 19451637].\nWe further explored this role experimentally. First, we evaluated the effects of NM-\nNTUs + light on the regulation of intracellular NADPH levels. The results revealed that\ncompared with that in the control NPCs, the NADPH content in the IL-1β-induced\ndegenerated NPCs was decreased. Compared with the IL-1β + NM-NTUs + dark\ntreatment, the IL-1β + NM-NTUs + light treatment significantly restored cellular\nNADPH levels (Response Figure 33a). Furthermore, by using a reactive oxygen species\n(ROS) assay kit, we found that the IL-1β + NM-NTUs + light group had reduced\nintracellular ROS levels compared with the IL-1β + NM-NTUs + dark group (Response\nFigures 33b, c). These findings support the conclusion that, in addition to producing\nenergy, NTUs may also help to mitigate cellular oxidative stress through the generation\nof NADPH.\nROS play major roles in inducing the ER stress response. Study has shown that\nROS originating from the ER and mitochondria disturb ER protein folding and generate\nER stress [PMID: 33558590]. Booth et al. demonstrated that the ER-mitochondrial\ninterface hosts a nanodomain of H O originating from mitochondrial cristae [PMID:\n2 2\n27397688]. Another study revealed that high mitochondrial ROS levels promote MAM\nformation and are associated with MAM-ROS and ER-ROS bursts [PMID: 41032619].\nTherefore, we hypothesize that NADPH produced by NTUs may indirectly affect the\ncontact between mitochondria and the ER through the regulation of redox homeostasis.\nAction: We have modified the description of NTU functions in the Introduction. We\nhave also added the abovementioned additional results and discussion to the revised\nmanuscript. The changes are highlighted (Page 4; Page 12; Page 23; Fig. S11).\n“Given the central position of the ER-mitochondrial network in cell metabolism and\nbecause NTUs participate in cellular metabolism by producing energy and reducing\nequivalents19, we aim to investigate whether NTUs can protect dysregulated organelle\ninteraction networks under pathological conditions.”\n“NTUs may also contribute to cellular metabolism through the generation of NADPH,\nwhich plays a critical role in redox homeostasis19,68. We further evaluated the effects of\nNM-NTUs + light on the regulation of intracellular NADPH levels. The results revealed\nthat compared with that in the control NPCs, the NADPH content in the IL-1β-induced\ndegenerated NPCs was decreased (Fig. S11a). The IL-1β + NM-NTUs + light treatment\nsignificantly restored cellular NADPH levels compared with the IL-1β + NM-NTUs +\ndark group (Fig. S11a). Furthermore, we found that IL-1β + NM-NTUs + light\ntreatment resulted in reduced intracellular ROS levels compared with the IL-1β + NM-\nNTUs + dark group (Fig. S11b and c). These findings supported the conclusion that, in\naddition to producing energy, NTUs might also help to mitigate cellular oxidative stress\nthrough the generation of NADPH. Studies have shown that the ER-mitochondrial\ninterface contains a nanodomain of H O originating from mitochondrial cristae69 and\n2 2\nthat high mitochondrial ROS levels promote MAM formation70. We hypothesized that\nNADPH produced by NTUs might indirectly affect the contact between mitochondria\nand the ER through the regulation of redox homeostasis.”\n“For intracellular NADPH quantification, cells from each group were lysed and\nanalysed using an NADPH assay kit (Beyotime) according to the manufacturer's\ninstructions.”\nResponse Figure 33\n(a) NADPH levels of NPCs in different groups (n = 6). (b) Representative flow\ncytometry plots. Intracellular reactive oxygen species (ROS) levels were detected using\nan ROS assay kit. Fluorescence emission was determined via flow cytometry.\n(c) Quantitative analysis of the DCF (FITC)-positive cells in (b) (n = 4). All quantitative\ndata are the mean ± s.d. P-values were calculated using one-way ANOVA (a, c).\nComment-28: “-Line 195: should be ‘IL1β’ group instead of ‘IL1β+NM-NTU’ group”\nResponse: We sincerely appreciate the reviewer’s careful reading. We have performed\na new RNA-seq experiment according to Comment-11, and the comparison here has\nalso been changed to compare the IL-1β + NM-NTU + dark group and the IL-1β + NM-\nNTU + light group.\nAction: The text has been corrected in the revised manuscript (Page 10).\n“Furthermore, in contrast to the IL-1β + NM-NTU + dark group, the IL-1β + NM-NTU\n+ light group presented lower levels of ROS-related genes (Sod2 and Prdx6) and\nmitochondrial apoptosis-related genes (Nptx1 and Bid) (Fig. 4f).”\nComment-29: “-Lines 204-206: The authors claim that ATP from NTU may enhance\nER Ca2+ storage. Please provide either citation or experimental data showing the\namount of ATP generated by NTU can affect ER Ca2+ storage”\nResponse: We thank the reviewer for this insightful comment, which prompted us to\nprovide experimental evidence to substantiate our claim regarding the role of NTU-\nderived ATP in enhancing ER Ca2+ storage. In the background, 3-(3,4-dichlorophenyl)-\n1,1-dimethylurea (DCMU) can block electron transfer between the primary quinone\nelectron accepter and the secondary quinone electron accepter on the reducing side of\nphotosystem II (PSII). This process interrupts the photosynthetic electron transport\nchain in photosynthesis and reduces ATP production by photosynthetic membranes\n[PMID: 39477444, 15618415]. Therefore, we used DCMU (10 μM) [PMID: 30952892]\nto disrupt light-dependent ATP synthesis by NM-NTUs. To measure ER Ca2+ storage,\nwe utilized the genetically encoded ER Ca2+ indicator (G-CEPIA1er) to specifically\nmonitor the ER intraluminal Ca2+ concentration ([Ca2+] ) [PMID: 24923787].\nER\nCompared with the NM-NTU + dark treatment, the NM-NTU + light treatment\nsignificantly increased [Ca2+] at 30 minutes (Response Figures 34a, b). Moreover, the\nER\ninhibition of ATP production in NM-NTUs + light by the DCMU significantly\nattenuated the NM-NTU-induced increase in [Ca2+] (Response Figures 34a, b).\nER\nThese results indicated that the ATP generated by NM-NTUs could increase ER Ca2+\nstorage. We are grateful to the reviewer for prompting us to strengthen this conclusion.\nAction: We have added the abovementioned additional experiments to the revised\nmanuscript. The changes are highlighted (Pages 10-11; Page 24; Fig. S9).\n“The ER is the primary store of cytosolic Ca2+, and the ATP produced by NTUs may\nenhance the ER Ca2+ storage function through energy supplementation. To validate this\nfinding, we utilized the genetically encoded ER Ca2+ indicator (G-CEPIA1er)61 to\nspecifically monitor the ER intraluminal Ca2+ concentration ([Ca2+] ) and used 3-(3,4-\nER\ndichlorophenyl)-1,1-dimethylurea (DCMU)62 to disrupt light-dependent ATP synthesis\nby NM-NTUs. The results showed that the NM-NTUs + light treatment significantly\nincreased [Ca2+] compared with the NM-NTUs + dark treatment in 30 minutes (Fig.\nER\nS9a and b). Moreover, the inhibition of ATP production in NM-NTUs + light by the\nDCMU significantly attenuated the NM-NTU-induced increase in [Ca2+] (Fig. S9a\nER\nand b). Next, we evaluated [Ca2+] using Fluo-4/AM dye. The NM-NTUs + light\nc\ntreatment decreased [Ca2+] compared with that in the NM-NTUs + dark group (Fig. 5a\nc\nand b). In summary, these results suggested that NM-NTUs might restore intracellular\nand ER Ca2+ homeostasis.”\n“Detection of ER Ca²⁺ concentration\nTo investigate the contribution of photosynthetic ATP to ER calcium storage, IH-NPCs\nwere transfected with pCMV G-CEPIA1er (Addgene# 58215), which encodes an ER\ncalcium indicator61. Photosynthetic ATP production in the NM-NTU was inhibited by\ntreatment with 10 μM DCMU. The experimental design included eight treatment groups\nanalysed at 0, 15, and 30-minute time points: control, IL-1β, IL-1β + NM-NTU + dark,\nand IL-1β + NM-NTU + light, with each condition further divided into subgroups either\ntreated with or without DCMU. Cells were imaged using an LCM (Nikon), and\nquantitative analysis of fluorescence intensity was performed using Fiji/ImageJ\nsoftware to assess [Ca2+] .”\nER\nResponse Figure 34\n(a) Representative images of [Ca2+] in IH-NPCs, as visualized by the G-CEPIA1er\nER\nreporter, following different NM-NTU treatments with or without 10 µM DCMU.\n(b) Quantitative analysis of the [Ca2+] measured in (a) (n = 20). All quantitative data\nER\nare the mean ± s.d. P-value was calculated using one-way ANOVA (b). Scale bar: 10\nµm (a).\nComment-30: “-Lines 231-241: Make a better transition to justify the investigation of\nER lipid composition. Add references regarding the connection between membrane\nfluidity to MAM.”\nResponse: We sincerely appreciate the reviewer’s insightful comment. ER‐localized\nenzymes synthesize the vast majority of cellular lipids. The ER has a major influence\non cellular lipid biomass and membrane biogenesis and balances the production of\ndifferent lipid categories, classes, and species [PMID: 29074503]. ER membrane lipid\ncomposition is altered in response to stressful conditions. For example, under prolonged\nnutrient and oxidative stress, the ER membrane lipid composition is remodelled to\ninvolve an increased proportion of saturated phospholipids that are less susceptible to\noxidation [PMID: 38395735]. Moreover, the acyl chain saturation of membrane lipids\nis crucial for membrane fluidity, with unsaturated lipid acyl chains enabling better\nfluidity and saturated lipid acyl chains forming nonfluid and packed structures [PMID:\n37652754, 29410529].\nWith respect to the connection between membrane fluidity and MAM, the study\nshowed that the loss of the p97-UBXD8 complex increased the number of\nmitochondrial–ER contact sites (MERCS) by reducing membrane lipid unsaturation\nand global membrane fluidity [PMID: 36746962]. UBXD8 KO cells exhibited greater\nabundances of PCs, PEs, DGs, and TG lipids with saturated fatty acyl tails, leading to\ndecreased global membrane fluidity, which in turn resulted in increased MERCS. This\naberrant contact phenotype could be rescued by unsaturated fatty acid supplementation.\nIn summary, Ganji et al. reported that increased membrane saturation leading to loss of\nfluidity may affect the dynamic association and disassociation between organelles,\nfurther leading to an increase in MERCS.\nTherefore, we investigated the lipidome of the ER to assess the relationships among\nER lipid composition, membrane fluidity, and ER–mitochondria contact, as well as the\nregulatory role of NM-NTUs in this process.\nAction: We have revised the manuscript for better translation and added a reference\nregarding the connection between membrane fluidity and MAM (Page 13).\n“ER‐localized enzymes synthesize most cellular lipids, and the ER has a major\ninfluence on cellular lipid biomass and membrane biogenesis71. ER membrane lipid\ncomposition is altered in response to stressful conditions. For example, under prolonged\nnutrient and oxidative stress, the ER lipid composition is remodelled to involve an\nincreased proportion of saturated phospholipids72. Moreover, acyl chain saturation of\nmembrane lipids is crucial for membrane fluidity, with unsaturated lipid acyl chains\nenabling better fluidity and saturated lipid acyl chains forming nonfluid and packed\nstructures73,74. Study has shown that increased membrane saturation/order leads to a\nloss of fluidity, which may affect the dynamic association and disassociation between\norganelles, further leading to an increase in MERCS42. Therefore, we investigated the\nlipidome of the ER to assess the relationships among ER lipid composition, membrane\nfluidity, and ER–mitochondria contact, as well as the regulatory role of NM-NTUs in\nthis process.”\nComment-31: “-Line 255: add ‘and’ before the last lipid category which is membrane\nlipid PC.”\nResponse: We thank the reviewer for the valuable suggestion. We have performed new\nlipidomic experiments according to Comment-15, and the original statement has been\ndeleted.\nComment-32: “-Line 284: mention the name of the method used to measure membrane\nfluidity”\nResponse: We thank the reviewer for pointing out this issue. We measured global\nmembrane fluidity in NPCs using a membrane fluidity kit (Abcam). Membrane fluidity\nwas quantified by measuring the ratio of excimer to monomer fluorescence of lipophilic\npyrene probes. With increasing membrane fluidity, the probe undergoes excimer\nformation [PMID: 36103824]. We have revised the manuscript to explicitly mention\nthe methodological details of the membrane fluidity measurement in the Results and\nMethods sections as suggested.\nAction: We have added these details to the corresponding Results and Methods sections.\nThe changes are highlighted (Page 15; Page 25).\n“Next, we measured global membrane fluidity in NPCs using a membrane fluidity kit.\nMembrane fluidity was quantified by measuring the ratio of excimer to monomer\nfluorescence of lipophilic pyrene probes80. With increasing membrane fluidity, the\nprobe underwent excimer formation. We found that NPCs in the IL-1β + NM-NTU +\nlight group exhibited greater membrane fluidity than did those in the IL-1β + NM-NTU\n+ dark group (Fig. 6p).”\n“Membrane fluidity detection\nThe global membrane fluidity of the NPCs was evaluated using a membrane fluidity\nkit (Abcam), which utilises fluorescent lipophilic pyrene probes that are incorporated\ninto the cell membrane. The monomeric pyrene probe undergoes excimer formation,\nshifting its emission from Em 400 nm to Em 470 nm (excitation at 360 nm). Membrane\nfluidity was quantified by determining the ratio of excimer (Em 470 nm) to monomer\n(Em 400 nm) fluorescence using a fluorescence microplate reader (BioTek).”\nComment-33: “-Line 347: ‘in front of’ is not a clear way to indicate position. Do\nauthors mean ‘ventral to’?”\nResponse: We sincerely appreciate the reviewer’s precise suggestion regarding\nanatomical terminology. We have replaced the term “in front of” with “ventral to” in\nthe revised manuscript.\nAction: We have replaced the relevant words. The change has been highlighted (Page\n17).\n“Furthermore, we simulated deep intervertebral disc illumination by surgically\nimplanting a PU-wp-LED ventral to the disc and injecting NM-NTUs into the disc using\nX-ray fluoroscopy (Fig. 9h and i).”\nComment-34: “-Line 437: Add wavelength of red light.”\nResponse: We thank the reviewer for the valuable comment. Red light from light-\nemitting diodes, peaking at 630 nm at an intensity of 80 μmol photons m−2 s−1, was used.\nWe have added the wavelength parameter to the revised manuscript.\nAction: The relevant information has been added to the Methods section (Page 23).\n“Rat NPCs were treated with IL-1β (20 ng mL−1) for 24 h and incubated with NM-\nNTUs (0.5 μg mL-1 for chlorophyll) for 6 h, with or without red light irradiation (from\nlight-emitting diodes, peaking at 630 nm at an intensity of 80 μmol photons m−2 s−1,\n30 min).”\nReviewer #4 (Remarks to the Author):\nGeneral comment: “I co-reviewed this manuscript with one of the reviewers who\nprovided the listed reports. This is part of the Nature Communications initiative to\nfacilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.”\nResponse: We thank the reviewer for your time and valuable input on our manuscript.\nWe sincerely appreciate the reviewer’s efforts and insights. We have tried our best to\nrevise the manuscript according to these valuable comments.\nPoint-to-Point Response:\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nComment-1: “1.It is necessary to clarify the duration after extracting and culturing\nNPCs from deteriorated IVDD before performing staining and imaging. This is because\nthe in vitro culture environment differs from the in vivo degenerative microenvironment,\nand if the culture time is too long, the cells may undergo changes?”\nResponse: We sincerely appreciate the reviewer’s insightful comment. We understand\nthe reviewer’s concern that in vitro culture may alter the phenotype of nucleus pulposus\ncells (NPCs) isolated from degenerated intervertebral discs. Studies have explored\nmethods for isolating and culturing NPCs to yield the greatest number of cells and\nmaintain viability and phenotype as close to those observed in vivo as possible [PMID:\n25749011, 19953600, 36994456]. These studies found that gene expression patterns in\nhuman NPCs were substantially affected by passage number/culture time. When the\ncell culture reached passage 2 (approximately 11–13 days), the NPCs showed\nsignificant changes in the expression of phenotypic markers (such as KRT18, CD24,\nand COL2A1).\nIn our study, we obtained degenerated human NPCs from deteriorated\nintervertebral disc tissue. Since the number of NPCs obtained directly from tissue\ndigestion is limited (approximately 104 to 105 per gram of tissue [PMID: 36994456]),\nwhich is insufficient for subsequent experiments, we cultured the NPCs in vitro for 5\ndays. On the basis of previous reports, the phenotype of human NPCs only changes\nsignificantly after approximately 11–13 days of in vitro culture [PMID: 25749011,\n36994456]; therefore, we chose a 5-day culture time to reduce the impact of phenotypic\nchanges on subsequent experiments.\nTo further evaluate the effects of 5 days of in vitro culture on the phenotypic\nchanges in degenerated human NPCs, we monitored the phenotypic profiles of\ndegenerated human NPCs cultured for 1–9 days. Referring to previous studies [PMID:\n25749011, 36994456], we examined the following NPC phenotypic markers: genes\n(CD24, KRT18, KRT19, SOX9, MIA, FN1, COL1A1, COL2A1, and COL3A1) and\nproteins (CD24 and KRT19). The results indicated that the gene and protein phenotypes\nof the degenerated NPCs remained stable during the first 5 days of culture, whereas\nchanges in the expression of certain markers began to emerge on day 7 (Response\nFigure 1a, b). Specifically, the gene expression of CD24 increased on both day 7 and\nday 9. The expression of KRT18, KRT19, SOX9, and MIA genes increased on day 7 but\ndecreased on day 9. Furthermore, the expression of FN1 gene decreased on day 9. The\nexpression of COL1A1, COL2A1, and COL3A1 genes decreased on day 7. On day 9,\nthe expression of COL1A1 and COL3A1 genes was upregulated, while that of COL2A1\nwas further downregulated (Response Figure 1a). The changes in the abundance of\nCD24 and KRT19 proteins over culture time tended to be similar to the changes in gene\nexpression (Response Figure 1b). In summary, these results indicate that NPCs\nextracted from degenerated nucleus pulposus tissue maintained a stable phenotype for\n5 days in culture but exhibited phenotypic changes and instability after 7 days of culture.\nTherefore, the 5-day in vitro culture time selected in this study did not significantly alter\nthe phenotype of degenerated human NPCs.\nAction: We have conducted new experiments to clarify the duration of phenotypic\nstability after the extraction and culture of degenerated human NPCs. The changes are\nhighlighted as follows (Pages 5-6; Pages 20-21; Fig. S2).\n“On the basis of previous reports, the phenotypic gene expression of human NPCs\nchanges significantly after approximately 11–13 days of in vitro culture38,39. To evaluate\nthe impact of in vitro culture on the changes in the phenotype of degenerated NPCs in\nthis study, we monitored the phenotypic profiles of degenerated human NPCs cultured\nfor 1–9 days. Referring to previous studies38,39, we examined the following NPC\nphenotypic markers: genes (CD24, KRT18, KRT19, SOX9, MIA, FN1, COL1A1,\nCOL2A1, and COL3A1) and proteins (CD24 and KRT19). The results indicated that the\nphenotype of degenerated NPCs remained stable during the first 5 days of culture,\nwhereas changes in the expression of certain markers began to emerge on day 7 (Fig.\nS2a and b). Therefore, a 5-day culture time was selected in this study to reduce the\nimpact of phenotypic changes on subsequent experiments.”\n“Human NP tissues were obtained from human participants. Cultures of primary human\nNPCs were maintained under standard conditions (5% CO and 37 °C), and the medium\n2\nwas changed every 72 hours. The human NPCs were passaged on the sixth day. To\nevaluate the impact of in vitro culture on the phenotypic changes in degenerated human\nNPCs, cell samples were collected every other day from day 1 to day 9 of culture. These\nsamples were subsequently used for gene and protein analysis of NPC phenotypic\nmarkers.”\n(a) Quantitative analysis of gene expression levels of NPC phenotypic markers at\ndifferent culture time points (n = 3). (b) Immunofluorescence staining and\nquantification of CD24 and KRT19 expression at different culture time points (n = 3).\nAll quantitative data are the mean ± s.d. P values were calculated using one-way\nANOVA (a, b). Scale bar: 50 μm (b).\nComment-2: “2.Why does the Dil fluorescence appear in the nucleus in the Figure\n3i?”\nResponse: We sincerely appreciate the reviewer’s careful observation of the DiI\nfluorescence signal in the nucleus shown in Figure 3i. We apologize for the lack of\nclarity in our previous explanation. To address this concern, we repeated the experiment\nand provided three-dimensional images as well as cross-sectional images. Three-\ndimensional reconstruction revealed that some NTUDiI (NTUs labelled with DiI) were\npositioned above the nucleus (Response Figure 2a). Confocal laser scanning\nmicroscopy (CLSM) uses pinholes to eliminate out-of-focus emissions, achieving\noptical sectioning (Response Figure 2b) [PMID: 29927100]. When tiny spheres are\nbeing imaged, the light passing through the optical system undergoes diffraction,\nresulting in a point source image that is much larger than the actual size of the object.\nDiffraction causes light to spread along the lateral and axial optical axes of the\nmicroscope, with the axial image plane more pronounced [PMID: 22082987]. A smaller\npinhole can minimize diffraction rings and improve resolution, whereas a larger pinhole\ncan receive more light and enhance contrast. There is a trade-off between these two\nfactors [PMID: 31876974]. In our earlier experiments, we used a larger pinhole size,\nwhich led to the projection of NTUDiI located above or below the nucleus onto the\nnuclear plane (Response Figure 2c). We found that when pinholes of an appropriate size\nwere used, NTUDiI did not localize to the nucleus (Response Figure 2d). However, with\na slightly larger pinhole, the fluorescence from the same NTUDiI appeared to overlap\nwith that of the nucleus in the cross-sectional view of the same cell (Response Figure\n2e). Therefore, we conclude that the previous image was an artefact and not nuclear\nlocalization of the NTUs. In our initial submission, we used a larger pinhole size, which\ncaused artefacts because of overlapping fluorescence signals during imaging. We\nsincerely apologize for this. To resolve this issue, we repeated the experiment, used a\nsuitable pinhole size, and replaced the original image with a clearer, more representative\nimage in which no NTUDiI fluorescence was detected in the cell nucleus (Response\nFigure 3a). Moreover, the results indicated that the cell membrane coating enabled the\nNTUs to bypass lysosomal degradation (Response Figure 3a, b).\nAction: We have replaced Figure 3i from the original paper with more representative\nimages (Fig. 3i).\nResponse Figure 2\n(a) Three-dimensional reconstruction of the NTUDiI (arrows) localized in the rat NPCs.\n(b) Optical path diagram of a confocal microscope with a smaller pinhole, where out-\nof-focus light (represented by dashed lines) is blocked. The red dots represent the\nNTUDiI. (c) Optical path diagram of a confocal microscope with a slightly larger pinhole,\nallowing some out-of-focus light to reach the detector. The red dots represent the\nNTUDiI. (d, e) Cross-sectional images of NTUDiI (arrows) in the same cell acquired with\na smaller (d) and a larger (e) confocal pinhole. Scale bars: 5 μm (a, d, e).\nResponse Figure 3\n(a) Colocalization and intensity profiles of NTUs or NM-NTUs with lysosomes. (b)\nMander’s coefficients (M1) of (a). The M1 quantifies the fraction of the signal intensity\nof NTUs that colocalizes with lysosomes relative to the total signal intensity of NTUs.\nn = 3 biological replicates, the same cells (n = 20) were acquired and analysed. All\nquantitative data are the mean ± s.d. P value was calculated using a two-tailed Student’s\nt-test (b). Scale bar: 5 μm (a).\nReviewer #2 (Remarks to the Author):\nComment: “The authors have supplemented the necessary experiments as required,\nand my concerns have been resolved. I think the article is suitable for acceptance.”\nResponse: Thank you for your time and effort in the review process.\nReviewer #3 (Remarks to the Author):\nComment: “\nDear authors,\nThanks for your extensive work to answer our comments. We are very impressed and\nthink that the revised version of the manuscript is substantially improved.\nAttached please find recommendations for rephrasing some parts of the manuscript.”\nResponse: We thank the reviewer for your time and valuable input on our manuscript.\nBased on your suggestion, we have rephrased parts of the manuscript, as detailed below.\n1. The sentence “The endosymbiosis that gave rise to mitochondria restructured the\ndistribution of DNA in relation to bioenergetic membranes, thereby allowing\neukaryotes to significantly increase the number of gene expressed compared with\nprokaryotes, based on which the diversity and complexity of life have evolved” has\nbeen revised to “The endosymbiotic event that gave rise to mitochondria enabled a\nsubstantial expansion in gene expression and protein synthesis in eukaryotes relative to\nprokaryotes. This increase was driven by the emergence of specialized bioenergetic\nmembranes and enhanced ATP-producing capacity, which together provided the\nenergetic foundation for the biological diversity and complexity4,5.” (Page 2)\n2. The sentence “Malate/oxaloacetate translocators together with malate\ndehydrogenases (malate valves) connect chloroplasts and mitochondria in plant cells.\nWhen plants are subjected to conditions such as high light, high CO , and cold stress,\n2\nthe activities of the enzymes of the malate valves change to balance the ATP/NAD(P)H\nratio required in subcellular compartments and therefore balance metabolic fluxes\nunder stress conditions” has been revised to “Malate/oxaloacetate translocators and\nmalate dehydrogenases form the malate valve, which metabolically links chloroplasts\nand mitochondria by transferring excess reducing equivalents from the chloroplast to\nmaintain stromal redox balance. Under stresses such as high light, elevated CO , cold,\n2\ndrought, or salinity, malate valve activity adjusts to maintain ATP/NAD(P)H\nhomeostasis, limit ROS formation, and support stress resilience8-10.” (Page 3)\n3. The sentence “The increase in fatty acid unsaturation in membrane lipids enables the\nmembranes to maintain their functional fluidity and is essential for plants to survive\nduring cold stress11-14. To adapt to chilling or freezing temperatures, plants can utilise\nchloroplast-localized fatty acid desaturase (FAD8) to alter the unsaturation of\nmembrane lipids. The cold-inducible ω3 fatty acid desaturase FAD8 is responsible for\ntrienoic fatty acid production in plants15,16. In Arabidopsis and maize, the overall level\nof FAD8 mRNA is elevated at relatively low temperatures17,18, indicating the\nconnection between chloroplast-localized FAD8 and fatty acid desaturation in cold\nenvironments.” has been revised to “Additionally, plants also enhance the unsaturation\nof fatty acids in membrane lipids that preserves membrane fluidity and sustains the\nactivity of essential membrane-associated proteins under cold stress11-18. Together,\nthese redox-balancing and membrane-stabilizing mechanisms enable plants to maintain\nmetabolic function and tolerate abiotic stress.” (Page 3)\n4. The sentence “Animal cells exhibit ER–mitochondria contact disruption under stress”\nhas been revised to “Animal cells exhibit ER–mitochondria contact alterations under\nstress”. (Page 4)\n5. The sentence “To validate the findings of transcriptomic analyses and study cellular\nchanges of human NPCs, we established primary culture of normal and degenerated\nhuman NPCs.” has been added to the manuscript to make a connection between\ntranscriptomic data and the MTT assay. (Page 5)\n6. The sentence “These results also suggested that the in vitro model matched IVDD,\nwhere a large percentage of NPCs were alive and that the findings in the following\nseries of experiments were not due to a change in cell number but rather cellular\nmetabolic changes.” has been added after the cell viability results. (Page 6)\n7. A short conclusion “Taken together, the above results indicated that in the\nintervertebral disc microenvironment, NM-NTUs were preferentially taken up by NPCs.\nAfter entering NPCs, NTUs could evade lysosomal digestion and probably did not\nphysically interact with MERCS.” has been added at the end of the NM-NTU\ncharacterization. (Page 9)\n8. The sentence “These results indicated that nonfunctional NM-NTUs (without light\nillumination) did not change the bioenergetics of cells and that light exposure was\nnecessary for NM-NTUs to generate ATP in mammalian cells.” has been added to\ndescribe the biological meaning of experiments on ATP generated by NM-NTUs. (Page\n10)\n9. The sentence “In addition, we compared differentially expressed genes from the\ntranscriptomes of normal and degenerative human NPCs, as well as from NPCs with\nand without IL-1β treatment. The results suggested that IL-1β treatment upregulated\nthe Adamts7 (matrix degradation-related gene63) and Il1b genes and downregulated the\nCol2a1 (matrix synthesis-related gene) and Sesn3 (protection against oxidative stress64)\ngenes. These differentially expressed genes were identical in the human transcriptome\nand exhibited consistent trends in the degenerative group, strengthening the validity of\nthe IL-1β-induced NPC degeneration model.” has been added to strengthen the validity\nof the IL-1β model. (Pages 10-11)\nReviewer #4 (Remarks to the Author):\nComment: “I co-reviewed this manuscript with one of the reviewers who provided the\nlisted reports. This is part of the Nature Communications initiative to facilitate training\nin peer review and to provide appropriate recognition for Early Career Researchers who\nco-review manuscripts.”\nResponse: We thank the reviewer for your time and valuable input on our manuscript.\nPoint-to-Point Response:\nREVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\nComment: “I appreciate the authors’ careful responses and revisions. All of my\ncomments have been satisfactorily addressed, and I have no further comments at this\nstage.”\nResponse: Thank you for your time and effort in the review process.\nReviewer #3 (Remarks to the Author):\nComment: “We think that the authors have replied very well and made the suggested\nchanges. We have no further comments.”\nResponse: We thank the reviewer for your time and valuable input on our manuscript.\nReviewer #4 (Remarks to the Author):\nComment: “I co-reviewed this manuscript with one of the reviewers who provided the\nlisted reports. This is part of the Nature Communications initiative to facilitate training\nin peer review and to provide appropriate recognition for Early Career Researchers who\nco-review manuscripts.”\nResponse: We thank the reviewer for your time and valuable input on our manuscript."}, {"round": 2, "reviewer_comments": {"reviewer_1": "1.It is necessary to clarify the duration after extracting and culturing NPCs from deteriorated IVDD before performing staining and imaging. This is because the in vitro culture environment differs from the in vivo degenerative microenvironment, and if the culture time is too long, the cells may undergo changes？\n2.Why does the Dil fluorescence appear in the cell nucleus in the Figure 3i?", "reviewer_2": "The authors have supplemented the necessary experiments as required, and my concerns have been resolved. I think the article is suitable for acceptance.", "reviewer_3": "Dear authors,\nThanks for your extensive work to answer our comments. We are very impressed and think that the revised version of the manuscript is substantially improved.\nAttached please find recommendations for rephrasing some parts of the manuscript.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Reviewer #1 (Remarks to the Author):\nComment-1: “1.It is necessary to clarify the duration after extracting and culturing\nNPCs from deteriorated IVDD before performing staining and imaging. This is because\nthe in vitro culture environment differs from the in vivo degenerative microenvironment,\nand if the culture time is too long, the cells may undergo changes?”\nResponse: We sincerely appreciate the reviewer’s insightful comment. We understand\nthe reviewer’s concern that in vitro culture may alter the phenotype of nucleus pulposus\ncells (NPCs) isolated from degenerated intervertebral discs. Studies have explored\nmethods for isolating and culturing NPCs to yield the greatest number of cells and\nmaintain viability and phenotype as close to those observed in vivo as possible [PMID:\n25749011, 19953600, 36994456]. These studies found that gene expression patterns in\nhuman NPCs were substantially affected by passage number/culture time. When the\ncell culture reached passage 2 (approximately 11–13 days), the NPCs showed\nsignificant changes in the expression of phenotypic markers (such as KRT18, CD24,\nand COL2A1).\nIn our study, we obtained degenerated human NPCs from deteriorated\nintervertebral disc tissue. Since the number of NPCs obtained directly from tissue\ndigestion is limited (approximately 104 to 105 per gram of tissue [PMID: 36994456]),\nwhich is insufficient for subsequent experiments, we cultured the NPCs in vitro for 5\ndays. On the basis of previous reports, the phenotype of human NPCs only changes\nsignificantly after approximately 11–13 days of in vitro culture [PMID: 25749011,\n36994456]; therefore, we chose a 5-day culture time to reduce the impact of phenotypic\nchanges on subsequent experiments.\nTo further evaluate the effects of 5 days of in vitro culture on the phenotypic\nchanges in degenerated human NPCs, we monitored the phenotypic profiles of\ndegenerated human NPCs cultured for 1–9 days. Referring to previous studies [PMID:\n25749011, 36994456], we examined the following NPC phenotypic markers: genes\n(CD24, KRT18, KRT19, SOX9, MIA, FN1, COL1A1, COL2A1, and COL3A1) and\nproteins (CD24 and KRT19). The results indicated that the gene and protein phenotypes\nof the degenerated NPCs remained stable during the first 5 days of culture, whereas\nchanges in the expression of certain markers began to emerge on day 7 (Response\nFigure 1a, b). Specifically, the gene expression of CD24 increased on both day 7 and\nday 9. The expression of KRT18, KRT19, SOX9, and MIA genes increased on day 7 but\ndecreased on day 9. Furthermore, the expression of FN1 gene decreased on day 9. The\nexpression of COL1A1, COL2A1, and COL3A1 genes decreased on day 7. On day 9,\nthe expression of COL1A1 and COL3A1 genes was upregulated, while that of COL2A1\nwas further downregulated (Response Figure 1a). The changes in the abundance of\nCD24 and KRT19 proteins over culture time tended to be similar to the changes in gene\nexpression (Response Figure 1b). In summary, these results indicate that NPCs\nextracted from degenerated nucleus pulposus tissue maintained a stable phenotype for\n5 days in culture but exhibited phenotypic changes and instability after 7 days of culture.\nTherefore, the 5-day in vitro culture time selected in this study did not significantly alter\nthe phenotype of degenerated human NPCs.\nAction: We have conducted new experiments to clarify the duration of phenotypic\nstability after the extraction and culture of degenerated human NPCs. The changes are\nhighlighted as follows (Pages 5-6; Pages 20-21; Fig. S2).\n“On the basis of previous reports, the phenotypic gene expression of human NPCs\nchanges significantly after approximately 11–13 days of in vitro culture38,39. To evaluate\nthe impact of in vitro culture on the changes in the phenotype of degenerated NPCs in\nthis study, we monitored the phenotypic profiles of degenerated human NPCs cultured\nfor 1–9 days. Referring to previous studies38,39, we examined the following NPC\nphenotypic markers: genes (CD24, KRT18, KRT19, SOX9, MIA, FN1, COL1A1,\nCOL2A1, and COL3A1) and proteins (CD24 and KRT19). The results indicated that the\nphenotype of degenerated NPCs remained stable during the first 5 days of culture,\nwhereas changes in the expression of certain markers began to emerge on day 7 (Fig.\nS2a and b). Therefore, a 5-day culture time was selected in this study to reduce the\nimpact of phenotypic changes on subsequent experiments.”\n“Human NP tissues were obtained from human participants. Cultures of primary human\nNPCs were maintained under standard conditions (5% CO and 37 °C), and the medium\n2\nwas changed every 72 hours. The human NPCs were passaged on the sixth day. To\nevaluate the impact of in vitro culture on the phenotypic changes in degenerated human\nNPCs, cell samples were collected every other day from day 1 to day 9 of culture. These\nsamples were subsequently used for gene and protein analysis of NPC phenotypic\nmarkers.”\n(a) Quantitative analysis of gene expression levels of NPC phenotypic markers at\ndifferent culture time points (n = 3). (b) Immunofluorescence staining and\nquantification of CD24 and KRT19 expression at different culture time points (n = 3).\nAll quantitative data are the mean ± s.d. P values were calculated using one-way\nANOVA (a, b). Scale bar: 50 μm (b).\nComment-2: “2.Why does the Dil fluorescence appear in the nucleus in the Figure\n3i?”\nResponse: We sincerely appreciate the reviewer’s careful observation of the DiI\nfluorescence signal in the nucleus shown in Figure 3i. We apologize for the lack of\nclarity in our previous explanation. To address this concern, we repeated the experiment\nand provided three-dimensional images as well as cross-sectional images. Three-\ndimensional reconstruction revealed that some NTUDiI (NTUs labelled with DiI) were\npositioned above the nucleus (Response Figure 2a). Confocal laser scanning\nmicroscopy (CLSM) uses pinholes to eliminate out-of-focus emissions, achieving\noptical sectioning (Response Figure 2b) [PMID: 29927100]. When tiny spheres are\nbeing imaged, the light passing through the optical system undergoes diffraction,\nresulting in a point source image that is much larger than the actual size of the object.\nDiffraction causes light to spread along the lateral and axial optical axes of the\nmicroscope, with the axial image plane more pronounced [PMID: 22082987]. A smaller\npinhole can minimize diffraction rings and improve resolution, whereas a larger pinhole\ncan receive more light and enhance contrast. There is a trade-off between these two\nfactors [PMID: 31876974]. In our earlier experiments, we used a larger pinhole size,\nwhich led to the projection of NTUDiI located above or below the nucleus onto the\nnuclear plane (Response Figure 2c). We found that when pinholes of an appropriate size\nwere used, NTUDiI did not localize to the nucleus (Response Figure 2d). However, with\na slightly larger pinhole, the fluorescence from the same NTUDiI appeared to overlap\nwith that of the nucleus in the cross-sectional view of the same cell (Response Figure\n2e). Therefore, we conclude that the previous image was an artefact and not nuclear\nlocalization of the NTUs. In our initial submission, we used a larger pinhole size, which\ncaused artefacts because of overlapping fluorescence signals during imaging. We\nsincerely apologize for this. To resolve this issue, we repeated the experiment, used a\nsuitable pinhole size, and replaced the original image with a clearer, more representative\nimage in which no NTUDiI fluorescence was detected in the cell nucleus (Response\nFigure 3a). Moreover, the results indicated that the cell membrane coating enabled the\nNTUs to bypass lysosomal degradation (Response Figure 3a, b).\nAction: We have replaced Figure 3i from the original paper with more representative\nimages (Fig. 3i).\nResponse Figure 2\n(a) Three-dimensional reconstruction of the NTUDiI (arrows) localized in the rat NPCs.\n(b) Optical path diagram of a confocal microscope with a smaller pinhole, where out-\nof-focus light (represented by dashed lines) is blocked. The red dots represent the\nNTUDiI. (c) Optical path diagram of a confocal microscope with a slightly larger pinhole,\nallowing some out-of-focus light to reach the detector. The red dots represent the\nNTUDiI. (d, e) Cross-sectional images of NTUDiI (arrows) in the same cell acquired with\na smaller (d) and a larger (e) confocal pinhole. Scale bars: 5 μm (a, d, e).\nResponse Figure 3\n(a) Colocalization and intensity profiles of NTUs or NM-NTUs with lysosomes. (b)\nMander’s coefficients (M1) of (a). The M1 quantifies the fraction of the signal intensity\nof NTUs that colocalizes with lysosomes relative to the total signal intensity of NTUs.\nn = 3 biological replicates, the same cells (n = 20) were acquired and analysed. All\nquantitative data are the mean ± s.d. P value was calculated using a two-tailed Student’s\nt-test (b). Scale bar: 5 μm (a).\nReviewer #2 (Remarks to the Author):\nComment: “The authors have supplemented the necessary experiments as required,\nand my concerns have been resolved. I think the article is suitable for acceptance.”\nResponse: Thank you for your time and effort in the review process.\nReviewer #3 (Remarks to the Author):\nComment: “\nDear authors,\nThanks for your extensive work to answer our comments. We are very impressed and\nthink that the revised version of the manuscript is substantially improved.\nAttached please find recommendations for rephrasing some parts of the manuscript.”\nResponse: We thank the reviewer for your time and valuable input on our manuscript.\nBased on your suggestion, we have rephrased parts of the manuscript, as detailed below.\n1. The sentence “The endosymbiosis that gave rise to mitochondria restructured the\ndistribution of DNA in relation to bioenergetic membranes, thereby allowing\neukaryotes to significantly increase the number of gene expressed compared with\nprokaryotes, based on which the diversity and complexity of life have evolved” has\nbeen revised to “The endosymbiotic event that gave rise to mitochondria enabled a\nsubstantial expansion in gene expression and protein synthesis in eukaryotes relative to\nprokaryotes. This increase was driven by the emergence of specialized bioenergetic\nmembranes and enhanced ATP-producing capacity, which together provided the\nenergetic foundation for the biological diversity and complexity4,5.” (Page 2)\n2. The sentence “Malate/oxaloacetate translocators together with malate\ndehydrogenases (malate valves) connect chloroplasts and mitochondria in plant cells.\nWhen plants are subjected to conditions such as high light, high CO , and cold stress,\n2\nthe activities of the enzymes of the malate valves change to balance the ATP/NAD(P)H\nratio required in subcellular compartments and therefore balance metabolic fluxes\nunder stress conditions” has been revised to “Malate/oxaloacetate translocators and\nmalate dehydrogenases form the malate valve, which metabolically links chloroplasts\nand mitochondria by transferring excess reducing equivalents from the chloroplast to\nmaintain stromal redox balance. Under stresses such as high light, elevated CO , cold,\n2\ndrought, or salinity, malate valve activity adjusts to maintain ATP/NAD(P)H\nhomeostasis, limit ROS formation, and support stress resilience8-10.” (Page 3)\n3. The sentence “The increase in fatty acid unsaturation in membrane lipids enables the\nmembranes to maintain their functional fluidity and is essential for plants to survive\nduring cold stress11-14. To adapt to chilling or freezing temperatures, plants can utilise\nchloroplast-localized fatty acid desaturase (FAD8) to alter the unsaturation of\nmembrane lipids. The cold-inducible ω3 fatty acid desaturase FAD8 is responsible for\ntrienoic fatty acid production in plants15,16. In Arabidopsis and maize, the overall level\nof FAD8 mRNA is elevated at relatively low temperatures17,18, indicating the\nconnection between chloroplast-localized FAD8 and fatty acid desaturation in cold\nenvironments.” has been revised to “Additionally, plants also enhance the unsaturation\nof fatty acids in membrane lipids that preserves membrane fluidity and sustains the\nactivity of essential membrane-associated proteins under cold stress11-18. Together,\nthese redox-balancing and membrane-stabilizing mechanisms enable plants to maintain\nmetabolic function and tolerate abiotic stress.” (Page 3)\n4. The sentence “Animal cells exhibit ER–mitochondria contact disruption under stress”\nhas been revised to “Animal cells exhibit ER–mitochondria contact alterations under\nstress”. (Page 4)\n5. The sentence “To validate the findings of transcriptomic analyses and study cellular\nchanges of human NPCs, we established primary culture of normal and degenerated\nhuman NPCs.” has been added to the manuscript to make a connection between\ntranscriptomic data and the MTT assay. (Page 5)\n6. The sentence “These results also suggested that the in vitro model matched IVDD,\nwhere a large percentage of NPCs were alive and that the findings in the following\nseries of experiments were not due to a change in cell number but rather cellular\nmetabolic changes.” has been added after the cell viability results. (Page 6)\n7. A short conclusion “Taken together, the above results indicated that in the\nintervertebral disc microenvironment, NM-NTUs were preferentially taken up by NPCs.\nAfter entering NPCs, NTUs could evade lysosomal digestion and probably did not\nphysically interact with MERCS.” has been added at the end of the NM-NTU\ncharacterization. (Page 9)\n8. The sentence “These results indicated that nonfunctional NM-NTUs (without light\nillumination) did not change the bioenergetics of cells and that light exposure was\nnecessary for NM-NTUs to generate ATP in mammalian cells.” has been added to\ndescribe the biological meaning of experiments on ATP generated by NM-NTUs. (Page\n10)\n9. The sentence “In addition, we compared differentially expressed genes from the\ntranscriptomes of normal and degenerative human NPCs, as well as from NPCs with\nand without IL-1β treatment. The results suggested that IL-1β treatment upregulated\nthe Adamts7 (matrix degradation-related gene63) and Il1b genes and downregulated the\nCol2a1 (matrix synthesis-related gene) and Sesn3 (protection against oxidative stress64)\ngenes. These differentially expressed genes were identical in the human transcriptome\nand exhibited consistent trends in the degenerative group, strengthening the validity of\nthe IL-1β-induced NPC degeneration model.” has been added to strengthen the validity\nof the IL-1β model. (Pages 10-11)\nReviewer #4 (Remarks to the Author):\nComment: “I co-reviewed this manuscript with one of the reviewers who provided the\nlisted reports. This is part of the Nature Communications initiative to facilitate training\nin\nin peer review and to provide appropriate recognition for Early Career Researchers who\nco-review manuscripts.”\nResponse: We thank the reviewer for your time and valuable input on our manuscript.\nPoint-to-Point Response:\nREVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\nComment: “I appreciate the authors’ careful responses and revisions. All of my\ncomments have been satisfactorily addressed, and I have no further comments at this\nstage.”\nResponse: Thank you for your time and effort in the review process.\nReviewer #3 (Remarks to the Author):\nComment: “We think that the authors have replied very well and made the suggested\nchanges. We have no further comments.”\nResponse: We thank the reviewer for your time and valuable input on our manuscript.\nReviewer #4 (Remarks to the Author):\nComment: “I co-reviewed this manuscript with one of the reviewers who provided the\nlisted reports. This is part of the Nature Communications initiative to facilitate training\nin peer review and to provide appropriate recognition for Early Career Researchers who\nco-review manuscripts.”\nResponse: We thank the reviewer for your time and valuable input on our manuscript."}, {"round": 3, "reviewer_comments": {"reviewer_1": "I appreciate the authors’ careful responses and revisions. All of my comments have been satisfactorily addressed, and I have no further comments at this stage.", "reviewer_3": "We think that the authors have replied very well and made the suggested changes. We have no further comments.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": ""}]

Decision letter