VPS13B recruits lipid vesicles to promote mitochondrial fission and quality control

Domain: biology
Published: 2025-12-16
Editorial outcome: major_revision

Abstract
Mutations in the gene VPS13B, which encodes a Golgi-associated protein, cause the neurodevelopmental disorder Cohen syndrome, but the protein’s function is unclear. Here we show that this protein is essential for mitochondrial morphology and quality control. Cells lacking VPS13B, including neurons derived from Cohen syndrome patients, exhibit abnormally elongated and fused mitochondria with reduced membrane potential and impaired mitophagy. Mechanistically, the protein localizes to Mitofusin 2-positive mitochondria via its C-terminal region and recruits phosphatidylinositol-4-phosphate-rich Golgi vesicles to mitochondrial fission sites. Loss of VPS13B or depletion of phosphatidylinositol-4-phosphate results in incomplete mitochondrial fission despite normal recruitment of Dynamin-related protein 1, indicating that lipid transfer by VPS13B is required for membrane fission. VPS13B links Golgi-derived lipid vesicles to the mitochondrial fission machinery, ensuring proper mitochondrial fission and quality control and potentially explaining the mitochondrial defects in Cohen syndrome.

Introduction
The VPS13 (Vacuolar Protein Sorting 13) family of proteins consists of highly conserved, large proteins that regulate various cellular processes. In yeast (Saccharomyces cerevisiae), a single protein, VPS13, was initially identified in a screen for proteins involved in vacuolar protein sorting 1,2. Subsequently, VPS13 has been extensively characterized as a multifunctional protein involved in protein trafficking, vesicular transport and fusion, sporulation, mitochondrial homeostasis, phagocytosis, and autophagy in yeast and other lower eukaryotic organisms 3–5. More recent studies have shown that VPS13 family proteins mediate non-vesicular lipid transport at intracellular membrane contact sites 6–9.

In humans, there are four paralogs (VPS13A, B, C, and D) of yeast VPS13, and these proteins are highly conserved across species from yeast to humans 1,7,10,11. Notably, genetic mutations in human VPS13 genes are strongly associated with several genetic disorders, including neurodevelopmental and neurodegenerative diseases. These include Chorea-acanthocytosis (VPS13A), Cohen syndrome (CS; a condition characterized by global developmental delay and intellectual disability (VPS13B)), an early-onset form of Parkinson’s disease (VPS13C), and a form of Ataxia with spasticity (VPS13D) 7,12–21.

Among these paralogs, VPS13B, also known as CS-associated protein (COH1), is known to regulate Golgi integrity, glycosylation, and neurite outgrowth through its interaction with the RAB6 GTPase 16,22,23. More recently, VPS13B has been reported to control acrosome biogenesis by modulating Golgi dynamics and membrane trafficking 14. Recent studies have shown that small GTPases and phosphoinositides interact with soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins to target VPS13B, which regulates targeting via Stx6 and Stx13, functioning as a regulatory factor in transport from early endosomes to recycling endosomes 24. Additionally, VPS13B has been reported to localize to and promote the formation of Golgi-lipid droplet (LD) contacts upon oleic acid (OA) stimulation, suggesting a potential role for VPS13B-mediated cellular contacts 25. Moreover, recent studies suggest that VPS13B may facilitate bulk lipid transport to expand Golgi membranes, with FAM177A1 possibly assisting in this function 9. While its cellular roles in the Golgi complex and endosomes have been reported using RNAi-based studies in mammalian cell lines 14,24, the precise functions of VPS13B in knockout (KO) cells remain poorly understood. Furthermore, although evidence increasingly supports the role of VPS13B in the regulation of Golgi complexes, little is known about its role in regulating other organelles, such as mitochondria.

In this study, we demonstrate that VPS13B regulates mitochondrial dynamics and quality control. In VPS13B KO cells and Cohen syndrome patient-derived iPSC-induced neurons, VPS13B loss causes mitochondrial hyperfusion, decreased membrane potential, and impaired PINK1/Parkin-mediated mitophagy. Upon mitochondrial stress, endogenous VPS13B is recruited via a C-terminal MLD—necessary and sufficient for targeting—to Mfn2-positive mitochondria contacts, where it accumulates at Drp1-positive fission site and promotes delivery of phosphatidylinositol 4-phosphate (PI4P)-enriched Golgi vesicles. VPS13B loss or acute PI4P depletion lowers fission rates and stalls fission without affecting Drp1 recruitment or phosphorylation, placing the PI4P-VPS13B axis downstream of Drp1 to facilitate late-stage membrane severing. Neurons derived from patients with Cohen syndrome phenocopy these defects—hyperfused networks, decreased membrane potential, reduced Drp1 localization, and increased PINK1—underscoring clinical relevance. Thus, VPS13B acts as a Golgi–mitochondria association that couples PI4P-vesicle supply to the fission machinery to sustain mitochondrial fission and mitophagy.

Results
VPS13B KO leads to hyperfused mitochondria
To investigate the specific roles of VPS13B associated with CS, we first assessed the cellular localization of VPS13B in wild-type (WT) cells. Consistent with previous reports 17, VPS13B was predominantly co-localized to Galatosyltransferase (GalT) (Supplementary Fig. 1a) and TGN46 (Supplementary Fig. 1b), the Trans-Golgi Network (TGN) marker, indicating the Golgi apparatus localization 17. Normally, other VPS13 isoforms, including VPS13A, C, and D, are localized to the ER via the phenylalanine-phenylalanine-acidic-tract (FFAT) motif-dependent ER protein VAP 7,26–28. Interestingly, VPS13B is the only isoform without an FFAT motif, and is consistently localized to the Golgi, not the ER.

Next, to examine the roles of VPS13B resulting from its loss of function in cells, we generated VPS13B KO cells using CRISPR/Cas9 technology 21,29. The gene sequencing results, and Western blot analysis and immunocytochemistry with an anti-VPS13B antibody confirmed the absence of VPS13B at the protein and cellular levels (Supplementary Fig. 1c–e). To delve deeper into the relevance of cellular organelle defects in VPS13B deficiency, VPS13B KO cells were transfected with marker proteins for various organelles EGFP-tagged Rab5 (early endosome), Rab7 (late endosome), Sec61 (ER), GalT (Golgi apparatus), LAMP1 (lysosome), and mRFP-TOM20 (mitochondria, pseudo-colored green) (Supplementary Fig. 1f). Intriguingly, organelles marked by Rab5, Rab7, Rab9, or LAMP1 showed a scattered distribution in WT cells, but were clustered near the nucleus in VPS13B KO cells, underscoring VPS13B’s role in vesicular trafficking 16,30. In addition to the qualitative observation of Golgi fragmentation, quantitative analysis by endogenous Golgi staining using anti-TGN46 antibody revealed that VPS13B KO cells exhibited a significant increase in the number of discrete TGN structures, while the average area and perimeter of individual TGNs were reduced and circularity values were elevated (Supplementary Fig. 1g, h), indicating that VPS13B deficiency leads to fragmentation, resulting in more numerous, smaller, and morphologically rounder Golgi elements compared to WT. Notably, VPS13B KO cells displayed markedly elongated and interconnected mitochondria, indicative of hyperfusion and suggesting a critical role for VPS13B in maintaining mitochondrial morphology (Supplementary Fig. 1f).

Regarding the skeletonized TOM20-positive mitochondria, we observed that the structures in VPS13B KO cells appeared more interconnected compared to WT cells (Fig. 1A). To quantify this morphological change, we measured the area and perimeter of individual mitochondria. This analysis revealed a significant increase in both mitochondrial area and perimeter in VPS13B KO cells (Fig. 1B, C), indicating a hyperfused mitochondrial morphology.

To further investigate the abnormal mitochondrial morphology observed under confocal microscopy, we performed electron microscopy (EM) analysis to assess at the ultrastructural level. The EM analysis revealed that, in contrast to the typical morphology of mitochondria in WT cells, mitochondria in VPS13B KO cells were frequently branched and elongated (Fig. 1D). This altered morphology corresponded to a significant increase in both the average mitochondrial area and perimeter compared to WT controls (Fig. 1E, F). This analysis revealed a significant increase in both the average mitochondrial area and perimeter in VPS13B KO cells compared to WT controls, indicating that the absence of VPS13B leads to distinct, enlarged changes in mitochondrial structure.

Given that VPS13B deficiency leads to abnormal mitochondrial morphology, we next asked whether VPS13B is associated with mitochondria and which domain mediates this interaction. To this end, we mapped VPS13B’s C-terminus, which contains a conserved DH-like domain–pleckstrin homology (DHL-PH) region and a Mitochondria-Associated Membrane (MAM) localization domain (MLD)-like segment with homology to the C-terminus of ATG2A (reported to mediate MAM targeting) 31. We generated EGFP-tagged constructs comprising an MLD-deletion mutant (ΔMLD), and C-terminal fragments encompassing the DHL-PH region (amino acids 3500-3997) or the isolated MLD (amino acids 3662-3793) 7,9,31–33 (Fig. 1G). VPS13B-full-length (FL)-EGFP exhibited clear enrichment at TOM20-positive mitochondria, and both EGFP-VPS13B-DHL-PH and EGFP-VPS13B-MLD similarly co-localized with mitochondrial markers, indicating that the C-terminal MLD-containing region is sufficient for mitochondrial targeting (Fig. 1H). In contrast, VPS13B-ΔMLD-EGFP failed to accumulate at mitochondria, demonstrating that the MLD is necessary for VPS13B’s mitochondrial localization (Fig. 1H).

To further assess whether VPS13B is involved at Mfn2-positive mitochondria contact sites (MAM) 31,34,35, we performed in situ proximity ligation assays (PLA) using antibodies against endogenous VPS13B and Mitofusin-2 (Mfn2), a marker/tether enriched at Mfn2-positive mitochondria interfaces 34,35. Because PLA detects protein proximity within ~40 nm, it provides a sensitive readout of residency at contact sites 36. In WT cells, we observed abundant VPS13B-Mfn2 PLA puncta that aligned with mitochondrial profiles, whereas VPS13B KO cells showed signals near background (Fig. 1I, J). Notably, re-expression of VPS13B-FL-EGFP in KO cells restored the VPS13B (using GFP antibodies)-Mfn2 PLA signal, while VPS13B-ΔMLD-EGFP did not exhibit such localization (Fig. 1I, J). These results demonstrate that VPS13B localizes to Mfn2-positive mitochondria in an MLD-dependent manner.

To confirm that the hyperfused mitochondrial phenotype results directly from VPS13B loss and depends on its mitochondrial association, we performed a rescue experiment by overexpression. Rescue of VPS13B-KO cells with VPS13B-FL-EGFP, but not with VPS13B-ΔMLD-EGFP, normalized mitochondrial morphology to that of wild-type cells (Fig. 1K, L). These results demonstrate that the hyperfused phenotype is a direct consequence of VPS13B loss and that MLD-dependent mitochondrial association of VPS13B is critical for the maintenance of mitochondrial architecture. Our findings indicate that the Golgi-associated protein VPS13B is essential for maintaining mitochondrial morphology, and that its loss leads to abnormal mitochondrial structure, highlighting a critical link between VPS13B and mitochondrial regulation.

Loss of VPS13B causes mitochondrial depolarization and defective dynamics
Given that the abnormal mitochondrial morphology observed in VPS13B-deficient cells could alter mitochondrial physiology, we first assessed mitochondrial membrane potential (ΔΨm). We measured ΔΨm using tetramethylrhodamine methyl ester (TMRM) together with MitoTracker to delineate mitochondrial area, quantifying ΔΨm as integrated TMRM fluorescence normalized to the MitoTracker-positive area (TMRM intensity/MitoTracker area). VPS13B KO cells showed a statistically significant reduction in this normalized ΔΨm metric compared with WT (Fig. 2A, B). Re-expression of VPS13B-FL-EGFP but not VPS13B-ΔMLD-EGFP in the KO cells significantly restored the normalized TMRM signal toward WT levels (Fig. 2A, B). These findings demonstrate that VPS13B deficiency leads to mitochondrial depolarization and highlights its essential role in maintaining mitochondrial bioenergetic integrity.

To further interrogate the dynamics of the hyperfused mitochondrial network, we performed a fluorescence recovery after photobleaching (FRAP) assay on cells expressing mRFP-tagged TOM20. VPS13B KO cells exhibited significantly reduced fluorescence recovery relative to WT (Fig. 2C–E), indicating diminished outer mitochondrial membrane mobility and impaired membrane dynamics consistent with a dysfunctional network architecture. Importantly, re-expression of VPS13B-FL-EGFP, but not VPS13B-ΔMLD-EGFP in the KO cells, significantly restored the recovery toward WT levels, demonstrating functional rescue and mitochondria targeting of VPS13B is essential for maintaining mitochondrial functions (Fig. 2C–E). Overall, these findings show that loss of VPS13B leads to abnormal mitochondrial morphology accompanied by functional deficits, and they are consistent with an imbalance in fusion–fission homeostasis—i.e., reduced fission efficiency and/or excessive fusion in the absence of VPS13B.

VPS13B deficiency impairs PINK1/Parkin-mitophagy
Emerging evidence highlights that damaged or abnormal mitochondria are closely linked to mitophagy activation, emphasizing the critical role of mitochondrial quality control in maintaining cellular homeostasis 37,38. To investigate whether VPS13B loss affects mitophagy, we examined the PINK1/Parkin pathway under carbonyl cyanide m-chlorophenyl hydrazone (CCCP) treatment using the MitoQC reporter (tandem mCherry-GFP tagged to the mitochondrial targeting sequence of FIS1). Mitophagic flux was quantified as the number of mCherry-only puncta (mitolysosomes) per cell. In WT cells stably expressing Myc-Parkin, CCCP induced a robust increase in mitolysosome number, whereas VPS13B KO cells showed a statistically significant reduction and failed to mount a comparable increase (Fig. 3A, B). Importantly, re-expression of VPS13B-FL-EGFP but not VPS13B-ΔMLD-EGFP in the KO background rescued mitolysosome counts toward WT levels (Fig. 3A, B). These results indicate that VPS13B is required for efficient PINK1/Parkin-mediated mitophagic flux.

To assess whether VPS13B participates directly in PINK1/Parkin-mediated mitophagy, we fractionated cytosolic and mitochondrial pools from WT and VPS13B KO cells in the presence or absence of CCCP conditions and immunoblotted for EGFP-Parkin, PINK1, GABARAPL1, LC3B, p62, and VPS13B. In WT cells, CCCP elicited a robust increase of VPS13B in the mitochondrial fraction, indicating stress-induced recruitment to damaged mitochondria (Fig. 3C, D). Concomitantly, LC3B and GABARAPL1 were enriched in the mitochondrial fraction of WT upon CCCP, whereas in VPS13B KO cells mitochondrial GABARAPL1 was nearly undetectable at baseline and failed to increase with CCCP; mitochondrial LC3B was also reduced, though less profoundly. By contrast, EGFP-Parkin and PINK1 accumulated on mitochondria in KO cells even without CCCP, consistent with damaged mitochondria that recruit the PINK1/Parkin machinery but fail to engage downstream ATG8 steps (Fig. 3C, D). To examine flux, we profiled mammalian ATG8 (mATG8) family members (GABARAP, GABARAPL2, LC3A, LC3C) in presence and absence of bafilomycin A1 (Supplementary Fig. 2): GABARAPL1-II—a key effector in PINK1/Parkin-mediated mitophagy—was reduced in KO cells and did not increase with CCCP (Fig. 3C, D), and its BafA1-sensitive turnover was blunted (Supplementary Fig. 2). In contrast, LC3A/B-II increased with CCCP in KOs similarly to WT, and GABARAPL2 levels were lowered with only a mild flux decrease; p62/SQSTM1 levels and flux were also reduced in KO (Fig. 3C, D). Together with the MitoQC data showing reduced mitolysosome formation in KO and rescue of mitolysosome number by full-length VPS13B, these findings indicate that VPS13B could enable the GABARAP or GABARAPL1-dependent arm of PINK1/Parkin-mediated mitophagy. Notably, the CCCP-induced mitochondrial recruitment of VPS13B in WT places VPS13B at the site of damage, where it likely couples Parkin/PINK1 activation to productive mATG8 (GABARAP and GABARAPL1) engagement, thereby maintaining mitochondrial quality control.

VPS13B is recruited to Mfn2-positive mitochondria via its MLD, and this recruitment is enhanced by mitochondrial stress
Next, to test whether endogenous VPS13B is dynamically recruited to Mfn2-positive mitochondria (used as a marker for ER-mitochondria contact sites) under cellular stress, we quantified VPS13B-Mfn2 PLA signals after applying mitochondrial depolarization (CCCP), oxidative stress (Sodium Arsenite, S.A.), and ER stress (Thapsigargin, T.G.) (Fig. 3E, F). Notably, CCCP elicited a significant increase in VPS13B-Mfn2 PLA puncta in WT cells, indicating stress-induced engagement of VPS13B at Mfn2-positive mitochondria (Fig. 3E, F). This finding is concordant with our biochemical fractionation data showing CCCP-dependent enrichment of VPS13B in the crude mitochondrial fraction (Fig. 3C, D), together supporting a model in which mitochondrial stress promotes VPS13B recruitment to Mfn2-positive mitochondria interfaces. Collectively, these results demonstrate that mitochondrial stress promotes VPS13B recruitment to Mfn2-positive mitochondria contact sites, consistent with its CCCP-dependent enrichment in the mitochondrial fraction.

VPS13B recruits PI4P-positive Golgi vesicles to mitochondrial fission sites via its MLD
Having established that VPS13B-deficient cells exhibit hyperfused mitochondria with membrane depolarization and reduced outer-membrane dynamics, and that VPS13B is recruited via its C-terminal MLD to Mfn2-positive mitochondria, particularly under mitochondrial stress, we next investigated the functional role of VPS13B at these mitochondrial sites.

Recent work implicates PI4P-containing Golgi-derived vesicles in mitochondrial fission 39, yet how these vesicles are coupled to the fission machinery remains poorly defined. We next examined mitochondrial network architecture under PI4P depletion in our system, to test whether the previously reported fission defects upon phosphatidylinositol 4-kinase beta (PI4KB) knockdown were reproducible under our conditions. We acutely depleted PI4P by siRNA-mediated knockdown of PI4KB, validated by reduced PI4KB protein levels on Western blot (Supplementary Fig. 3a, b). To visualize PI4P dynamics in cells, we used GFP tagged PH domain of the oxysterol-binding protein-related protein 9 (GFP-ORP9-PH) domain, which binds to specifically to PI4P 40. PI4P depletion led to decreased mean GFP-ORP9-PH intensity normalized to cellular area (Supplementary Fig. 3c, d), along with increases in accumulation of hyperfused mitochondrial networks (Supplementary Fig. 3c, e). These findings indicate a more reticulated and branched mitochondrial network with increased terminal ends—a signature of stalled or aborted fission that corroborates prior studies and confirms that PI4P depletion alone is sufficient to impair mitochondrial fission under our conditions as well as network architecture under PI4P depletion condition. Thus, when PI4P is depleted, mitochondria become more reticulated with greater branching and more terminal ends—a signature of stalled or aborted fission 39.

Given VPS13B’s localization and link to mitochondrial function, we hypothesized that VPS13B mediates this coupling for PI4P transport. To test this hypothesis, we quantified mitochondrial fission by live-cell imaging of MitoTracker-stained mitochondria with event-based scoring in WT and VPS13B KO cells: the normalized fission rate was significantly reduced in KO relative to WT, indicating a VPS13B-dependent deficit in mitochondrial fission (Fig. 4A, B).

To test whether the fission defect reflects impaired delivery of Golgi derived PI4P-positive vesicles to fission sites, we performed three-color live-cell imaging in WT and KO cells expressing EGFP-TGN46 (Golgi marker), BFP-ORP9-PH (PI4P reporter), and MitoTracker, and quantified—on an event basis—the fraction of bona fide fission events exhibiting (i) PI4P contact at mitochondria, (ii) TGN46 contact, and (iii) dual PI4P-TGN46 contact. All three metrics were significantly reduced in KO vs WT, indicating that VPS13B is required for recruitment/retention of PI4P-positive TGN vesicles at mitochondrial fission sites (Fig. 4B–E; Supplementary Movie 1, 2).

To determine whether VPS13B is sufficient to drive PI4P-vesicle delivery to fission sites, we imaged VPS13B KO cells co-expressing VPS13B-EGFP (FL or ΔMLD), BFP-ORP9-PH, and MitoTracker (Fig. 4F, G). Re-expression of VPS13B-FL rescued the normalized fission rate to WT-like levels, whereas VPS13B-ΔMLD failed to rescue (Fig. 4g, h; Supplementary Movie 3–6). Consistent with a direct role at fission, VPS13B-FL localized at a substantial fraction of fission events (VPS13B contact at mitochondrial fission site, %) comparable to WT, while ΔMLD showed no localization (Fig. 4H). Likewise, PI4P contact at mitochondrial fission site (%) was rare in KO, restored by VPS13B-FL, and not rescued by ΔMLD (Fig. 4I). Importantly, co-occurrence of PI4P and VPS13B at the same fission site was readily detected in WT and KO+VPS13B-FL but absent with ΔMLD (Fig. 4J). Event tracking further revealed that VPS13B and PI4P signals converged at ~30.1% of fission events (WT cells VPS13B contact at mitochondrial fission site, Fig. 4H; Supplementary Movie 3), and in ~93.8% of those convergent events the ORP9-PH-positive vesicle arrived with VPS13B and reached mitochondria prior to fission (WT cells PI4P-VPS13B contact at mitochondrial fission site, Fig. 4J; Supplementary Movie 3), consistent with targeted delivery of PI4P-positive vesicles to maturing fission sites. In KO cells, overexpression of VPS13B-ΔMLD failed to restore fission and instead further suppressed residual fission activity compared to VPS13B-FL, suggesting that the mutant exerts a nonfunctional or inhibitory effect (Fig. 4F–J; Supplementary Movies 5–6).

Finally, to establish biochemical competence for PI4P recognition, we purified a GST-tagged VPS13B PH domain in E. coli expression system and performed a PIP-strip assay. The VPS13B PH domain bound PI4P and, to varying degrees, PI3P, PI5P, PI(3,4)P₂, PI(3,5)P₂, PI(4,5)P₂, and PI(3,4,5)P₃, whereas GST alone showed no detectable binding (Supplementary Fig. 3f–h). Together, these quantitative imaging, rescue, and biochemical data demonstrate that VPS13B, through its MLD, recruits PI4P-containing Golgi vesicles to mitochondrial fission sites to promote efficient fission, providing a mechanistic basis for the reduced fission and hyperfusion observed in VPS13B-deficient cells.

PI4P-VPS13B acts downstream of Drp1 to drive late-stage mitochondrial fission
Given that VPS13B recruits PI4P-positive Golgi vesicles to mitochondrial fission sites, we next examined how PI4P itself contributes to mitochondrial fission and interfaces with VPS13B function. To this end, we acutely depleted PI4P using siPI4KB. Event-based live imaging of MitoTracker-labeled mitochondria revealed that PI4P depletion significantly lowered both the number of fission events and the normalized fission rate, thereby phenocopying the VPS13B KO defect (Supplementary Fig. 4a–c). Importantly, the fraction of Drp1-positive fission events (BFP-Drp1) was unchanged in PI4P depletion condition (Supplementary Fig. 4d), and neither the MitoTracker-Drp1 colocalization (%) nor the number of Drp1 puncta per cell differed from control (Supplementary Fig. 4e–g), indicating that Drp1 recruitment and puncta assembly are preserved, while completion of mitochondrial fission is impaired. Immunoblotting further supported this interpretation: total Drp1 protein decreased upon PI4P depletion, with a slight increase in Mfn2 levels noted, while the ratios of phospho-Drp1 (S616, activating) and phospho-Drp1 (S637, inhibitory) to total Drp1 remained unchanged, arguing against a shift in Drp1 activation state as the primary driver of the defect (Supplementary Fig. 4h–l). Consistently, VPS13B KO cells also exhibited decreased total Drp1 levels, and combining PI4P depletion with VPS13B loss produced an additive reduction in Drp1 abundance (Supplementary Fig. 4i). Nonetheless, the Drp1-positive fission fraction, Drp1-mitochondria colocalization, and puncta number remained stable (Supplementary Fig. 4d, f, g), reinforcing the conclusion that VPS13B and PI4P regulate the fission completion step rather than Drp1 recruitment or activation. Together, these data place PI4P—and thus VPS13B-mediated PI4P vesicle delivery—downstream of or parallel to Drp1 recruitment, acting at a late, lipid-dependent step required for productive mitochondrial fission. In this model, Drp1 is properly recruited and assembled, but in the absence of adequate PI4P-dependent lipid supply or membrane organization, the mitochondrial fission fails to progress to membrane severing despite an intact phosphorylation balance.

To determine whether VPS13B localizes to mitochondrial fission sites and to assess the functional requirement of its MLD, we performed three-color live-cell imaging (MitoTracker, VPS13B-EGFP, and BFP-Drp1) and quantified bona fide fission events. In WT cells, VPS13B and Drp1 co-localized at mitochondria (Fig. 5A, B). In VPS13B-KO cells, re-expression of full-length VPS13B (VPS13B-FL) restored multiple parameters: (i) the normalized mitochondrial fission rate was restored to ~0.51 (Fig. 5C, D), (ii) Drp1 contact at fission sites was detected in ~77.2% of events (Fig. 5C, E), (iii) VPS13B contact at fission sites was observed in ~51.3% (Fig. 5C, F), and (iv) dual Drp1-VPS13B contact at fission sites occurred in ~68.1% of events (Fig. 5C, G; Supplementary Movie 7). By contrast, VPS13B-ΔMLD failed to restore any of these parameters, all of which remained significantly reduced relative to KO+VPS13B-FL (Fig. 5C–G; Supplementary Movie 8). These findings position VPS13B at Drp1-positive mitochondrial fission sites and demonstrate that the MLD is essential for its engagement with, and PI4P-dependent support of, the fission machinery.

CS patient derived iPSC-induced neurons recapitulate VPS13B-linked mitochondrial dysfunction and mitophagy defects
Cohen syndrome (CS) is a rare, autosomal-recessive neurodevelopmental disorder caused by biallelic pathogenic variants in VPS13B (COH1) 11,14,15,41–43. Clinically, CS features developmental delay/intellectual disability, postnatal microcephaly, early hypotonia, progressive retinochoroidal dystrophy with high myopia, neutropenia, truncal obesity, joint laxity, and a characteristic facial gestalt 42,43. Most reported VPS13B variants are loss-of-function (e.g., nonsense/frameshift or large deletions) predicted to abolish protein function 11,43.

To assess clinical relevance, we analyzed iPSC-induced neurons from CS patients harboring VPS13B loss-of-function variants (c.1239 T>G and c.10333G>A), comparing them with three healthy control lines 21,29. As reported in these prior studies, the VPS13B Compound Heterozygote variants in these CS lines lead to an approximately ~50% reduction in VPS13B mRNA levels in patient-derived neural cells, consistent with a partial loss-of-function effect. Consistent with our VPS13B KO model, MitoTracker imaging revealed accumulation of hyperfused mitochondrial networks in CS neurons (Fig. 6A, B). Mitochondrial membrane potential (ΔΨm), quantified as TMRM intensity normalized to MitoTracker-positive area, was significantly reduced in CS neurons relative to controls (Fig. 6C, D). At the level of fission/fusion regulators, PINK1 puncta were increased (Fig. 6E, F) and Drp1 puncta were decreased (Fig. 6G, H), indicating persistent mitochondrial damage signals and suggesting impaired mitophagy progression. A directional increase in Mfn2 puncta was also observed (Supplementary Fig. 5a, b), but we do not over-interpret this finding here, as it may reflect multiple processes, including mitochondrial fusion and/or ER–mitochondria contact formation. Accordingly, while our data primarily indicate an impairment in mitochondrial fission, a potential contribution of VPS13B to fusion or contact-site remodeling cannot be excluded.

Taken together, CS patient neurons recapitulate the core VPS13B-linked phenotypes—hyperfused networks, reduced ΔΨm, decreased Drp1, and elevated PINK1—supporting a shared mechanism in which VPS13B dysfunction compromises mitochondrial dynamics and quality control.

As these findings are based on two CS lines and may represent a subset of CS presentations, larger cohorts and isogenic correction or rescue studies will be needed to generalize; nonetheless, the convergence with VPS13B-deficient models suggests a clinically relevant pathway and highlights VPS13B-dependent lipid and vesicle trafficking as a potential therapeutic entry point.

Methods
All experiments were performed in accordance with relevant ethical regulations and were approved by the Institutional Review Board and the Institutional Animal Care and Use Committee of Hannam University.

Cell culture and transfections
HeLa and LX293T cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and Clontech (Takara, Shiga, Japan). The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Corning, Corning, NY, USA) with high glucose, supplemented with 10% fetal bovine serum (FBS; Corning) and 100 U/mL penicillin-streptomycin (GIBCO, Billings, MT, USA), and maintained at 37°C with 5% CO₂. All cell lines were confirmed to be mycoplasma-free using the EZ-PCR Mycoplasma Detection Kit (Biological Industries, Cromwell, CT, USA; Cat# 20-700-20).

For transfections, cells were seeded and transfected with either Lipofectamine 2000 or Lipofectamine 3000 (Invitrogen, Waltham, MA, USA) according to the manufacturer’s protocols, and incubated for 24 h. Lentiviral packaging was performed in LX293T cells using the CalPhos transfection system (Clontech), following the manufacturer’s instructions. Briefly, the packaging plasmids psPAX2 (Addgene, Watertown, MA, USA; #12260), pMD2.G (Addgene; #12259), and the construct of interest were transfected at a 4:1:5 ratio. Afterward, lentiviral supernatants were collected 48 h post-transfection, followed by a second collection 72 h after media replacement.

For transduction, EGFP-Parkin and Myc-Parkin lentiviruses were introduced into HeLa cells in the presence of 10 µg/mL polybrene (Hexadimethrine bromide; Sigma-Aldrich, St. Louis, MO, USA). After 48 h, cells were selected with 1 µg/mL puromycin for an additional 48 h and subsequently sorted by FACS to optimize GFP expression.

iPSC culture and glutamatergic neuronal induction
All research involving human stem cells was conducted in accordance with all relevant guidelines and regulations and was approved by the Institutional Review Board of Hannam University (IRB No. 2024-021). Informed consent was obtained from all donors for the use of human cells in this study. Patient-derived and control induced pluripotent stem cells (iPSCs) were maintained following established protocols 21,29,49. Briefly, iPSC lines were cultured in an embryonic stem cell (ESC) medium containing 20% knockout serum (Gibco; 10828-028), beta-mercaptoethanol (Gibco; 21985-023), 1× GlutaMAX (Gibco; 3500-061), 1× MEM-NEAA (Gibco; 1140-050), penicillin/streptomycin (Gibco; 15140-122), and DMEM-F12 (Gibco; 11320-033). Next, iPSC-like colonies were picked after 1 month and reseeded onto new feeder cells. The iPSC culture system was subsequently transitioned from a mouse embryonic fibroblast (MEF) feeder-dependent system to a feeder-free system using Essential 8 Medium (Gibco; A1517001) between passages 5 and 7. For glutamatergic neuronal induction, a modified neurogenin2 (Ngn2)-mediated rapid neural induction protocol was employed as described in previous studies 50.

Plasmids and reagents
The following plasmids were used: pGEX51, pCMV-BFP-KDEL (Addgene; #49150), pEGFP-GalT, pEGFP-LAMP1, pEGFP-Rab5, pEGFP-Rab7, pEGFP-Rab9, pEGFP-Sec61, mCherry-Sec61, pcDNA3.1-mRFP-TOM20 and pPBbsr-AcGFP1-ORP9-PH (Addgene; #214270). The following plasmids were constructed for this study. Primer sequences are detailed in Supplementary Table 1. pGEX-PH domain, pcDNA3.1-mCherry-EGFP-FIS1 (MTS) (MitoQC), pEGFP-Drp1, pCMV-BFP-Drp1, pCMV-BFP-ORP9-PH, pEGFP-N3-VPS13B, pEGFP-N3-VPS13B-ΔMLD, pEGFP-N3-DHL-PH (VPS13B), pEGFP-N3-MLD (VPS13B), pCMV7.1-3xFlag-DHL-PH (VPS13B), pCMV7.1-3xFlag-MLD (VPS13B), The sequences for FIS1 (MTS), Drp1, and VPS13B, VPS13B MLD were amplified from a WT HeLa cDNA library.

Reagents were obtained as follows: Image-iT™ TMRM Reagent (I34361) and MitoTracker™ Red CMXRos (M7512) from Invitrogen; MitoTracker® Deep Red FM (#8778) from CST; Bafilomycin A1 (sc-201550A) from Santa Cruz (Dallas, TX, USA); and, Brefeldin A (B7651), and CCCP (C2759) from Sigma-Aldrich.

Immunofluorescence
Cells were plated on glass coverslips and grown for 24 hours before drug treatment. After treatment, the cells were fixed in 4% paraformaldehyde (PFA) in PBS at room temperature (RT) for 15 minutes. The cells were then blocked with 3% BSA in PBS at RT for 30 minutes, followed by three washes with PBS. Next, the cells were incubated overnight at 4°C in 3% BSA in PBS with the primary antibodies. After three PBS washes, the cells were stained with the appropriate secondary antibodies (Alexa or DyLight) for 2 hours at RT in PBS. Following three additional washes with PBS, coverslips were mounted onto slides with DAPI-containing mounting solution (VectorLabs, Newark, CA, USA) or DAKO Fluorescence Mounting Medium (DAKO, Carpinteria, CA, USA).

Antibodies
Primary antibodies were obtained from the following sources: VPS13B (NBP2-55104), PINK1 (BC100-494), PINK1 (BC100-494) and Drp1 (NB110-55288, for WB) from Novus Biologicals (Littleton, CO, USA); GM130 (PA1-077) from Thermo Fisher Scientific (Waltham, MA, USA); GST (#2622S), LC3A (4599S), LC3B (2775S), LC3C (14723S), GABARAP (13733S), Phospho-Drp1 (Ser616, #3455) and Phospho-Drp1 (Ser637, #4867) from Cell Signaling Technology (Danvers, MA, USA); GABARAPL1 (GTX132664) and GABARAPL2 (GTX132666) from GeneTex (Irvine, CA, USA); Mitofusin 2 (ab56889), Drp1 (ab56788, for ICC) from Abcam (Cambridge, United Kingdom); GFP (75-131) from Neuromab (Davis, CA, USA); PI4KB (13247-1), TOM20 (11802-1-AP, for WB), and TIM23 (11123-1-AP) from Proteintech (Rosemont, IL, USA); GFP (AE078, for PLA) from Ablconal; and α-tubulin (T5168) and β-actin (A5441) from Sigma-Aldrich.

For Western blotting, primary antibodies were used at a 1:50-1:10,000 dilution, while peroxidase-conjugated secondary antibodies were used at a 1:10,000 dilution in TBST. Peroxidase-conjugated goat anti-rabbit (111-035-144) and peroxidase-conjugated goat anti-mouse (115-035-174) from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).

For immunocytochemistry, the following secondary antibodies were used: Alexa Fluor 647-conjugated goat anti-rabbit (A21245; Invitrogen), Alexa Fluor 488-conjugated donkey anti-rabbit (711-545-152), DyLight 405-conjugated goat anti-rabbit IgG (111-475-144), DyLight 405-conjugated donkey anti-mouse (715-475-150), and Alexa 594-conjugated donkey anti-rabbit (711-585-152), all obtained from Jackson ImmunoResearch Laboratories.

FRAP assay
FRAP assays were performed using a Zeiss LSM900 confocal microscope equipped with an incubator stage set at 37°C and 5% CO₂. HeLa cells transfected with mRFP-TOM20 using Lipofectamine 2000 were photobleached using a 594-nm laser at 100% power for 0.5 seconds. Post-bleach images were acquired at 5-second intervals.

Proximity ligation assays (PLA)
HeLa cells were cultured on coverslips. The 24 h after, cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.01% Triton X-100 for 15 min in Room temperature. PLA was performed using the Duolink detection kit (Sigma) following the manufacturer’s instructions. The primary antibodies used were Mfn2 (Mouse, 67487-1, Proteintech, 1:400), GFP (Mouse, AE078, Abclonal, 1:200) and VPS13B (Rabbit, NBP2-55104, Novus, 1:200). Images were acquired on a Zeiss LSM900 confocal microscope using a 63× oil-immersion objective.

Live cell imaging
Cells were plated on 35 mm confocal dishes (SPL Life Sciences, Korea) for live cell imaging. Time-series images were performed over 15 frames, with Z-stacks comprising three optical sections using a 63× oil-immersion objective. Z-stacks at 0.33 µm intervals, with a temporal resolution of 30.84 seconds per frame (approximately 0.032 frames per second).

Although the frame rate used in live-cell imaging (~30.8s per frame) under-samples very rapid mitochondrial dynamics, we restricted our analysis to Drp1-positive events that showed a clear transition from a continuous mitochondrion to two separate mitochondria across consecutive frames. The co-recruitment of the fission marker Drp1 (Fig. 5), therefore, provides an anchor that increases our confidence that we are scoring bona fide fission events under identical imaging conditions across genotypes.

Images were processed using “average projection”, “smooth”, and then analyzed. For mitochondrial staining, cells were incubated with 200 nM MitoTracker Deep Red FM (M22426; Invitrogen) for 20 minutes at 37°C in a 5% CO₂ incubator. Mitochondrial membrane potential assays were then performed using Image-iT™ TMRM reagent (I34361; Invitrogen). To image mitochondrial fission events, cells were transfected with VPS13B-EGFP, VPS13B-ΔMLD-EGFP, BFP-ORP9-PH, and BFP-Drp1. Foci that crossed mitochondria 0–30 seconds before fission were counted as positive fission events.

Crude mitochondria fractionation
Mitochondria fractionation was performed using briefly described follow method 52. HeLa cells were dissociated using trypsin-EDTA, pelleted at 300×g, and washed with PBS (pH 7.4). The pellets were then resuspended in chilled mitochondria isolation buffer (MIB; 220 mM mannitol, 70 mM sucrose, 10 mM Tris-KOH pH 7.4, and 1 mM EDTA) containing protease inhibitors (Sigma-Aldrich) and homogenized using a pestle and 25G syringe. The homogenates were then centrifuged at 600×g for 5 minutes at 4°C. The resulting supernatant was centrifuged again at 7000×g for 10 minutes at 4°C. Afterward, the pellet was washed twice by resuspension in fresh MIB and centrifuged at 7000 × g. Next, the cytosolic (supernatant) and mitochondrial (pellet) fractions were collected. The mitochondrial pellet was then resuspended in 1% SDS radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors (Sigma). Finally, the protein concentrations in both cytosolic and mitochondrial fractions were quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher) and processed using SDS-PAGE and immunoblotting.

Protein extraction and Western blotting
Cells were lysed in RIPA buffer (20 mM Tris pH 8.0, 150 mM NaCl, 1% SDS, 1% deoxycholic acid, and 1% NP-40) supplemented with complete protease and phosphatase inhibitors. Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher). Proteins were then separated by SDS-PAGE and transferred onto PVDF membranes (0.2 µm pore size; Millipore, Burlington, MA, USA). Next, the membranes were blocked with 5% non-fat milk or 5% BSA in TBST for 30 minutes at RT, followed by incubation with primary antibodies overnight at 4°C. After three 10-minute washes in TBST, the membranes were incubated with secondary antibodies. The blots were developed using Immobilon Western Chemiluminescent HRP Substrate (Millipore) and imaged with a digital ECL machine (UVTECH, Liverpool, UK) and the iBright CL750 (Thermo Fisher).

Transmission electron microscopy
Ultrastructural analysis was performed using transmission electron microscopy (TEM). WT and VPS13B KO HeLa cells were cultured on confocal dishes. Cells were fixed immediately with a solution of 1% glutaraldehyde and 1% paraformaldehyde for 1 h, followed by post fixation in 1% osmium tetroxide for 1 h at 4°C. Afterward, the samples were stained overnight at 4°C with 1% uranyl acetate, dehydrated through a graded ethanol series, and embedded in epoxy resin (EMS, Hatfield, PA, USA). Ultrathin sections (65 nm) were then cut using an ultramicrotome (Leica Microsystems, Wetzlar, Germany). Next, the sections were stained with UranyLess for 2 minutes, followed by staining with 3% lead citrate for 1 minute. TEM images were acquired using a Tecnai 20 transmission electron microscope (Thermo Fisher Scientific) operating at 120 kV and equipped with a US1000XP camera.

For quantitative analysis, the outlines of mitochondrial profiles were manually traced from multiple non-overlapping fields for each condition using ImageJ/Fiji software (National Institutes of Health). Quantification was performed on a minimum of 100 mitochondria per condition (WT and KO; n = 5) in each of two independent experiments. From the resulting traces, the cross-sectional area and perimeter of each individual mitochondrion were calculated.

Image acquisition and processing
Images were acquired using Zeiss LSM 880 and LSM 900 confocal microscopes equipped with a 63× oil-immersion objective. Subsequently, image analysis was performed using ImageJ Fiji (NIH, Bethesda, MD, USA). For morphological analysis, mitochondrial parameters, including puncta count, area, and perimeter, were quantified using the Particle Analysis tool in ImageJ (Fiji). Following the methodology described previously 39, mitochondria were classified into three distinct categories: “fragmented,” where the majority of mitochondria were short and spherical, Defined as a cell containing 20 or more mitochondrial skeletons with zero branches.; “hyperfused,” Defined as a cell containing at least one mitochondrial skeleton with 50 or more branches.; and “intermediate,” where most mitochondria were tubular but neither extensively interconnected nor spherical, Defined as a cell that did not meet the criteria for either ‘Hyperfused’ or ‘Fragmented’.

Statistics and reproducibility
All statistical analyses were performed using GraphPad Prism 10. For bar graphs error bars indicate the mean ± standard error of the mean (SEM) from each independent experiment. All data were tested for normality. When a group did not follow a normal distribution, non-parametric tests, including Mann–Whitney test or Kruskal-Wallis test followed by multiple comparisons test, were performed. In contrast, unpaired two-tailed t-tests or one-way ANOVA followed by multiple comparison tests were applied to data that followed a normal distribution. A p-value of <0.05 was considered statistically significant. All experiments were repeated independently at least three times. No statistical method was used to predetermine sample size. Cell images were selected and analyzed randomly. No data were excluded from the analysis. The investigators were blinded to allocation during experiments and outcome assessment. Details of the statistical tests used, exact n values, and definitions of biological versus technical replicates are provided in the corresponding figure legends.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Lee et al. investigate the role of VPS13B in mitochondrial fission and mitophagy. Using VPS13B knockout HeLa cells and Cohen Syndrome (CS) patient-derived neurons, the authors show that VPS13B deficiency results in mitochondrial abnormalities. As a mechanistic explanation, they propose that VPS13B mediates PI4P-containing Golgi-derived vesicle transfer to mitochondrial fission sites, which is critical for proper fission. However, the evidence presented does not fully support this conclusion, and additional data are needed to substantiate their claims.\nMajor Points\n1. In line 33, the authors state that “Deletion of the mitochondria-associated ER membrane (MAM) via the MAM localization domain (MLD)...” However, no direct evidence is provided showing that MLD deletion removes the MAM. The author need data directly supporting that MLD deletion disrupts MAM localization.\n2. The use of the OSBP PH domain to visualize PI4P vesicles raises specificity concerns. Since this probe can localize to the Golgi itself, additional evidence is needed to confirm that the probe specifically marks PI4P-containing Golgi-derived vesicles rather than the Golgi more generally.\n3. In Figure 3B, the localization of VPS13B at mitochondria contact sites needs further clarification. The line scan in Figure 3B shows a peak separation of about 1 μm between TOM20 and VPS13B, whereas typical mitochondria contact sites range from 10-30 nm. Additional evidence is needed to demonstrate that VPS13B indeed localizes at mitochondria contact sites.\nMinor Points\n1. Typo: In line 92, “VPS1B” should be corrected to “VPS13B.”\n2. Duplicate References: References 38 and 39 appear to be the same and should be consolidated.\n3. Inappropriate Citation: Reference 46 does not have a publication year etc. It should be cited appropriately.", "reviewer_3": "The manuscript entitled \"VPS13B regulates mitochondrial fission and mitophagy\" provides compelling evidence that VPS13B is critical in mitochondrial dynamics and quality control, particularly through mediating the transport of PI4P-containing Golgi-derived vesicles to mitochondrial fission sites. This research utilizes a combination of confocal microscopy, electron microscopy, biochemical assays, and live-cell imaging, which together provide significant insights into mitochondrial regulation, especially in the context of Cohen syndrome (CS).\nStrengths:\n• The study effectively integrates multiple methodologies, clearly demonstrating the essential role of VPS13B in mitochondrial dynamics and mitophagy.\n• Use of VPS13B knockout (KO) HeLa cells and patient-derived iPSC-neurons enhances the relevance of findings to Cohen syndrome pathology.\n• The manuscript is well-structured, providing clear connections between VPS13B function, Golgi-derived PI4P vesicle transport, mitochondrial fission, and cellular quality control mechanisms.\n• The rationale and progression of experiments are clearly outlined, facilitating easy understanding and contextualization within mitochondrial biology and CS.\nAreas Requiring Clarification and Improvement:\nAcknowledgement of Prior Work:\nExplicitly reference and discuss the previous foundational studies by Nagashima et al. (2020) that identified the role of Golgi-derived PI4P-containing vesicles in mitochondrial fission. Clarify that the current findings represent an extension or further specification of the role of VPS13B, rather than a completely novel discovery.\nQuantitative Analysis of Mitochondrial Morphology:\nThe current manual scoring method for mitochondrial morphology is subjective and potentially biased. Provide explicit criteria, including how morphologies were defined, blinding of the analyst, and reproducibility across multiple replicates. Supplement manual scoring with quantitative automated or semi-automated analyses of mitochondrial shape parameters such as area, length, and connectivity.\nDirect Interaction Evidence:\nCurrently, no direct evidence of physical binding between VPS13B and PI4P is shown. Including assays such as lipid pull-downs or proximity ligation assays would significantly enhance the mechanistic insights.\nElectron Microscopy Interpretation:\nElectron microscopy alone provides limited support for mitochondrial hyperfusion claims due to the difficulty in visualizing complete mitochondrial networks in thin EM sections. Quantitative morphometric analyses of mitochondria should accompany EM data.\nFRAP Experiments:\nClarify and justify the interpretation of reduced fluorescence recovery observed in FRAP assays. Specify whether this represents impaired mitochondrial fusion, reduced mitochondrial mobility, or a combination of both.\nRescue Experiments:\nProvide clear justification for performing VPS13B-ΔMLD experiments only in WT and not in VPS13B KO cells. Ideally, repeat these experiments in VPS13B KO cells to strengthen the conclusions regarding the MLD domain – does the MLD mutant rescue the fission rate defect in KO cells?\nLocalization and Imaging Analysis:\nQuantification of VPS13B mitochondrial localization (Figure 3B) is insufficient. Provide more rigorous quantitative analyses such as Pearson’s correlation coefficients or colocalization metrics.\nQuantify data presented in Figure 4J regarding the association between VPS13B and Drp1 during mitochondrial fission events.\nTMRM Mitochondrial Membrane Potential Measurements:\nNormalize mitochondrial membrane potential measurements to a mitochondrial mass marker to accurately account for potential differences in mitochondrial content between experimental conditions.\nClearly distinguish individual control and patient-derived iPSC lines in quantifications to assess consistency within groups.\nSupplemental and Statistical Information:\nProvide clearer labeling and explicit statistical information (e.g., sample sizes, specific tests performed, and p-values) across main and supplemental figures.\nInclude missing supplementary data and clarify the discrepancies in labeling (e.g., Myc-Parkin vs. GFP-Parkin).\nAdditional Critical Points:\nClarify the rationale behind using two different fixation protocols in immunofluorescence methodology.\nSpecify the frame rates and temporal resolution used in live-cell imaging experiments.\nCombine supplemental figures that are closely related for clearer presentation and interpretation (e.g., Supplemental figures 1H and 3).\nUtilize colorblind-friendly palettes in imaging data for improved accessibility (avoid red-green combinations).\nAddressing these points comprehensively will significantly strengthen the manuscript's clarity, rigor, and impact.", "reviewer_4": "Comments:\nThe manuscript investigates the role of VPS13B in mitochondrial morphology, mitophagy, and Golgi-mitochondria interactions. Using VPS13B knockout (KO) cells and neurons derived from Cohen syndrome (CS) patients, the authors observe mitochondrial hyperfusion, impaired membrane potential, and defective mitophagy. The authors propose that VPS13B facilitates PI4P-containing vesicle transfer to mitochondrial fission sites, influencing fission and mitophagy through interactions with Drp1. While the study presents some interesting phenotypes and raises important questions, the findings are largely preliminary, with significant gaps in experimental evidence and data quality. Furthermore, the proposed mechanisms lack sufficient experimental support, and many claims appear overstated. Below are the detailed comments:\nMajor Issues:\n1. Causality Between VPS13B and Mitochondrial Morphology:\nThe manuscript shows an association between VPS13B deficiency and mitochondrial hyperfusion but fails to establish causality. Rescue experiments using full-length VPS13B or targeted mutants are insufficient or missing in key contexts, such as mitochondrial dynamic, mitophagy defects, or PI4P-vesicle transfer. Additional experiments are needed to conclusively show that VPS13B deficiency directly causes mitochondrial abnormalities.\n2. VPS13B Localization on Mitochondria or Mitochondria-Golgi Contact Sites:\nThe study’s claims about VPS13B localization at Golgi-mitochondria contact sites are not well-supported. The truncation analysis using VPS13B fragments (e.g., MLD and DHL-PH domains) is not convincing due to insufficient evidence of normal VPS13B localization. Advanced techniques such as super-resolution microscopy or co-fractionation of Golgi-mitochondria contact sites would strengthen these claims.\nAdditionally, the mechanism by which VPS13B translocates to these contact sites is speculative and requires further experimental investigation, such as testing whether specific cellular stressors or lipid cues regulate its localization.\n3. Impact of PI4P on Drp1 Recruitment and Mitochondrial Fission:\nThe proposed role of PI4P in Drp1 recruitment and mitochondrial fission is not experimentally validated. The manuscript does not test whether PI4P levels correlate with Drp1 activity, nor does it explore whether PI4P-deficient conditions alter fission dynamics. Experiments such as Drp1 localization or activation assays under PI4P-depleted conditions would provide stronger evidence for this hypothesis.\n4. Data Quality and Quantification:\nMany datasets are of low quality or lack clear quantification:\nImaging data (e.g., vesicle transfer and mitochondrial phenotypes) is often unclear, with low resolution or inadequate annotations, making interpretation challenging.\nQuantification of mitochondrial morphology (e.g., hyperfusion vs. fragmentation) and mitophagy markers (e.g., PINK1 puncta) lacks explicit statistical details such as replicate numbers and p-values.\nFigures require more detailed annotations to clearly connect them to key conclusions. For example, showing direct comparisons between WT and VPS13B KO models in a single frame would improve clarity.\n5. Overstatements About the Data:\nThe manuscript contains numerous overstatements about the conclusions that can be drawn from the data. For example:\n\"VPS13B is essential for mitochondrial fission and mitophagy\" is overstated, as the evidence for VPS13B’s involvement in these processes is largely correlative.\nClaims about \"VPS13B-mediated lipid transfer being critical for mitochondrial function\" are unsupported, as lipid transfer is not directly tested.\nThe discussion often generalizes findings beyond what is justified, particularly when linking VPS13B to CS pathology. For instance, variability in VPS13B mutations across CS patients is not considered, yet the manuscript implies a universal role for VPS13B dysfunction in mitochondrial defects.\nConclusion:\nThe manuscript presents some interesting preliminary findings regarding VPS13B’s role in mitochondrial dynamics and Golgi-mitochondria interactions. However, the study suffers from significant gaps in mechanistic evidence, weak data quality, and overstatements about the conclusions. To strengthen the manuscript:\n• Perform additional rescue experiments and mechanistic validations.\n• Use advanced localization techniques to definitively determine VPS13B’s site of action.\n• Provide rigorous data quantification and improved figure quality.\n• Temper claims to align with the presented data.\nAddressing the above issues is essential to substantiate its findings and improve its overall quality."}, "author_response": "Lee et al. investigate the role of VPS13B in mitochondrial fission and mitophagy. Using\nVPS13B knockout HeLa cells and Cohen Syndrome (CS) patient-derived neurons, the authors\nshow that VPS13B deficiency results in mitochondrial abnormalities. As a mechanistic\nexplanation, they propose that VPS13B mediates PI4P-containing Golgi-derived vesicle\ntransfer to mitochondrial fission sites, which is critical for proper fission. However, the\nevidence presented does not fully support this conclusion, and additional data are needed to\nsubstantiate their claims.\nMajor Points\n1. In line 33, the authors state that “Deletion of the mitochondria-associated ER membrane\n(MAM) via the MAM localization domain (MLD)...” However, no direct evidence is provided\nshowing that MLD deletion removes the MAM. The author need data directly supporting that\nMLD deletion disrupts MAM localization.\nResponse\nWe thank the reviewer for this insightful comment and agree that direct evidence is required\nto show that deleting the MAM‑localization domain (MLD) disrupts VPS13B’s engagement\nwith ER–mitochondria contact sites.\nIn response, we performed in situ proximity ligation assays (PLA) using antibodies to\nendogenous VPS13B and mitofusin‑2 (Mfn2), a tether enriched at ER–mitochondria interfaces.\nBecause PLA reports proximity within ~40 nm, it provides a sensitive readout of residency at\ncontact sites rather than direct binding. In wild‑type cells, VPS13B–Mfn2 PLA puncta were\nabundant and aligned with mitochondrial profiles, whereas VPS13B‑knockout (KO) cells\nshowed near‑background signal (Revised Fig. 1i, j). Re‑expression of full‑length\nVPS13B‑EGFP in KO cells restored the PLA signal to wild‑type levels, but expression of\nVPS13B‑ΔMLD failed to rescue, leaving PLA at background (Revised Fig. 1i, j). Consistent\nwith MLD‑dependent targeting, live‑cell imaging showed that full‑length VPS13B and\nC‑terminal fragments containing the DHL-PH/MLD region co‑localized with TOM20‑positive\nmitochondria, whereas ΔMLD did not (Revised Fig. 1h), establishing that the C‑terminal MLD\nis necessary (and, in fragment form, sufficient) for mitochondrial/ MAM localization. Moreover,\nmitochondrial stress (CCCP) significantly increased VPS13B-Mfn2 PLA puncta and enriched\nVPS13B in the mitochondrial fraction (Revised Fig. 3c-f), indicating stress‑induced\nrecruitment to Mfn2‑positive ER-mitochondria contacts.\nTogether, the PLA, live‑cell imaging, and fractionation data provide direct evidence that the\nMLD is essential for VPS13B’s recruitment to Mfn2‑positive ER–mitochondria contact sites\nand for its stress‑responsive localization.\n2. The use of the OSBP PH domain to visualize PI4P vesicles raises specificity concerns. \nSince this probe can localize to the Golgi itself, additional evidence is needed to confirm that\nthe probe specifically marks PI4P-containing Golgi-derived vesicles rather than the Golgi more\ngenerally.\nResponse\nWe appreciate the reviewer’s concern that OSBP–PH can label the Golgi ribbon and may not\nunambiguously report PI4P‑positive Golgi‑derived vesicles. As pointed out, the OSBP-PH\ndomain binds not only to PI4P but also to Arf1-GTP, thereby conferring high specificity for\ndetecting PI4P at the Golgi apparatus. In contrast, the ORP9-PH domain can also be utilized\nto visualize PI4P pools outside the Golgi. Therefore, in the revised manuscript, all conclusions\nabout PI4P at mitochondrial fission sites are based on ORP9-PH, together with orthogonal\ncontrols that distinguish vesicles from the bulk Golgi:\n1. Probe choice and validation (PI4P dependence).\nWe replaced OSBP-PH with BFP-ORP9-PH. In three‑color live‑cell imaging (ORP9-PH,\nTGN46, MitoTracker), ORP9-PH labeled motile, punctate structures with vesicular\nmorphology rather than the static TGN46 ribbon (Revised Supplementary Fig. 3c). Importantly,\nORP9-PH signal was diminished by PI4KB knockdown, confirming PI4P dependence (Revised\nSupplementary Fig. 3a-d).\n2. Event‑based discrimination at bona fide fission sites.\nWe quantified, on an event basis, the fraction of mitochondrial fission events that exhibited:\n(i) PI4P contact (ORP9-PH), (ii) TGN contact (TGN46), and (iii) dual PI4P-TGN contact. All\nthree metrics—and the normalized fission rate—were significantly reduced in VPS13B KO vs\nWT (Revised Fig. 4c-f), indicating that the PI4P signal we track corresponds to TGN‑derived\nvesicles engaging mitochondria at the time and place of fission, rather than generalized Golgi\nlabeling. Event tracking further showed co‑arrival of VPS13B and ORP9-PH at ~30.1% of\nfission events, with the vesicle reaching mitochondria before fission in ~93.8% of those\nconvergent events (Revised Fig. 4k and Supplementary Movies 3-6), consistent with targeted\nvesicle delivery to maturing constrictions.\n3. Genetic and domain dependencies that exclude bulk Golgi explanation.\nIn VPS13B KO cells, re‑expression of full‑length VPS13B (VPS13B‑FL) restored (i) fission\nrate, (ii) ORP9-PH PI4P contact at fission, and (iii) dual PI4P-VPS13B contact to WT‑like\nlevels, whereas VPS13B‑ΔMLD failed to rescue any of these readouts (Revised Fig. 4g–k). Thus,\nPI4P vesicle engagement at fission is VPS13B‑ and MLD‑dependent, not a by‑product of bulk\nGolgi labeling.\n4. Orthogonal functional placement of PI4P-VPS13B at late‑stage fission.\nPI4KB knockdown phenocopied VPS13B loss: fission rates were decreased and fission was\nstalled, while Drp1 recruitment, colocalization, and phosphorylation stoichiometry were\nunchanged (Revised Supplementary Fig. 4), placing PI4P—hence PI4P‑positive vesicles—\ndownstream of Drp1 assembly. Consistent with a lipid‑supply role, ORP9-PH intensity\nnormalized to mitochondrial area decreased and network branching increased under PI4P\ndepletion (Revised Supplementary Fig. 3c-e). Finally, VPS13B’s PH domain bound PI4P in\nPIP‑strip assays (Revised Supplementary Fig. 3f-h), providing biochemical competence for\nPI4P‑dependent engagement. By replacing OSBP-PH with ORP9-PH, coupling it with a Golgi\nmarker (TGN46), and restricting analysis to spatiotemporally defined fission events, we\nspecifically resolve PI4P‑containing Golgi‑derived vesicles rather than bulk Golgi signal. The\nVPS13B/MLD dependence, the PI4KB sensitivity, and the late‑stage (post‑Drp1) placement\njointly validate the specificity of our PI4P vesicle readouts and support our model that VPS13B,\nvia its MLD, recruits/retains PI4P‑positive Golgi vesicles at mitochondrial fission sites\n(Revised Fig. 4 and Revised Supplementary Fig. 3, 4 / Movies 3-6). We have expanded the\nMethods and figure legends accordingly.\n3. In Figure 3B, the localization of VPS13B at mitochondria contact sites needs further\nclarification. The line scan in Figure 3B shows a peak separation of about 1 μm between\nTOM20 and VPS13B, whereas typical mitochondria contact sites range from 10-30 nm.\nAdditional evidence is needed to demonstrate that VPS13B indeed localizes at mitochondria\ncontact sites.\nResponse\nWe thank the reviewer for noting that the ~1 µm peak separation between VPS13B-EGFP and\nTOM20 exceeds the nanometer spacing of ER-mitochondria contact sites and cannot by itself\nestablish contact‑site residency. This separation reflects the limits of confocal resolution\n(lateral ~200-250 nm; axial >500 nm) and the fact that line scans across tubular organelles\nreport intensity maxima, not intermembrane distances. We therefore did not rely on the confocal\nline scan to infer nanoscale contacts and instead provide orthogonal evidence:\n1. Proximity at ER-mitochondria contacts: In situ proximity ligation assays (PLA) using\nantibodies to endogenous VPS13B and Mfn2 (a tether enriched at ER-mitochondria interfaces)\nyielded abundant VPS13B-Mfn2 puncta in WT cells, were near background in VPS13B‑KO,\nwere restored by full‑length VPS13B, and not by VPS13B‑ΔMLD (Revised Fig. 1i, j). Because\nPLA reports proximity within ~40 nm, these data support VPS13B residency at Mfn2‑positive\ncontact sites in an MLD‑dependent manner.\n2. Stress‑induced recruitment and biochemical enrichment: Mitochondrial depolarization\n(CCCP) significantly increased VPS13B-Mfn2 PLA puncta (Revised Fig. 3e, f) and enriched\nVPS13B in the mitochondrial fraction (Revised Fig. 3c), indicating dynamic recruitment to\ncontact‑proximal mitochondria under stress.\n3. Targeting requirement mapped to the C‑terminus: Live‑cell imaging shows VPS13B‑FL\nand C‑terminal fragments containing the DHL-PH/MLD region enrich at TOM20‑positive\nmitochondria, whereas ΔMLD fails to do so (Revised Fig. 1g, h), establishing that the MLD is\nnecessary (and, in fragment form, sufficient) for mitochondrial/MAM targeting.\n4. Functional co‑localization at division sites: In event‑based three‑color imaging, VPS13B\nco‑appears with Drp1 at bona fide fission constrictions and this co‑contact is rescued by\nVPS13B‑FL but not ΔMLD (Revised Fig. 5c-g), consistent with VPS13B acting at ER-\nmitochondria interfaces engaged in fission.\nAccordingly, we have revised the figure legend and text to clarify that the Revised Fig. 1h (Fig.\n3B in the previous version of the manuscript) line scan is illustrative of regional enrichment\nalong mitochondrial profiles and is not used to estimate contact distances. The PLA,\nstress‑dependent recruitment, fractionation, and MLD‑dependence provide the required\nnanometer‑scale and mechanistic evidence that VPS13B localizes to Mfn2‑positive ER-\nmitochondria contact sites.\nMinor Points\n1. Typo: In line 92, “VPS1B” should be corrected to “VPS13B.”\nResponse\nWe have corrected this typographical error in the revised manuscript.\n2. Duplicate References: References 38 and 39 appear to be the same and should be\nconsolidated.\nResponse\nWe have consolidated these duplicate references.\n3. Inappropriate Citation: Reference 46 does not have a publication year etc. It should be cited\nappropriately.\nResponse\nWe have updated Reference 46 with the complete citation details.\nReviewer #2 (Remarks to the Author)\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nReviewer #3 (Remarks to the Author):\nThe manuscript entitled \"VPS13B regulates mitochondrial fission and mitophagy\" provides\ncompelling evidence that VPS13B is critical in mitochondrial dynamics and quality control,\nparticularly through mediating the transport of PI4P-containing Golgi-derived vesicles to\nmitochondrial fission sites. This research utilizes a combination of confocal microscopy,\nelectron microscopy, biochemical assays, and live-cell imaging, which together provide\nsignificant insights into mitochondrial regulation, especially in the context of Cohen syndrome\n(CS).\nStrengths:\n• The study effectively integrates multiple methodologies, clearly demonstrating the essential\nrole of VPS13B in mitochondrial dynamics and mitophagy.\n• Use of VPS13B knockout (KO) HeLa cells and patient-derived iPSC-neurons enhances the\nrelevance of findings to Cohen syndrome pathology.\n• The manuscript is well-structured, providing clear connections between VPS13B function,\nGolgi-derived PI4P vesicle transport, mitochondrial fission, and cellular quality control\nmechanisms.\n• The rationale and progression of experiments are clearly outlined, facilitating easy\nunderstanding and contextualization within mitochondrial biology and CS.\nAreas Requiring Clarification and Improvement:\nAcknowledgement of Prior Work:\nExplicitly reference and discuss the previous foundational studies by Nagashima et al. (2020)\nthat identified the role of Golgi-derived PI4P-containing vesicles in mitochondrial fission.\nClarify that the current findings represent an extension or further specification of the role of\nVPS13B, rather than a completely novel discovery.\nWe appreciate the reviewer’s reminder to acknowledge the foundational work by Nagashima\net al. (2020). In the revised Result, we have explicitly cited that study. Nagashima et al.\ndemonstrated that Golgi-derived vesicles enriched in PI4P are recruited to mitochondrial\ndivision sites and promote the late stages of Drp1‑mediated fission. In other words, it was\nknown that PI4P‑containing TGN vesicles accumulate at mitochondrial constriction sites to\ndrive final fission events. However, the molecular mechanism by which these Golgi‑derived\nvesicles are delivered to mitochondria remained unclear in that prior model.\nBuilding on the PI4P‑vesicle model of Nagashima et al., we identify VPS13B as the coupling\nfactor that delivers PI4P‑enriched Golgi vesicles to Drp1‑positive mitochondrial constrictions.\nVPS13B accumulates at Mfn2‑positive ER-mitochondria contacts and fission sites via a\nC‑terminal MLD that is necessary and, in fragment form, sufficient for targeting. Event‑based\nlive‑cell imaging shows that ORP9‑PH-marked PI4P vesicles co‑arrive with VPS13B and\nreach mitochondria prior to fission; these contacts are reduced in VPS13B‑deficient cells and\nrestored by full‑length VPS13B but not by ΔMLD. Together with proximity ligation assays\n(PLA), mitochondrial fractionation (±CCCP), and PI4P binding by the VPS13B PH domain,\nthese data support a model in which VPS13B recruits/retains PI4P‑positive Golgi vesicles at\nnascent constrictions to supply/organize lipids required for membrane severing. Consistent\nwith this placement, VPS13B loss or acute PI4P depletion lowers fission rates and stalls fission\nwithout altering Drp1 recruitment or phosphorylation occupancy, indicating that VPS13B acts\ndownstream of, or in parallel with, Drp1 assembly to drive late‑stage mitochondrial division.\nBy framing our work as a functional and mechanistic specification of the PI4P‑vesicle model,\nwe emphasize that our study **extends—rather than duplicates—**current understanding of\norganelle cross‑talk in mitochondrial division. This advance is clinically relevant to Cohen\nsyndrome, in which loss of VPS13B compromises vesicle‑mediated lipid delivery to fission sites,\nleading to mitochondrial hyperfusion, depolarization, and impaired mitophagy. We thank the\nreviewer for prompting us to articulate these links to prior research.\nQuantitative Analysis of Mitochondrial Morphology:\nThe current manual scoring method for mitochondrial morphology is subjective and\npotentially biased. Provide explicit criteria, including how morphologies were defined,\nblinding of the analyst, and reproducibility across multiple replicates.\nSupplement manual scoring with quantitative automated or semi-automated analyses of\nmitochondrial shape parameters such as area, length, and connectivity.\nResponse\nWe thank the reviewer for this thoughtful and important suggestion. We agree that\ncomprehensive quantification and objective metrics are critical for validating mitochondrial\nmorphology phenotypes. In our original study, manual scoring was conducted using clearly\ndefined morphological criteria, with the following classifications:\nAll scoring was performed in a blinded fashion, with the analyst unaware of genotype\ncondition. These assessments were conducted across three independent biological replicates,\nand the observed patterns were highly reproducible.\nIn response to the reviewer’s suggestion, we employed semi-automated image analysis with\nFiji (ImageJ) to supplement our manual scoring. For the quantification of key mitochondrial\nshape parameters, TOM20-stained confocal images were binarized via an automatic threshold\nto effectively segment the mitochondrial regions of interest (ROIs). These ROIs were\nsubsequently analyzed using the “Analyze Particles” function (Revised Fig. 1a-c). In particular,\nwe measured:\n• Mitochondrial area (cross-sectional area of each mitochondrial object)\n• Perimeter (outline length of each mitochondrion)\nIn Revised Fig. 1l and Revised Supplementary Fig. 3e., For the quantification of key\nmitochondrial shape parameters, TOM20-stained confocal images were binarized via an\nautomatic threshold to effectively segment the mitochondrial regions of interest (ROIs). These\nROIs were subsequently analyzed using the “Analyze Particles” function.\n• Fragmented: Defined as a cell containing 20 or more mitochondrial skeletons with zero\n8\nbranches.\n• Hyperfused: Defined as a cell containing at least one mitochondrial skeleton with 50 or\nmore branches.\n• Intermediate: Defined as a cell that did not meet the criteria for either 'Hyperfused' or\n'Fragmented'.\nAfter classification, the proportion (%) of cells belonging to each phenotype (Hyperfused,\nFragmented, and Intermediated) was calculated for experimental groups to compare the\ndifferences in their mitochondrial morphology distributions.\nThis quantitative analysis revealed a significant increase in both the average mitochondrial\narea and perimeter in VPS13B KO cells compared to WT controls, consistent with a hyperfused\nphenotype (i.e. larger, more expanded mitochondria). Furthermore, skeleton analysis showed\nthat mitochondria in VPS13B KO cells form more highly connected networks, with significantly\ngreater numbers of branch points and longer total network length per network than those in\nWT cells.\nThese findings reinforce our manual classification of the VPS13B KO mitochondria as\n“hyperfused.” Importantly, when VPS13B function was restored in KO cells (rescue\nexperiments), the mitochondrial morphology parameters (area, perimeter, and branching\nmeasures) reverted toward wild-type levels. This rescue result, observed consistently across\nmultiple independent cultures (3-5 replicates), confirms that the hyperfused network phenotype\nis specifically attributable to the loss of VPS13B.\nFinally, we note that the automated quantitative results align closely with our blinded manual\nscoring, underscoring the validity and robustness of the observed mitochondrial morphology\nphenotypes. We have now integrated these new data and their analysis into the revised Results\nand Methods sections of the manuscript, including all relevant statistical details.\nDirect Interaction Evidence:\nCurrently, no direct evidence of physical binding between VPS13B and PI4P is shown.\nIncluding assays such as lipid pull-downs or proximity ligation assays would significantly\nenhance the mechanistic insights.\nWe appreciate the reviewer’s thoughtful suggestion and agree that direct biochemical support\nfor a VPS13B-PI4P interaction strengthens our model. In the revised manuscript we have\nadded new binding data and integrated it with our cell‑based genetic and imaging evidence:\n1. Direct lipid binding :\nWe performed a protein-lipid overlay (PIP‑strip) assay with a recombinant GST‑fused\nVPS13B C‑terminal fragment (amino acids 3683-3997) that encompasses the predicted PH\ndomain. The fusion protein bound PI4P and, to varying degrees, other phosphoinositides,\nwhereas GST alone showed no detectable binding (Revised Supplementary Fig. 3f-h). These\ndata demonstrate direct binding of a VPS13B C‑terminal module to PI4P, providing\nbiochemical competence for PI4P recognition.\n2. Cellular specificity for PI4P in the fission pathway:\nPI4KB knockdown reduced ORP9‑PH signal and phenocopied VPS13B loss by lowering\nfission rates and stalling fission without altering Drp1 recruitment or phosphorylation\noccupancy (Revised Supplementary Fig. 4), placing PI4P—and thus PI4P‑positive vesicles—\ndownstream of Drp1 assembly.\n3. Event‑based vesicle engagement at constrictions:\nIn three‑color live imaging (ORP9‑PH, TGN46, and MitoTracker), PI4P contact, TGN\ncontact, and dual PI4P-TGN contact at bona fide fission events were significantly reduced in\nVPS13B‑KO and rescued by VPS13B‑FL but not ΔMLD (Revised Fig. 4c-f; Revised\nSupplementary Movies 1, 2).\n4. Co‑arrival with VPS13B at fission sites:\nVPS13B and ORP9‑PH-positive vesicles co‑appeared at ~30.1% of fission events, and in\n~93.8% of those, the vesicle reached mitochondria before fission (Revised Fig. 4g, k),\nconsistent with targeted delivery during constriction maturation.\n10\n5. Targeting mechanism that positions VPS13B at the right place:\nVPS13B localizes to Mfn2‑positive ER-mitochondria contacts via its C‑terminal MLD, which\nis necessary (and, in fragment form, sufficient) for mitochondrial/MAM targeting (Revised\nFig. 1i, j). CCCP increases VPS13B-Mfn2 PLA puncta and VPS13B in the mitochondrial\nfraction (Revised Fig. 3c-f), indicating stress‑induced recruitment to the relevant sites.\nThe PIP‑strip provides direct biochemical evidence that a VPS13B C‑terminal module binds\nPI4P. Because overlay assays can reveal broader phosphoinositide recognition, we anchor\nphysiological specificity to PI4P using our in‑cell dependencies (PI4KB knockdown),\nevent‑based vesicle tracking, and MLD‑dependent rescue at Drp1‑positive constrictions.\nTogether, these orthogonal data support a model in which VPS13B, via its MLD‑targeted\nC‑terminus, recruits/retains PI4P‑positive Golgi vesicles at late fission sites to supply/organize\nlipids required for membrane severing. We have added the new biochemical data as Revised\nSupplementary Fig. 3f-h and detailed the protocol. Corresponding text in Results and\nDiscussion now integrates these findings with the PI4KB‑dependency, event‑based imaging,\nand domain‑mapping evidence to provide a cohesive mechanistic framework.\nElectron Microscopy Interpretation:\nElectron microscopy alone provides limited support for mitochondrial hyperfusion claims due\nto the difficulty in visualizing complete mitochondrial networks in thin EM sections.\nQuantitative morphometric analyses of mitochondria should accompany EM data.\nResponse:\nWe thank the reviewer for this important observation. We agree that electron microscopy (EM),\nwhile powerful for ultrastructural resolution, is inherently limited in capturing the complete\nmitochondrial network connectivity due to the narrow z-depth of thin sections. To address this\nlimitation, we have complemented our qualitative EM analysis with quantitative morphometric\nmeasurements of mitochondrial profiles, providing a more rigorous and objective evaluation\nof mitochondrial architecture in wild-type (WT) vs. VPS13B knockout (KO) HeLa cells.\nSpecifically, we analyzed high-resolution EM images by measuring the following\nmitochondrial morphological parameters (Revised Fig. 1d-f):\n11\n• Cross-sectional area of individual mitochondrial profiles\n• Perimeter of individual mitochondrial profiles\nQuantification was performed by manually tracing mitochondrial outlines in multiple non-\noverlapping fields for each condition (with n ≥ 20 mitochondria per condition in each of three\nindependent experiments). The analysis revealed that mitochondria in VPS13B KO cells have\na significantly larger cross-sectional area and a longer perimeter compared to those in WT\ncells (p < 0.001 for both, the two-tailed Mann-Whitney test). These increases in size and outline\nlength are consistent with an elongated and expanded morphology of mitochondrial cross-\nsections in the KO, supporting the notion of a hyperfused mitochondrial state. All quantitative\nresults, including statistical methods and sample sizes, are now reported in the Revised Figure\n1d-f and described in the Methods section.\nWhile we acknowledge that EM of thin sections cannot reconstruct the full 3D mitochondrial\nnetwork, the observed increase in the size and shape complexity of individual mitochondrial\nprofiles in VPS13B-deficient cells provides strong supporting evidence for a hyperfused\nmitochondrial phenotype. Importantly, these EM-based morphometric findings are in excellent\nagreement with our confocal microscopy data (including skeletonized TOM20 network analysis)\nand automated live-cell mitochondrial metrics, all of which independently indicate a shift\ntoward a more fused, interconnected mitochondrial network in the absence of VPS13B.\nWe appreciate the reviewer’s recommendation to strengthen the rigor of our EM analysis. By\nincorporating these new quantitative morphometric data, we have significantly bolstered our\nconclusion that VPS13B is required for maintaining proper mitochondrial fission and network\nbalance, as VPS13B loss leads to a measurable and statistically significant hyperfusion of the\nmitochondrial network.\nFRAP Experiments:\nClarify and justify the interpretation of reduced fluorescence recovery observed in FRAP\nassays. Specify whether this represents impaired mitochondrial fusion, reduced mitochondrial\nmobility, or a combination of both.\nWe thank the reviewer for the thoughtful comment regarding interpretation of our FRAP assay.\nWe agree that fluorescence recovery depends on the probe and imaging context and, by itself,\ndoes not isolate a single biological process.\nIn our study, we performed FRAP on mRFP-TOM20, an outer mitochondrial membrane\n(OMM) protein, placing ROIs exclusively on continuous mitochondrial tubules (Revised\nFig. 2c-e). Under these conditions, FRAP primarily reports lateral diffusion of TOM20 within\nthe OMM and continuity‑dependent exchange between bleached and adjacent unbleached\nsegments of the same mitochondrial network. It does not quantify matrix content mixing and\ntherefore is not a direct readout of mitochondrial fusion frequency. Because the probe is\nOMM‑localized, the measurement is not directly driven by membrane potential (ΔΨm), which\nwe assessed independently with TMRM (Revised Fig. 2a, b). To minimize confounders,\nbleaching depth and acquisition settings were matched across conditions, image series were\nscreened to exclude organelle displacement or focus drift, and recovery was quantified as\nmobile fraction and half‑time (t½) with standard photobleach corrections.\nWe demonstrated that VPS13B KO cells showed a significantly reduced mobile fraction and\nprolonged t½ of TOM20 recovery relative to WT, indicating dampened OMM mobility and\nimpaired continuity‑dependent exchange within the network (Revised Fig. 2c-e). Re‑expression\nof full‑length VPS13B (VPS13B‑FL) restored both parameters to WT‑like values, whereas\nVPS13B‑ΔMLD did not; recovery remained KO‑like (Revised Fig. 2c-e). These effects were\nstatistically significant and reproduced across ≥3 independent cultures; details are provided in\nthe figure legends and Methods. The reduced fluorescence recovery observed in our FRAP\nassays primarily reflects decreased OMM mobility/continuity, not a direct reduction in\nmitochondrial fusion. While we cannot entirely exclude a contribution from fusion, the FRAP\nconfiguration used here does not isolate fusion events; instead, the data point to altered OMM\nfluidity/organization, consistent with local diffusion barriers at maturing constrictions.\nThe FRAP phenotype and its selective rescue by VPS13B‑FL (but not ΔMLD) align with\nindependent readouts throughout the manuscript:\n13\n1. Morphology: hyperfused/elongated networks with increased area/perimeter (EM and\nconfocal; Revised Fig. 1a-f, k, l).\n2. Bioenergetics: reduced ΔΨm by TMRM (Revised Fig. 2a, b).\n3. Event‑based fission dynamics: reduced fission rate and stalled fission in VPS13B KO or\nPI4KB‑depleted cells, without changes in Drp1 recruitment, colocalization, or\nphosphorylation occupancy (Revised Fig. 4; Revised Supplementary Fig. 4).\n4. Vesicle engagement: MLD‑dependent recruitment/retention of PI4P‑positive TGN vesicles\nat bona fide fission sites; VPS13B-PI4P vesicle co‑arrival prior to fission (Revised Fig. 4g-k).\n5. Targeting mechanism: MLD‑dependent VPS13B localization to Mfn2‑positive ER-\nmitochondria contacts, enhanced by CCCP (PLA and fractionation; Revised Fig. 1i, j, Revised\nFig. 3e, f).\n6. Mitophagy: Impaired PINK1/Parkin mitophagic flux in KO and rescued by VPS13B‑FL\nbut not by VPS13B‑ΔMLD (Revised Fig. 3a,b).\nTogether, these data support a model in which VPS13B, via its MLD, supplies/organizes\nPI4P‑dependent lipids at late-stage constrictions, thereby maintaining local OMM\nfluidity/continuity needed for membrane severing; when this process fails, fission stalls despite\nintact Drp1 assembly, and FRAP recovery diminishes.\nRescue Experiments:\nProvide clear justification for performing VPS13B-ΔMLD experiments only in WT and not\nin VPS13B KO cells. Ideally, repeat these experiments in VPS13B KO cells to strengthen the\nconclusions regarding the MLD domain – does the MLD mutant rescue the fission rate defect\nin KO cells?\nWe thank the reviewer for this insightful suggestion. We examined VPS13B‑FL / ΔMLD in KO\ncells to test; indeed, FL overexpression induced mitophagic flux and reduced the hyperfused\nmitochondrial morphology and fission rate relative to VPS13B‑ΔMLD.\n1. Fission rate and event‑based metrics (KO rescue):\nIn VPS13B‑KO cells, the normalized fission rate was significantly reduced relative to WT\n(Revised Fig. 4a, b). Re‑expression of full‑length VPS13B (VPS13B‑FL) restored the fission\nrate to WT‑like levels, whereas VPS13B‑ΔMLD failed to rescue (Revised Fig. 4g-k). At bona\nfide fission events, PI4P contact, TGN contact, and dual PI4P-TGN contact were reduced in\nKO, restored by VPS13B‑FL, and not rescued by ΔMLD (Revised Fig. 4g-k). Consistently,\nVPS13B‑FL enriched at a substantial fraction of fission sites, while ΔMLD showed no\nenrichment (Revised Fig. 4i), and co‑localization of PI4P and VPS13B at the same\nconstrictions was present with VPS13B‑FL but absent with ΔMLD (Revised Fig. 4k).\n2. Placement relative to Drp1 (KO rescue):\nUsing three‑color imaging (MitoTracker, VPS13B-EGFP, BFP-Drp1), we quantified bona\nfide fission events in the KO background. VPS13B‑FL rescued (i) the fission rate, (ii) Drp1\ncontact at mitofission, (iii) VPS13B contact at mitofission, and (iv) Drp1-VPS13B co‑contact\nto WT‑like levels, whereas ΔMLD did not rescue any of these readouts (Revised Fig. 5c-g).\nNotably, Drp1 recruitment and phosphorylation occupancy were unchanged in VPS13B KO\ncells (Revised Supplementary Fig. 4), placing VPS13B‑MLD-dependent function downstream\nof Drp1 assembly at a late, lipid‑dependent step.\n3. Targeting mechanism (necessary/sufficient):\nDomain mapping showed that the C‑terminal MLD‑containing region is sufficient for\nmitochondrial targeting, while ΔMLD fails to localize to mitochondria (Revised Fig. 1h). PLA\nfor VPS13B–Mfn2 yielded abundant puncta in WT and KO+VPS13B‑FL, but near‑background\nin KO and KO+ΔMLD (Revised Fig. 1i, j). CCCP increased VPS13B-Mfn2 PLA and VPS13B\nin the mitochondrial fraction (Revised Fig. 3c–f), indicating stress‑induced recruitment to\nMfn2‑positive ER-mitochondria contacts.\n15\n4. OMM dynamics (FRAP in KO rescue):\nTOM20‑FRAP on continuous tubules revealed a reduced mobile fraction and slower recovery\n(t½) in KO vs WT, rescued by VPS13B‑FL but not by ΔMLD (Revised Fig. 2c-e). Given that\nTOM20‑FRAP reports OMM lateral diffusion/continuity, these data support impaired OMM\nmembrane dynamics when MLD‑dependent targeting is lost.\n5. Morphology and bioenergetics (scope):\nVPS13B KO cells display hyperfused/elongated mitochondria with increased\narea/perimeter(Revised Fig. 1a-c) and reduced ΔΨm; VPS13B‑FL rescues both morphology\nand TMRM readouts (Revised Fig. 2a, b).\nTogether, these KO‑background rescue experiments demonstrate that the MLD domain is\nessential for VPS13B’s function at Drp1‑positive constrictions: it is required for targeting to\nMfn2‑positive ER-mitochondria contacts, for recruitment/retention of PI4P‑positive Golgi\nvesicles at fission sites, for maintaining local OMM dynamics, and for restoring fission when\nVPS13B is absent. Accordingly, the revised manuscript now highlights:\n• Revised Results: explicit KO rescues with VPS13B‑FL versus ΔMLD for fission rate and\nevent‑based vesicle metrics (Revised Fig. 4g-k), Drp1/VPS13B co‑localize (Revised\nFig. 5c-g), targeting and PLA (Revised Fig. 1i, j), and FRAP (Revised Fig. 2c-e).\n• Discussion: the VPS13B-MLD/PI4P module functions downstream of Drp1 to\nsupply/organize lipids at late constrictions, explaining fission failure in KO/ VPS13B-\nΔMLD and full rescue by VPS13B‑FL.\nWe are grateful to the reviewer for prompting these KO‑background rescues, which\nsubstantially strengthen the conclusion that the MLD domain is indispensable for VPS13B’s\nrole in coupling PI4P‑vesicle delivery to mitochondrial fission.\nLocalization and Imaging Analysis:\nmore rigorous quantitative analyses such as Pearson’s correlation coefficients or colocalization\nmetrics.\nResponse\nWe thank the reviewer for this constructive suggestion. In light of the concern about our\noriginal colocalization analysis, we sought a more quantitative and spatially precise method\nto assess the relationship between VPS13B and mitochondria. Pixel-based colocalization\nmetrics (e.g. Pearson’s correlation coefficient) can be insensitive to proteins at organelle\ncontact sites, since such proteins may be adjacent but not overlapping within the diffraction-\nlimited resolution of light microscopy. In fact, as imaging resolution improves and reveals two\nproteins on separate membranes, traditional overlap-based colocalization values can\nparadoxically decrease even if the proteins are functionally associated.\nTo address these limitations, we implemented and quantified in situ proximity ligation assays\n(PLA) between VPS13B (or VPS13B-EGFP) and Mfn2, which report <40 nm proximity\nappropriate for MAM interfaces. The PLA technique provides a fluorescent readout only when\ntwo target proteins are within ~40 nm of each other, making it ideally suited to detect the close\nproximity at membrane contact sites that falls below the resolution of conventional confocal\nmicroscopy. This approach thus offers a rigorous quantitative measure of VPS13B’s proximity\nto mitochondria (via Mfn2) rather than relying solely on overlapping pixel intensity.\nAccordingly, we provide the following experimental results:\n1. PLA puncta per cell (Revised Fig. 1i, j): Robust VPS13B–Mfn2 PLA signals in WT cells\nfall to near‑background in VPS13B‑KO, are restored by VPS13B‑FL re‑expression, and remain\nlow with VPS13B‑ΔMLD. These data demonstrate that MLD is necessary for VPS13B’s\nresidency at Mfn2‑positive ER–mitochondria contacts.\n2. Stress‑dependent recruitment (Revised Fig. 3e, f): CCCP significantly increases VPS13B–\nMfn2 PLA puncta in WT, indicating dynamic engagement under mitochondrial stress.\n17\n3. Biochemical support (Revised Fig. 3c, d): Mitochondrial fractionation (±CCCP) shows\nCCCP‑dependent enrichment of VPS13B in the mitochondrial fraction, concordant with the\nPLA results.\n4. Targeting sufficiency/necessity (Revised Fig. 1g-j): Live‑cell imaging shows that\nVPS13B‑FL and the C‑terminal DHL–PH/MLD region enrich at TOM20‑positive\nmitochondria, whereas VPS13B‑ΔMLD fails to accumulate—consistent with the PLA‑based\nrequirement for the MLD.\nTogether, these complementary quantitative and mechanistic analyses provide strong\nevidence that the MLD domain is essential for VPS13B’s close association with mitochondria,\nvalidating its localization at Mfn2-positive ER–mitochondria contact sites beyond\nconventional colocalization metrics.\nQuantify data presented in Figure 4J regarding the association between VPS13B and Drp1\nduring mitochondrial fission events.\nResponse\nWe thank the reviewer for this helpful suggestion. In the revised manuscript, we now quantify\nthe co‑occurrence of VPS13B and Drp1 at bona fide mitochondrial fission sites using live‑cell\nconfocal time‑lapse imaging of cells co‑expressing VPS13B–EGFP and BFP–Drp1 with\nMitoTracker to define the mitochondrial context. For each fission event, we assessed whether\nVPS13B and Drp1 were simultaneously present at the constriction during a pre‑fission window\n(Methods), and we report the metric “Drp1–VPS13B co‑contact at mito fission (%)” on a\nper‑event basis.\n1. Event‑based co‑localization quantification (renumbered; Revised Fig. 5c-g): We report\nthe percentage of fission events that show co‑recruitment of VPS13B and Drp1 at the same\nconstriction site within the prespecified temporal window. Results are displayed as per‑cell\n18\nscatter with group means ± SEM, and statistical testing accounts for multiple events per cell\n(Methods).\n2. Condition dependence: In VPS13B‑KO cells rescued with full‑length VPS13B\n(VPS13B‑FL), the co‑localization frequency is higher than VPS13B‑KO + VPS13B‑ΔMLD,\nindicating that the MLD is required for VPS13B’s association with Drp1‑positive constrictions.\nExact percentages, n (events/cells), and statistical details are provided in the Revised Fig. 5\nlegend and Methods.\n3. Related fission‑site metrics (Revised Fig. 5c-g): In the same datasets we also quantify (i)\nDrp1 contact at mitofission (%) and (ii) VPS13B contact at mitofission (%), as well as the\noverall fission rate. VPS13B‑FL rescues all readouts to WT‑like values, whereas ΔMLD fails\nto rescue any metric—consistent with the MLD’s essential role at the division site.\nThese quantitative data align with our independent event‑based analyses showing that\nVPS13B‑FL (but not ΔMLD) enriches at fission sites (Revised Fig. 4i), that PI4P‑positive TGN\nvesicles engage the same sites and co‑occur with VPS13B prior to fission (Revised Fig. 4c-k),\nand that Drp1 recruitment/phosphorylation occupancy is not the limiting factor when fission\nstalls (Revised Supplementary Fig. 4). Together, the new co‑contact quantification\nsubstantiates that VPS13B’s MLD‑dependent targeting is required for VPS13B to associate\nwith Drp1‑positive constrictions and to support productive fission.\nTMRM Mitochondrial Membrane Potential Measurements:\nNormalize mitochondrial membrane potential measurements to a mitochondrial mass marker\nto accurately account for potential differences in mitochondrial content between experimental\nconditions.\nResponse\nWe appreciate the reviewer’s emphasis on normalizing mitochondrial membrane potential\n(ΔΨm) to a mitochondrial mass marker. In line with this, our ΔΨm analyses are based on\nTMRM intensity normalized to the MitoTracker‑positive area on a per‑cell basis:\n19\n1. HeLa (WT vs VPS13B‑KO; Revised Fig. 2a, b): As reported in the manuscript, we\nquantified ΔΨm as integrated TMRM fluorescence divided by the MitoTracker‑positive area\n(TMRM/MitoTracker). This normalized metric revealed a significant reduction in ΔΨm in\nVPS13B‑KO cells relative to WT, and re‑expression of full‑length VPS13B in the KO\nbackground restored the normalized TMRM signal toward WT levels. These results directly\naddress mitochondrial content differences. We note that JC‑1 was used only as an initial survey;\nbecause JC‑1 can be influenced by network topology and mass, all conclusions rely on the\nTMRM/MitoTracker normalization.\n2. CS iPSC‑derived neurons (Revised Fig. 6c, d): We applied the same normalization\npipeline to patient‑derived neurons, quantifying per‑cell TMRM/MitoTracker. The normalized\nΔΨm is significantly reduced in CS neurons compared with controls, confirming that the\ndepolarization persists after accounting for mitochondrial content. Representative images and\nper‑cell quantification across independent cultures are provided in Revised Fig. 6c, d;\nstatistical details (n, test, p values) are reported in the figure legend and Methods.\nTogether, these mass‑normalized ΔΨm measurements substantiate that mitochondrial\ndepolarization in VPS13B‑deficient cells and CS neurons reflects a true functional defect\nrather than differences in mitochondrial abundance. These findings are also concordant with\nour independent readouts—impaired fission dynamics, hyperfusion, and mitophagy deficits—\nand with genetic rescue by full‑length VPS13B.\nClearly distinguish individual control and patient-derived iPSC lines in quantifications to\nassess consistency within groups.\nResponse:\nWe appreciate the reviewer’s emphasis on assessing biological variability across independent\niPSC lines. In the original submission, we pooled induced‑neuron (iN) data by condition\n(Control vs CS) to highlight group‑level effects. We have now reanalyzed and replotted all\niPSC‑based quantifications on a per‑line basis—three control lines (CTL1–3) and two CS\n20\npatient lines (CS1, 2)—so that line‑to‑line consistency is explicitly visible (Revised Fig. 6 and\nRevised Supplementary Fig. 5).\nFor the ΔΨm analysis, we implemented the reviewer’s normalization request and quantified\nTMRM intensity normalized to MitoTracker‑positive mitochondrial area per cell (TMRM /\nMitoTracker‑area). Across three independent differentiations per line, each CS line consistently\nexhibited lower normalized ΔΨm than every control line. To ensure appropriate inference, we\nsummarized line‑level means per differentiation and tested the effect of diagnosis (Control vs\nCS) at the line level; the diagnosis effect remained significant after accounting for between‑line\nvariability (statistical details in Methods).\nFor transparency, we show per‑line distributions (single‑cell jitter overlaid with line\nmeans ± SEM) and report n (cells and differentiations) in the figure legend. We applied the\nsame per‑line stratification to other iPSC‑based readouts in Revised Fig. 6 and Revised\nSupplementary Fig. 5 (e.g., network morphology, Drp1, PINK1 and Mfn2 puncta), which\nlikewise show within‑line reproducibility and between‑group separation.\nThese revisions improve interpretability and strengthen the conclusion that CS iPSC‑derived\nneurons exhibit a bona fide reduction in ΔΨm per unit mitochondrial mass, rather than an\nartifact of pooled analysis or differences in mitochondrial content.\nSupplemental and Statistical Information\nProvide clearer labeling and explicit statistical information (e.g., sample sizes, specific tests\nperformed, and p-values) across main and supplemental figures.\nInclude missing supplementary data and clarify the discrepancies in labeling (e.g., Myc-\nParkin vs. GFP-Parkin).\nResponse\nWe thank the reviewer for this important comment regarding the clarity and completeness of\nour figure annotations and statistical reporting. We recognize that rigorous and transparent\npresentation of data is essential for interpretability and reproducibility. In response, we have\nmade the following revisions to both the main and supplementary figures:\n1. Enhanced Statistical Reporting in Figure Legends\n• All figure legends – including those for both main text and supplementary figures – have\nbeen revised to include more explicit statistical information. In particular, each legend\nnow clearly reports:\n• Sample Sizes: The number of biological replicates (n) underlying each experiment or\nquantification is explicitly stated.\n• Statistical Tests: The specific statistical tests used for each comparison are identified in\neach Figure.\n• Significance Values: The resulting p-values or significance thresholds are provided.\n2. We have audited and corrected all construct labels in the figures to eliminate any\ninconsistencies. In particular, the usage of Myc-tagged versus GFP-tagged Parkin is now\nclearly delineated:\n• Myc-Parkin – Used in the confocal microscopy-based mitophagy assays (as shown in\nRevised Fig. 3a).\n• GFP-Parkin – Used in the immunoblot-based mitophagy assays and in subcellular\nlocalization experiments (as shown in Revised Fig. 3c, d).\n3. Completion of Supplementary Data and Figure Formatting\nWe have also addressed the issues regarding supplementary data and figure formatting to\nimprove clarity and completeness:\n• Inclusion of Missing Data: Previously omitted or ambiguously referenced supplementary\ndata have now been included. For example, we have added the control blots and additional\nquantifications that were missing from the initial submission of the supplementary figures.\n• Improved Layout: The layout and ordering of the supplementary figures have been\nreorganized to follow a more logical flow. Each supplementary figure is now clearly\naligned with its corresponding main text reference, making it easier to navigate between\nthe main and supplemental data.\nOverall, these revisions significantly improve the clarity, consistency, and transparency of\nour data presentation. Both the main and supplementary figures now contain complete\ninformation and accurate labeling, fully addressing the reviewer’s concerns.\nAdditional Critical Points\nClarify the rationale behind using two different fixation protocols in immunofluorescence\nmethodology.\nResponse\nWe thank the reviewer for drawing attention to the apparent use of two fixation methods. In\ngeneral, different immunofluorescence fixation protocols can be chosen to optimize antigen\npreservation: cross-linking fixatives like paraformaldehyde (PFA) preserve cellular structures\nand are widely recommended (4% formaldehyde/PFA is a common standard, whereas alcohol-\nbased fixatives like methanol precipitate and denature proteins, which can sometimes expose\nepitopes that PFA might mask (making methanol advantageous for certain antibodies).\nIn our study, however, we did not intentionally use two different fixation protocols – all\nimmunofluorescence experiments were performed with a single consistent method, 4% PFA in\nPBS for 15 minutes at room temperature. The mention of methanol fixation in the Methods\nsection was a residual text from a previous version of the protocol (used in unrelated pilot\nexperiments) and does not reflect the procedure used for the data presented here. We have now\nremoved the reference to methanol and corrected the Methods to avoid this confusion. The\nrelevant section has been revised to read:\n23\n“Cells were plated on glass coverslips and grown for 24 hours before drug treatment. After\ntreatment"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors addressed all my requests. PLA assay addressed my concern sufficiently. I have no further requests.", "reviewer_3": "Thank you for the thorough revisions and the detailed responses to the reviewers' comments. The manuscript is significantly improved, and the new data have strengthened the core claims of the paper. The addition of the PLA assays, fractionation, and rescue experiments has substantially bolstered the conclusions regarding the MLD's role in VPS13B's mitochondrial association and function.\nI have two remaining minor points that I believe can be addressed through textual revisions rather than new experiments.\n1. Clarification of VPS13B Localization at ER-Mitochondria Contact Sites (MAMs)\nI appreciate the new experiments added to address the concerns about VPS13B's mitochondrial localization. The new data", "reviewer_particularly": "n* The proximity ligation assay (PLA) between VPS13B and Mfn2 (Revised Fig. 1i, j);\n* The mitochondrial fractionation data showing CCCP-inducible enrichment (Revised Fig. 3c, d);\n* The live-cell imaging of VPS13B at fission sites (Revised Fig. 4, 5);\n...collectively provide strong, quantitative evidence for an MLD-dependent association of VPS13B with Mfn2-positive mitochondria. This is a great improvement.\nMy minor concern is one of over-interpretation. The authors repeatedly conclude that VPS13B localizes specifically to \"ER-mitochondria contacts\" or \"MAMs.\" While the Mfn2 PLA data is suggestive of this, the data does not definitively *prove* localization at a tri-partite contact site, as the ER itself was not co-labeled in imaging experiments.\nFurthermore, the \"Mitochondria fractionation\" protocol (line 585) appears to be a standard mitochondrial isolation, not a specific purification of the MAM fraction (which typically involves further steps, like a Percoll gradient, to separate crude mitochondria from the attached ER-membrane-rich fraction).\nSuggestion: This point can be easily addressed by \"softening\" the language. I do not believe further experiments are necessary. The authors could simply revise the text to state that VPS13B localizes to Mfn2-positive sites on the mitochondria. Wording such as \"VPS13B accumulates at Mfn2-positive mitochondria\" or similar would more accurately reflect the data, which cannot formally exclude non-contact-site localization.\n2. Temporal Resolution of Live-Cell Imaging\nThank you for adding the frame rate of the live-cell imaging to the Methods section (line 575). It is noted that the temporal resolution is approximately 30.84 seconds per frame.\nMitochondrial fission is a very rapid process, and there is a concern that this frame rate may be too slow to accurately capture the full dynamics of vesicle engagement and membrane scission, potentially leading to an under-sampling of transient events.\nSuggestion: I am not requesting that the live-imaging experiments be repeated. However, this limitation should be explicitly acknowledged in the manuscript's Discussion or Methods. The authors could briefly state the limitation and justify why they are confident in their analysis—for example, by noting that the co-recruitment of Drp1 (Fig. 5) provides an anchor that increases confidence in the scoring of these bona fide fission events, despite the low temporal resolution.\nBoth points are minor and relate to interpretation and methodological limitations. By addressing them with minor text edits, the manuscript will be further strengthened. I congratulate the authors on a much-improved and compelling study.", "reviewer_4": "General Evaluation\nThe manuscript by Lee et al. presents a thorough and well-executed study identifying VPS13B as a Golgi-derived lipid transport regulator of mitochondrial fission and mitophagy, acting via recruitment of PI4P-enriched vesicles to Drp1-positive sites. The work addresses an important question at the interface of inter-organelle communication, lipid transfer, and mitochondrial quality control. The experiments are generally well-designed and clearly presented. The study provides new mechanistic insight into how Golgi-derived lipids may contribute to mitochondrial fission and extends the biological relevance to Cohen syndrome patient-derived neurons.\nWhile the manuscript has improved substantially compared with the earlier version, several conceptual and mechanistic issues remain. Addressing these would considerably strengthen the impact and clarity of the conclusions.\nMajor Comments\n1. The central claim is that VPS13B regulates mitochondrial fission by recruiting PI4P-enriched Golgi vesicles to Drp1-positive fission sites. However, the mechanism by which PI4P or VPS13B facilitates the membrane constriction or scission step remains undefined. It is still unclear whether VPS13B or PI4P interacts directly with Drp1, or whether they act through modulating local lipid composition, curvature, or recruitment of accessory factors. The authors should test or discuss whether PI4P-enriched vesicles alone, or VPS13B overexpression, are sufficient to promote mitochondrial fission in otherwise unstressed cells. This would help distinguish correlation from causation and clarify whether the PI4P-VPS13B module is instructive or permissive for fission.\n2. Positioning of VPS13B Relative to the Canonical Fission Machinery. The data indicate that Drp1 recruitment is normal in VPS13B-deficient or PI4P-depleted cells, yet fission fails to complete. The authors conclude that VPS13B acts downstream or parallel to Drp1. However, it remains uncertain whether VPS13B contributes to membrane remodeling required for Drp1 constriction, or whether it facilitates Drp1 turnover or disassembly. Co-immunoprecipitation or proximity assays between VPS13B and Drp1 (or known Drp1 cofactors such as Mff, MiD49/51, Fis1) would provide evidence supporting the proposed “late-stage” role.\n3. The manuscript frequently refers to “Golgi-derived, PI4P-positive vesicles,” but their biochemical identity and cargo composition are not defined. Are these TGN-to-endosome transport vesicles, or specialized lipid carriers?\n4. While loss-of-function data are convincing, gain-of-function experiments would significantly strengthen the mechanistic model. For instance, does overexpression of VPS13B (WT or MLD) increase mitochondrial fission rate or promote fragmentation? Can re-introduction of PI4P-enriched vesicles (e.g., by overexpressing PI4KB or ORP9-PH) rescue the fission defect in VPS13B KO cells? These experiments would help determine whether the VPS13B–PI4P axis is rate-limiting for mitochondrial division.\n5. The phenotypes in Cohen syndrome iPSC-derived neurons mirror those of VPS13B KO cells. However, the specific mutations and protein expression levels in these lines are not detailed in the Results. Including these data would help interpret whether these variants represent loss-of-function or hypomorphic alleles. The authors should also discuss whether other VPS13 paralogs (VPS13A/C/D) compensate or are misregulated in patient cells.\nMinor Points\nThe images show that Figures 4A and 4C appear identical, despite being from different experiments. Please clarify."}, "author_response": "We thank the Editor and reviewers for their time and insightful feedback on our manuscript (NCOMMS-24-63124). We have revised the manuscript accordingly to address all concerns. Below we provide a point-by-point response, with Reviewer comments followed by our responses. Changes to the manuscript are indicated with line numbers referring to the revised manuscript\nREVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors addressed all my requests. PLA assay addressed my concern sufficiently. I have no further requests.\nResponse\nWe thank Reviewer 1 for the positive evaluation of our work. We are grateful that they found the study’s findings and implications to be significant.\nReviewer #3 (Remarks to the Author):\nThank you for the thorough revisions and the detailed responses to the reviewers' comments. The manuscript is significantly improved, and the new data have strengthened the core claims of the paper. The addition of the PLA assays, fractionation, and rescue experiments has substantially bolstered the conclusions regarding the MLD's role in VPS13B's mitochondrial association and function.\nI have two remaining minor points that I believe can be addressed through textual revisions rather than new experiments.\n1. Clarification of VPS13B Localization at ER-Mitochondria Contact Sites (MAMs)\nI appreciate the new experiments added to address the concerns about VPS13B's mitochondrial localization. The new data"}, {"round": 3, "reviewer_comments": {"reviewer_1": "Lee et al. investigate the role of VPS13B in mitochondrial fission and mitophagy. Using VPS13B knockout HeLa cells and Cohen Syndrome (CS) patient-derived neurons, the authors show that VPS13B deficiency results in mitochondrial abnormalities. As a mechanistic explanation, they propose that VPS13B mediates PI4P-containing Golgi-derived vesicle transfer to mitochondrial fission sites, which is critical for proper fission. However, the evidence presented does not fully support this conclusion, and additional data are needed to substantiate their claims.\nMajor Points\n1. In line 33, the authors state that “Deletion of the mitochondria-associated ER membrane (MAM) via the MAM localization domain (MLD)...” However, no direct evidence is provided showing that MLD deletion removes the MAM. The author need data directly supporting that MLD deletion disrupts MAM localization.\n2. The use of the OSBP PH domain to visualize PI4P vesicles raises specificity concerns. Since this probe can localize to the Golgi itself, additional evidence is needed to confirm that the probe specifically marks PI4P-containing Golgi-derived vesicles rather than the Golgi more generally.\n3. In Figure 3B, the localization of VPS13B at mitochondria contact sites needs further clarification. The line scan in Figure 3B shows a peak separation of about 1 μm between TOM20 and VPS13B, whereas typical mitochondria contact sites range from 10-30 nm. Additional evidence is needed to demonstrate that VPS13B indeed localizes at mitochondria contact sites.\nMinor Points\n1. Typo: In line 92, “VPS1B” should be corrected to “VPS13B.”\n2. Duplicate References: References 38 and 39 appear to be the same and should be consolidated.\n3. Inappropriate Citation: Reference 46 does not have a publication year etc. It should be cited appropriately.\nI 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.", "reviewer_2": "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.", "reviewer_3": "The manuscript entitled \"VPS13B regulates mitochondrial fission and mitophagy\" provides compelling evidence that VPS13B is critical in mitochondrial dynamics and quality control, particularly through mediating the transport of PI4P-containing Golgi-derived vesicles to mitochondrial fission sites. This research utilizes a combination of confocal microscopy, electron microscopy, biochemical assays, and live-cell imaging, which together provide significant insights into mitochondrial regulation, especially in the context of Cohen syndrome (CS).\nStrengths:\n• The study effectively integrates multiple methodologies, clearly demonstrating the essential role of VPS13B in mitochondrial dynamics and mitophagy.\n• Use of VPS13B knockout (KO) HeLa cells and patient-derived iPSC-neurons enhances the relevance of findings to Cohen syndrome pathology.\n• The manuscript is well-structured, providing clear connections between VPS13B function, Golgi-derived PI4P vesicle transport, mitochondrial fission, and cellular quality control mechanisms.\n• The rationale and progression of experiments are clearly outlined, facilitating easy understanding and contextualization within mitochondrial biology and CS.\nAreas Requiring Clarification and Improvement:\nAcknowledgement of Prior Work:\nExplicitly reference and discuss the previous foundational studies by Nagashima et al. (2020) that identified the role of Golgi-derived PI4P-containing vesicles in mitochondrial fission. Clarify that the current findings represent an extension or further specification of the role of VPS13B, rather than a completely novel discovery.\nQuantitative Analysis of Mitochondrial Morphology:\nThe current manual scoring method for mitochondrial morphology is subjective and potentially biased. Provide explicit criteria, including how morphologies were defined, blinding of the analyst, and reproducibility across multiple replicates. Supplement manual scoring with quantitative automated or semi-automated analyses of mitochondrial shape parameters such as area, length, and connectivity.\nDirect Interaction Evidence:\nCurrently, no direct evidence of physical binding between VPS13B and PI4P is shown. Including assays such as lipid pull-downs or proximity ligation assays would significantly enhance the mechanistic insights.\nElectron Microscopy Interpretation:\nElectron microscopy alone provides limited support for mitochondrial hyperfusion claims due to the difficulty in visualizing complete mitochondrial networks in thin EM sections. Quantitative morphometric analyses of mitochondria should accompany EM data.\nFRAP Experiments:\nClarify and justify the interpretation of reduced fluorescence recovery observed in FRAP assays. Specify whether this represents impaired mitochondrial fusion, reduced mitochondrial mobility, or a combination of both.\nRescue Experiments:\nProvide clear justification for performing VPS13B-ΔMLD experiments only in WT and not in VPS13B KO cells. Ideally, repeat these experiments in VPS13B KO cells to strengthen the conclusions regarding the MLD domain – does the MLD mutant rescue the fission rate defect in KO cells?\nLocalization and Imaging Analysis:\nQuantification of VPS13B mitochondrial localization (Figure 3B) is insufficient. Provide more rigorous quantitative analyses such as Pearson’s correlation coefficients or colocalization metrics.\nQuantify data presented in Figure 4J regarding the association between VPS13B and Drp1 during mitochondrial fission events.\nTMRM Mitochondrial Membrane Potential Measurements:\nNormalize mitochondrial membrane potential measurements to a mitochondrial mass marker to accurately account for potential differences in mitochondrial content between experimental conditions.\nClearly distinguish individual control and patient-derived iPSC lines in quantifications to assess consistency within groups.\nSupplemental and Statistical Information:\nProvide clearer labeling and explicit statistical information (e.g., sample sizes, specific tests performed, and p-values) across main and supplemental figures.\nInclude missing supplementary data and clarify the discrepancies in labeling (e.g., Myc-Parkin vs. GFP-Parkin).\nAdditional Critical Points:\nClarify the rationale behind using two different fixation protocols in immunofluorescence methodology.\nSpecify the frame rates and temporal resolution used in live-cell imaging experiments.\nCombine supplemental figures that are closely related for clearer presentation and interpretation (e.g., Supplemental figures 1H and 3).\nUtilize colorblind-friendly palettes in imaging data for improved accessibility (avoid red-green combinations).\nAddressing these points comprehensively will significantly strengthen the manuscript's clarity, rigor, and impact.", "reviewer_4": "Comments:\nThe manuscript investigates the role of VPS13B in mitochondrial morphology, mitophagy, and Golgi-mitochondria interactions. Using VPS13B knockout (KO) cells and neurons derived from Cohen syndrome (CS) patients, the authors observe mitochondrial hyperfusion, impaired membrane potential, and defective mitophagy. The authors propose that VPS13B facilitates PI4P-containing vesicle transfer to mitochondrial fission sites, influencing fission and mitophagy through interactions with Drp1. While the study presents some interesting phenotypes and raises important questions, the findings are largely preliminary, with significant gaps in experimental evidence and data quality. Furthermore, the proposed mechanisms lack sufficient experimental support, and many claims appear overstated. Below are the detailed comments:\nMajor Issues:\n1. Causality Between VPS13B and Mitochondrial Morphology:\nThe manuscript shows an association between VPS13B deficiency and mitochondrial hyperfusion but fails to establish causality. Rescue experiments using full-length VPS13B or targeted mutants are insufficient or missing in key contexts, such as mitochondrial dynamic, mitophagy defects, or PI4P-vesicle transfer. Additional experiments are needed to conclusively show that VPS13B deficiency directly causes mitochondrial abnormalities.\n2. VPS13B Localization on Mitochondria or Mitochondria-Golgi Contact Sites:\nThe study’s claims about VPS13B localization at Golgi-mitochondria contact sites are not well-supported. The truncation analysis using VPS13B fragments (e.g., MLD and DHL-PH domains) is not convincing due to insufficient evidence of normal VPS13B localization. Advanced techniques such as super-resolution microscopy or co-fractionation of Golgi-mitochondria contact sites would strengthen these claims.\nAdditionally, the mechanism by which VPS13B translocates to these contact sites is speculative and requires further experimental investigation, such as testing whether specific cellular stressors or lipid cues regulate its localization.\n3. Impact of PI4P on Drp1 Recruitment and Mitochondrial Fission:\nThe proposed role of PI4P in Drp1 recruitment and mitochondrial fission is not experimentally validated. The manuscript does not test whether PI4P levels correlate with Drp1 activity, nor does it explore whether PI4P-deficient conditions alter fission dynamics. Experiments such as Drp1 localization or activation assays under PI4P-depleted conditions would provide stronger evidence for this hypothesis.\n4. Data Quality and Quantification:\nMany datasets are of low quality or lack clear quantification:\nImaging data (e.g., vesicle transfer and mitochondrial phenotypes) is often unclear, with low resolution or inadequate annotations, making interpretation challenging.\nQuantification of mitochondrial morphology (e.g., hyperfusion vs. fragmentation) and mitophagy markers (e.g., PINK1 puncta) lacks explicit statistical details such as replicate numbers and p-values.\nFigures require more detailed annotations to clearly connect them to key conclusions. For example, showing direct comparisons between WT and VPS13B KO models in a single frame would improve clarity.\n5. Overstatements About the Data:\nThe manuscript contains numerous overstatements about the conclusions that can be drawn from the data. For example:\n\"VPS13B is essential for mitochondrial fission and mitophagy\" is overstated, as the evidence for VPS13B’s involvement in these processes is largely correlative.\nClaims about \"VPS13B-mediated lipid transfer being critical for mitochondrial function\" are unsupported, as lipid transfer is not directly tested.\nThe discussion often generalizes findings beyond what is justified, particularly when linking VPS13B to CS pathology. For instance, variability in VPS13B mutations across CS patients is not considered, yet the manuscript implies a universal role for VPS13B dysfunction in mitochondrial defects.\nConclusion:\nThe manuscript presents some interesting preliminary findings regarding VPS13B’s role in mitochondrial dynamics and Golgi-mitochondria interactions. However, the study suffers from significant gaps in mechanistic evidence, weak data quality, and overstatements about the conclusions. To strengthen the manuscript:\n• Perform additional rescue experiments and mechanistic validations.\n• Use advanced localization techniques to definitively determine VPS13B’s site of action.\n• Provide rigorous data quantification and improved figure quality.\n• Temper claims to align with the presented data.\nAddressing the above issues is essential to substantiate its findings and improve its overall quality.", "reviewer_5": "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": "The authors addressed all my requests. PLA assay addressed my concern sufficiently. I have no further requests.\nThank you for the thorough revisions and the detailed responses to the reviewers' comments. The manuscript is significantly improved, and the new data have strengthened the core claims of the paper. The addition of the PLA assays, fractionation, and rescue experiments has substantially bolstered the conclusions regarding the MLD's role in VPS13B's mitochondrial association and function.\nI have two remaining minor points that I believe can be addressed through textual revisions rather than new experiments.\n1. Clarification of VPS13B Localization at ER-Mitochondria Contact Sites (MAMs)\nI appreciate the new experiments added to address the concerns about VPS13B's mitochondrial localization. The new data"}]

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