Structural organization of p62 filaments and the cellular ultrastructure of calcium-rich p62-enwrapped lipid droplet cargo

Domain: physics
Published: 2025-11-28
Editorial outcome: minor_revision

Abstract
The selective autophagy receptor p62/SQSTM1 is known to form higher-order filaments in vitro and to undergo liquid-liquid phase separation when mixed with poly-ubiquitin. Here, we determine the full-length cryo-EM structure of p62 and elucidate a structured double helical filament scaffold composed of the PB1-domain associated with the flexible C-terminal part and the solvent-accessible major groove. At different pH values and upon binding to soluble LC3, LC3-conjugated membranes and poly-ubiquitin, we observe p62 filament re-arrangements in the form of structural unwinding, disassembly, lateral association and bundling, respectively. In the cellular environment, under conditions of ATG5 knockdown leading to stalled autophagy, we imaged high-contrast layers consisting of p62 oligomers enwrapping lipid droplets by cryogenic electron tomography in situ, which we identified as calcium as well as phosphorus by compositional spectroscopy analysis. Together, we visualize the cellular ultrastructure of p62 oligomers with high calcium content as a potential early stage of autophagy.

Introduction
Autophagy is a conserved cellular process that degrades and recycles cytoplasmic material such as aggregated proteins, damaged organelles and pathogens to maintain intracellular homeostasis1,2. While autophagy operates at a basal level for routine maintenance, it can also be activated by stress factors like nutrient deprivation, hypoxia and DNA damage3. There are three main autophagy pathways: macroautophagy, microautophagy and chaperone-mediated autophagy. In macroautophagy (from here on simply referred to as autophagy), an autophagosome forms within the cytoplasm to enclose cellular material that is subsequently degraded and recycled after fusing with a lysosome. A series of defined autophagy core machinery complexes mediate the progression of cellular autophagy: Atg1/ULK1 kinase, PI-3 kinase, ATG9 lipid scramblase, the ATG2-ATG18 lipid transfer complex and two ubiquitin-like conjugation systems4. The recognition of cargo can proceed in a non-selective or in a selective manner by either enclosing the bulk stochastically or through specific binding by selective autophagy receptors, respectively5.

Selective autophagy requires cargo receptors that recognize poly-ubiquitinated cargo and interact with Atg8 family proteins6,7. The Atg8 family contains ubiquitin-like proteins: Atg8 in yeast and microtubule-associated protein 1 light chain 3 (LC3), as well as gamma-aminobutyric acid type A receptor-associated protein (GABARAP) in mammals8. The conjugation of Atg8 proteins to the lipid membrane is essential for phagophore membrane expansion1 and is mediated by two autophagy-specific ubiquitin-like conjugation systems: ATG12-ATG5 and the LC3 conjugation system9. Selective autophagy receptors, like p62/SQSTM1 (from here on: p62)10, contain an LC3-interacting region (LIR) motif that facilitates interaction with Atg8 proteins and a ubiquitin-binding domain11,12. At the early stages of autophagy, selective autophagy receptors like p62 thereby bridge ubiquitinated cargo via conjugated Atg8 to the forming autophagosome. Thus far, a series of additional autophagy receptors have been identified with LIR motifs as well as ubiquitin binding abilities in order to confer specificity and redundancy in the cargo recognition and removal13. For instance, for mitophagy, NBR1, optineurin, NDP52 and TAXBP1 have been characterized in addition to p6214. While p62 knock-outs are viable, increased p62 levels have been found to provide long-term benefits and promote proteostasis and longevity in response to stress in C. elegans15.

p62 has a key role in selective autophagy by recognizing poly-ubiquitinated cargo and delivering it to the autophagosome10. Originally, p62 was described as an interaction partner for tyrosine-protein kinase Lck16, while it was also shown to be involved in multiple pathways, including Wnt, Nrf2, mTOR and NF-κB signaling17. In the NF-κB pathway, p62 can interact with MEKK3, MEK5, aPKCs18 and tumor necrosis factor receptor-associated factor 6 (TRAF6) as a mediator in tumorigenesis19. Moreover, p62 actively promotes the spatio-temporal organization of autophagy downstream effectors such as FIP200 at the cargo site20. p62 is a multi-domain protein with a Phox1 and Bem1p (PB1) domain, followed by a ZZ-domain and an intrinsically disordered region (IDR) up to the C-terminal ubiquitin-binding associated (UBA) domain (Fig. 1a). The PB1 domain (1–102) is responsible for p62 polymerization into long flexible filaments21,22, and the short region 103–122 linking PB1 and ZZ domain has been shown to support polymerization21 further supported by the formation of disulfide conjugates23. The PB1 domain was shown to be critical for its function in autophagy, as polymerization-deficient mutations excluded p62 from the autophagosome formation site24. The IDR contains several relevant interaction motifs, including the LIR and KEAP1-interacting region (KIR) motif25. The C-terminal UBA domain captures poly-ubiquitinated cargo26. The autophagy receptor NBR1 is structurally the most similar to p62 amongst the autophagy receptors, as it is closely evolutionarily related and directly interacts with p62 via its PB1 domain, albeit it is unable to form higher-order oligomers or filaments22,27.

p62 is a well-studied cargo receptor that recognizes and removes various cellular cargoes: aggregated proteins in aggrephagy10,28, damaged peroxisomes in pexophagy29 and lipid droplets in lipophagy30,31. For lipophagy, the size of the removed lipid droplets is smaller than one µm, while larger lipid droplets were shown to be predominantly degraded by lipolysis32. At the same time, lipid droplets are a special cargo in the context of autophagy as they can also serve as a direct or indirect lipid source for the growing phagophore mediated by direct contact in addition to the ATG9A vesicles or ATG2 lipid bridges33–36. Lipid droplets were also found to serve as direct substrates for non-canonical autophagy during prolonged starvation after LC3-conjugation37.

p62 has also been shown to undergo liquid-liquid phase separation38 induced by poly-ubiquitin interaction with multiple polymerized p62 entities in vitro39,40. However, in vivo, p62 phase-separated condensates recruit many autophagy regulators that can affect the mobility of the separated phase and, as a result, autophagy efficiency. For instance, Keap1 over-expression was observed to increase the rigidity of p62 condensates, thus promoting autophagy41. NBR1 enhances p62 condensate formation and poly-ubiquitin affinity42. In addition to binding partners, the oligomeric state of p62 and post-translational modifications can affect the phase separation properties, e.g., as the IDR of p62 harbors the KIR motif that, when post-translationally modified, has been shown to enhance the interaction with Keap143. The quaternary structural organization of p62 presents another level of regulation for cellular phase separation, as phosphorylation of the PB1 domain by PKA was identified and shown to disrupt PB1 domain interactions44.

In order to improve our detailed structural understanding of purified p62, we determined the cryo-electron microscopy (cryo-EM) structure of full-length p62. The structure revealed the double helical organization of an N-terminal PB1-domain scaffold, while the more poorly resolved partially disordered C-terminal part resided in the lumen and the major groove of the assembly. Binding studies of LC3b and poly-ubiquitin showed that p62 filaments can undergo structural re-arrangements of disassembly or filament bundling, respectively. When p62 filaments were added to LC3b-conjugated membranes, they associated laterally and also unwound their regular helical structures into bubbles at physiological pH. Native ultrastructural imaging of p62-positive structures using cryo-electron tomography (cryo-ET) in ATG5-depleted RPE1 cells revealed stalled intermediate autophagy stages of p62-enwrapped lipid droplets. Interestingly, the surrounding p62 layer had a dark contrast and contained high levels of calcium and phosphorus based on energy-dispersive X-ray spectroscopy analysis (EDX). Proximity proteomics experiments showed nearby ER-resident calcium exporters. The high-resolution visualization of p62-enwrapped cargo together with calcium may present an important intermediate state of autophagy initiation.

Results
p62 forms a double-helical filament with a PB1 scaffold and a flexible C-terminus in the major groove
Previously, we determined the three-dimensional (3D) cryo-EM structure of the p62-PB1 domain arranged in a series of different filamentous assemblies22. In order to advance our structural understanding of full-length human p62, we optimized the previous preparations of full-length p62 for improved homogeneity. As previous studies suggested an effect of pH on the assembly conditions45, we compared the preparations at different pHs ranging from 6 to 8 (Supplementary Fig. 1a). Classifications of small cryo-EM data sets revealed that at pH 6.0 the majority of the filament classes were helically regular while at pH 8.0 a large fraction of them were found unwound resembling an open bubble (Supplementary Fig. 1b). Therefore, we recorded a full cryo-EM data set at pH 6.0 revealing long and flexible filaments with a width of approximately 15 nm (Fig. 1b). Using segmented helical reconstruction46–48, we set out to determine the structure of p62 filaments. Due to pitch heterogeneity between 130 and 160 Å (Fig. 1c, Supplementary Fig. 1c), we refined a more homogeneous subset with a 150 Å pitch and 13.6 units per turn (corresponding to 11.0 Å helical rise and a −26.1° helical rotation), including dihedral symmetry and obtained a 3D reconstruction at 4.5 Å global resolution (Supplementary Fig. 1d, e, Table 1). Consistent with the class averages, the local resolution varied significantly from a well-defined PB1 domain scaffold at 4.2 Å resolution up to over 9 Å for the more flexible regions (Fig. 1d). The cryo-EM structure revealed a left-handed double-helical filament with two antiparallel strands of PB1 domains in the full-length p62 assembly. The atomic model of the PB1 domain of p62 (PDB: 6TGY) fitted well in the best-defined scaffold density supporting β-strand separation (Fig. 1e left). At lower density thresholds, the ZZ domain envelope emerged connected to the PB1 domain, albeit at lower resolution (Fig. 1e right). The densities of the associated C-terminal domains were weak and poorly resolved as they did not follow helical symmetry, presumably due to molecular flexibility with respect to the PB1 strands, which is in agreement with the assigned structurally disordered region in residues 169–33821. Based on our p62 filament structure, we expanded the domains to match the PB1 density to the experimentally observed helical symmetry assembly (Fig. 1f). The resulting p62 double helix architecture is built on the scaffold of the PB1 domain, generating a major groove that can accommodate the flexible C-terminal part.

p62 filament interactions with LC3b and ubiquitin
To experimentally study the interactions of p62 filaments with LC3b and ubiquitin, we employed single-particle fluorescence microscopy in total internal reflection fluorescence (TIRF) mode and negative staining EM. We used the above-characterized preparations of p62 filaments at pH 8 as they were closer to physiological pH than the stable pH 6 filaments formed for structural analysis. Subsequently, we labeled them at lysine residues with multiple Cy5 dyes (p62 filaments-Cy5) while cyan fluorescent protein (CFP) was fused to LC3b (CFP-LC3b). Consistent with the observed p62 filaments, elongated p62 structures were visible in the Cy5 channel, rather mobile and diffusing above the surface in dilute nM concentrations on the coverslip (Fig. 2a, Supplementary Movie 1). Upon addition of equimolar amounts of CFP-LC3b, the same filament became visible in the CFP channel as well (Fig. 2b, Supplementary Movie 2), supporting that LC3b was bound to the p62 filaments. Upon addition of GST-4xUbiquitin (GST-4xUb) to the labeled filaments, we observed a rapid and spontaneous formation of µm-sized condensates that displayed minimal mobility (Fig. 2c, Supplementary Movie 3). Interestingly, the subsequent addition of CFP-LC3b to the formed condensates resulted in the appearance of smaller mobile diffusing fragments (Fig. 2d, Supplementary Movie 4).

The corresponding p62 filament samples in nM concentration were also imaged by negative staining EM. In the presence of CFP-LC3b, p62 filaments exhibited a distinctive decoration in comparison with the filaments alone (Fig. 2e, f, white arrows in insets). A quantitative analysis of the filament length distribution revealed a shift towards shorter filaments for CFP-LC3b (Fig. 2g), suggesting that interaction with LC3b induced depolymerization of p62 filaments. Similarly, in the presence of CFP-LC3b, GST-4xUb-induced condensates were disintegrated into smaller substructures (Fig. 2h, i). In support of previous studies49,50, our single-particle studies in fluorescence and electron microscopy performed in diluted nM concentration showed that poly-ubiquitinated cargoes induce condensate formation, while LC3b can promote disassembly of preformed p62 filaments as well as preformed condensates.

In order to molecularly assess the cryo-EM structure and the presented binding experiments, we generated a multimer AI-prediction of full-length p62 using AlphaFold3 (AF3)51, consisting of ten subunits that formed closed rings mediated by the well-characterized PB1 domain interactions (Supplementary Fig. 2a, Materials and Methods). In an expanded model matching the helical symmetry, the C-terminal UBA domain fits tightly to the major groove next to the PB1 domain that is consistent with the cryo-EM density (Supplementary Fig. 2b). When the model is seen in top view, the PB1 domain scaffold and UBA domain are located at the outer rim of the filament while the ZZ domain is located on the inside. Notably, parts of the IDR, including the LIR and KIR binding motifs, are positioned towards the filament’s outside, supporting the observed molecular access of p62 interacting partners through the major groove of the filament. To assess the interaction accessibility within the p62 filament in more detail, we generated a second p62 multimer model in the above-described way, this time with LC3b known to bind to the LIR motif of p6228. In the resulting model, LC3b is tightly fit in the filament’s major groove surface between the PB1 domain and covering the UBA domain (Supplementary Fig. 2c). The LIR of p62 is positioned outward and bound in the hydrophobic pocket 1 (HP1) and 2 (HP2) of LC3 in agreement with previously determined X-ray structures52. A third p62 AF3-model with bound ubiquitin revealed that ubiquitin also resided in the major groove, suggesting a competing mode between LC3b and ubiquitin binding (Fig. 2j). Together, the AF3-based integrative molecular model of the p62 filaments supports the observed accessibility to binding partners of ubiquitin and LC3b, while it also provides a molecular framework for the observed opposing higher-order structures of filament bundling and disassembly.

p62 filament membrane interactions
In order to more closely mimic the cellular conditions, we performed the interaction studies at higher µM concentrations of p62 and investigated the binding of GST-4xUb and LC3b maleimide-conjugated to small unilamellar vesicles (SUVs). Subsequently, we visualized the natively preserved ultrastructures using cryo-ET. To better characterize the fine ultrastructure of p62 filaments, we recorded tomograms using the Volta phase plate for detailed high-contrast visualization. The resulting 3D tomograms showed 15 nm-wide flexible p62 filaments consistent with repeating pitches of 14 nm along the filament exhibiting regular major groove indentations matching the pH 6 conditions described above (Fig. 3a). Closer inspection revealed overlapping filaments crossing underneath each other, branching filaments where individual PB1 scaffold strands are adopting a close to 90° bent as well as a cross-over reminiscent of Holliday junctions in double-stranded DNA. Due to the underfocus-induced white halo of the phase plate surrounding the filament density, we turned back to conventional cryo-imaging at underfocus and closer to physiological pH 7.4 for the tomograms matching the binding experiments above. Upon addition of GST-4xUb, p62 filaments were found to concentrate locally by forming bundles of parallel filaments supported by filament image segmentations (Fig. 3b). Bundle formation was accompanied by the formation of µm-sized 3D agglomerates that were often too thick to be imaged by cryo-ET while visible by negative staining EM (Supplementary Fig. 3) being consistent with the previous in vitro observations of phase separated p62 bodies49,50. When we incubated p62 filaments with LC3b-conjugated SUVs, we observed unbound filaments as in the p62-only control in proximity to SUVs as well as SUV bound filaments (Fig. 3c). When not bound, the p62 exhibited the typical filament appearance with a regular 15 nm pitch and major groove indentations as well as unwound stretches of the high pH 8 p62 sample alone (Fig. 3d). When p62 filaments were found connected to the SUV density, they were either laterally associated along the membrane of the SUVs or bound through their ends. As in solution, some laterally associated filaments were characterized by densities giving up the regular helical pitch 15 nm repeat, giving rise to more open and unwound stretches. The co-incubation of GST-4xUb and LC3b-conjugated SUVs resulted in less dense network of filament bundles in comparison with GST-4xUb alone as well as SUV bound filaments (Fig. 3e). Together, the cryo-ET visualization of purified p62 revealed the flexible properties of p62 filaments including cross-overs and branching, bundling of parallel p62 filaments in GST-4xUb bound condensates as well as the lateral association of the helical assembly on the surface of LC3b-conjugated membranes.

ATG5 depletion induces p62-positive lipid droplet accumulation in RPE1 cells, primary neurons and brain cortical tissues
In order to further our understanding of the structural organization of the autophagy receptor p62, we decided to visualize the cargo receptor in cells. Capturing rare and transient p62 punctae is challenging; therefore, we turned to a model system of ATG5 depletion that was shown to increase the size and number of p62 punctae28,53 along with increased numbers of lipid droplets54. We stalled autophagy by treating human RPE1 cells stably expressing mCherry-p62 with siRNA against ATG5 for 72 h. To confirm successful ATG5 siRNA knockdown, we performed a Western blot of the RPE1 cells showing a reduction in ATG5 expression levels by 90% in comparison with the control samples (Fig. 4a). To validate the phenotype upon ATG5 knockdown, we used confocal fluorescence microscopy to detect p62 punctae via mCherry-p62, as well as lipid droplets by using the lipid-droplet specific Lipi-Blue dye in conditions of no transfection, and transfection with ATG5 siRNA, respectively (Fig. 4b, c). As a control for our mCherry-p62 overexpression, we imaged lipid droplets in wild type (WT) RPE1 reference cells with Lipi-Blue after no transfection, mock transfection and transfection with ATG5 siRNA using confocal microscopy (Supplementary Fig. 4a–c). These experiments confirmed that ATG5 knockdown in human RPE1 cells resulted in the accumulation of p62-positive punctae and an increase in lipid droplet content, establishing the conditions of a cellular model to study the spatial association between p62 and lipid cargo.

In order to verify that our observations were not limited to adherent RPE1 cells, we extended our experiments to mouse primary neurons and brain cortical tissue. First, we isolated primary neuronal cells from conditional Atg5flox:CAG-CreTmx newborn mice, a model previously shown to exhibit robust and complete knockout (KO) of ATG555. After fixation, we labeled WT and KO astrocytes and neurons with an antibody against p62, and stained lipid droplets with Lipidspot488 (Fig. 4d–g). Second, we analyzed cortical brain sections from three-month-old WT and conditional ATG5 (cKO) mice, in which ATG5 deletion is driven by the neuronal-specific Scl32a1 promoter56. These sections were stained with antibodies against p62 and perilipin 1 (PLIN1), a protein associated with the surface of lipid droplets (Fig. 4h, i). We used confocal microscopy to image these samples and quantified the amount of lipid droplets per cell, the amount of p62 punctae per cell, the average size of the lipid droplets and the percentage of lipid droplets that co-localized with p62 signal in each cell. Both in human and mouse cells, we observed an increase in the amount of lipid droplets, while this effect was most pronounced in mouse primary KO astrocytes and cortical 3-month-old KO neurons (Fig. 4j). Additionally, as expected, we found a significant increase in the number of p62 punctae upon ATG5 KD or KO in the analyzed cells (Fig. 4k). In the absence of ATG5, the average size of the lipid droplets increased from 0.3 to 0.4 μm2, from 0.3 to 0.6 μm2, from 0.7 to 0.8 μm2, for mCherry-p62 RPE1, mouse astrocytes and neurons, respectively (Fig. 4l). The mouse cortical neurons were harder to quantify as they, on average, contained few, very large lipid droplets. Finally, we also detected a significant increase in the number of lipid droplets that co-localized with p62 punctae in those samples lacking ATG5 (Fig. 4m). Interestingly, in our mock-transfected RPE1 cells, we also observed a moderate increase in lipid droplet sizes (Supplementary Fig. 4d), possibly due to the fact that the cationic lipid mixtures used in transfection mixtures have been observed to induce autophagy, promoting LC3 conversion and p62 degradation57,58. Together with the autophagy-stalling effect of ATG5 siRNA, the lipids likely contribute to further enrichment of p62-positive structures. In conclusion, when transfecting RPE1 cells with ATG5 siRNA, and upon ATG5 KO in mouse primary astrocytes, mouse primary neurons and/or 3-month-old mouse cortex, we observed an accumulation of lipid droplets co-localizing with p62 punctae, as well as a size increase for lipid droplets. The characterized condition of ATG5 knockdown in human RPE1 cells may be well suited for the in situ visualization of p62 punctae adjacent to lipid droplet cargo, as autophagosome formation and trafficking to the lysosome is impaired.

p62 encapsulates lipid droplets by a discontinuous coat of variable thickness
Based on the established condition of accumulated co-localized p62 punctae and lipid droplets, we set out to perform detailed in situ structural analyses to study p62 during lipid droplet cargo recognition. RPE1 cells were grown under the same conditions described above and placed on micropatterned electron microscopy grids, vitrified, subsequently thinned to ~150 nm using a focused ion beam (FIB) and visualized using a scanning electron microscope (SEM) (Fig. 5a). To verify that the lamella contained mCherry-p62, we imaged the sample using an integrated cryo-fluorescence microscope and correlated it with the FIB-SEM image59 (Fig. 5b). When we visualized these p62 structures using super-resolution cryo-confocal microscopy, they showed circularly resolved mCherry-p62 signal on their rim suggesting the cargo encapsulation by the autophagy receptor p62 (Fig. 5c). At these sites, a total of 25 tomograms were recorded while those with green autofluorescence from lysosomes were not further analyzed. In the reconstructed tomograms, we identified characteristic structures made of a homogeneous texture of spherical shape suggestive of a lipid droplet60 (Fig. 5d) in agreement with the frequent co-localization of p62 and lipid droplet punctae demonstrated above. A dark and highly dense layer was found surrounding the droplet in addition to a single phospholipid bilayer. The homogeneously textured spheres were similar in size measuring between 510–530 nm (Fig. 5e). The surrounding layer, or coat, varied in thickness within a single structure as well as between structures ranging between 4 and 27 nm, with a mean of 12.6 nm (Fig. 5f and Supplementary Fig. 5). The distance between the surface of the sphere and the surrounding single lipid membrane was relatively uniform at 13.8 nm with some weaker protein densities filling this space while other densities connecting these two structures (Fig. 5g, h). The segmented tomograms revealed that when the layer was thin, patches or islands of dark contrast were found distributed around the surface of the lipid droplet (Supplementary Fig. 6). However, when the layer was thick, it was completely continuous. Next to the lipid droplet, several small vesicles between 35 and 52 nm in diameter were observed, consistent with previously described ATG9A vesicles35. In summary, ATG5 knockdown in human RPE1 cells yielded p62-positive spherical structures of 500 nm diameter composed of an inner lipid droplet and a p62-containing, dense layer of variable thicknesses covering the surface, surrounded by a single bilayer membrane.

Elemental analysis reveals calcium and phosphorus in the p62-positive layer
Given the strong observed intensity of the surrounding p62-positive layer with respect to the gray levels of the lipid droplet and cytosol, we wondered whether non-organic elements contributed to the high contrast. To address this question, we employed scanning transmission electron microscopy (STEM) coupled with energy-dispersive X-ray (EDX) spectroscopy to record element-specific X-rays from the mCherry-p62 RPE1 lamella treated with ATG5 siRNA investigated above (Fig. 6a–d). For this sample, we acquired STEM images on a high-angle annular dark-field (HAADF) detector and simultaneously recorded the back-scattered EDX signal (Fig. 6e). The EDX signal was averaged over the excised regions of the cytosol, lipid droplet core and the p62 coat (Supplementary Fig. 7). For the cytosol, we detected mostly carbon (C), nitrogen (N) with low and oxygen (O) with high intensity at approx. 300, 400 and 500 eV, respectively. For the lipid droplet, we observed enriched C in comparison with the cytosol, reduced N and O, consistent with the presence of triacylglycerides and sterols within the sphere of lipid droplets. In both regions, no inorganic elements were observed at higher energies. For the p62 coat, we found the C, N, O signal comparable to the cytosol with clear additional inorganic signals of magnesium (Mg), calcium (Ca) and phosphorus (P) at 1250, 2000 and 3800 eV, respectively, supporting the observation of the relatively strong contrast in the raw tomograms. The detected high levels of Ca and P suggest the presence of calcium phosphate. As a control, we focused on lipid droplets showing no p62 co-localization and obtained a very similar EDX signal of the core lipid droplet region while lacking the high levels of Ca and P in their surrounding (Supplementary Fig. 8). Other p62-positive single-membrane vesicular structures resembling lysosomes showed no differences in EDX profiles between the vesicular area and the cytosol (Supplementary Fig. 9).

Given the unexpected finding of inorganic elements located in the p62 coat, we tested whether the addition of Ca2+ ions had an effect on purified p62 filaments. When increasing the CaCl2 concentrations from 100 nM to 100 mM, we observed a bundling of p62 filaments at 100 mM CaCl2 in negative staining EM (Fig. 6f–h). Moreover, when testing other divalent cations such as Zn2+ and Mg2+, we found that the addition of ZnCl2 had a stronger clustering effect already visible at 100 µM, while the addition of MgCl2 did not show a significant bundling (Supplementary Fig. 10). Together, these experiments show that the observed p62-positive structures correspond to lipid droplets surrounded by a coat made of p62, including significant amounts of calcium (Fig. 6i) that also promotes the formation of p62 oligomer bundles in vitro and may explain the characteristic dense layer observed in the tomograms.

Proximity proteomics identifies calcium transporters in proximity to the p62 encapsulated lipid droplets
In order to provide further molecular insights into the observed p62-encapsulated lipid droplets, we turned to a proximity proteomics experiment61. In short, the miniTurbo enzyme was fused to mCherry-p62 and a stable RPE1 cell line was generated. Subsequently, proteins in the immediate proximity of p62 were biotinylated, pulled down and identified using mass spectrometry (Fig. 7a, b). The abundance of biotinylated proteins was estimated for the 72-h ATG5 knockdown (including mCherry-p62 and miniTurbo), i.e., T-mCh-p62 ATG5 KD, and compared with a control of endogenously expressed ATG5 (including mCherry-p62 and miniTurbo), i.e., T-mCh-p62, as well as a second control devoid of the miniTurbo enzyme (including mCherry-p62), i.e., mCh-p62. Western blot confirmed the absence of ATG5 in the T-mCh-p62 ATG5 KD sample, while ATG5 was detected in the mCh-p62 and T-mCh-p62 control samples (Fig. 7c). Consistently, we found neither ATG5 nor ATG16 in the proteomic data of the T-mCh-p62 ATG5 KD sample (ProteomeXchange-ID PXD066163). We detected several known interactors of p62 and other early autophagy proteins in both the T-mCh-p62 and T-mCh-p62 ATG5 KD samples: NBR1, ATG13, ZFYVE1/DFCP1 and TRAF6, while they were not present in the mCh-p62 control. Statistical analysis of relative peptide abundance between the T-mCh-p62 ATG5 KD and the T-mCh-p62 endogeneous ATG5 sample identified several other proteins related to autophagy that were only present in the absence of ATG5 (Fig. 7d). Most interestingly is the presence of SIRT1 and alpha-synuclein as these proteins have been described to be present on lipid droplets involved in lipophagy62,63. Analysis of protein enrichment and depletion between endogeneous ATG5 and ATG5 knockdown also identified proteins related to lipid droplets or lipid and steroid metabolism: TPRG1L64, SQLE and PIP4P1. Finally, we inspected proteins that were possibly related to the observed calcium accumulation around the p62-lipid droplet structures. Intracellular calcium chelators, such as calmodulin or calcineurin, were neither enriched nor depleted upon ATG5 knockdown. However, we identified a calcium channel and a regulator of calcium homeostasis in the ATG5 knockdown but not in the endogenous ATG5 sample: First, ITPR2, an ER-resident, inositol 1,4,5-trisphosphate-gated calcium channel and, second, MCUb, known as a negative regulator for the mitochondrial calcium uniporter. The former proteins, i.e., ITPR channels, release calcium from the ER and have been shown to affect autophagy65. The latter protein, i.e., MCUb, has been described to block calcium overload of nearby mitochondria, and has also been associated with the early stages of autophagy and mitophagy66–68. Together, our proximity proteomics experiments on the ATG5 knockdown induced p62-encapsulated lipid droplets revealed proteins such as SIRT1 and alpha-synuclein, linking lipid droplets to autophagy. Moreover, upon ATG5 knockdown, proteins involved in calcium transport from the ER, such as ITPR2 and MCUb, were found in close proximity to p62, supporting the notion that the observed calcium layer and nearby single phospholipid bilayer may represent calcium released from the ER.

Methods
Protein expression, purification of p62, 4x-linear-ubiquitin and LC3B
Full-length p62 (440 residues) was cloned into a pETM43 expression vector containing an N-terminal maltose-binding protein (MBP) tag and a C-terminal His-tag. Both tags featured a recognition sequence for HRV-3C protease, facilitating their removal after purification. 4x-linear-Ubiquitin, CFP-LC3B, and LC3B with an additional C-terminal cysteine residue were cloned into the pGEX expression vector containing an N-terminal Glutathione-S-Transferase (GST) tag with an HRV-3C protease cleavage site. For protein expression, E. coli BL21 (DE3) cells were used, with bacterial cultures grown at 37°C in Terrific Broth (TB) medium to an OD600 of ~2. Induction with IsoPropylThioGalactoside (IPTG) followed, and cells were left to grow overnight at 23°C. Cells were harvested, and the pellet was stored at −80°C until further use.

After thawing on ice, cells were resuspended in lysis buffer [50 mM HEPES, pH 8.0, 300 mM NaCl, 2.5 mM MgSO4, 10 µM ZnCl2, 0.5 mM TCEP and 1 tablet EDTA-free protease inhibitor cocktail] and lysed using a cell disruptor at 1.85 bar. Cell debris was removed by centrifugation, and the cleared lysate was applied to a Ni-NTA affinity column. The column was washed twice with lysis buffer containing 10 mM and 20 mM imidazole, respectively, and the protein was eluted with lysis buffer containing 300 mM imidazole. Eluted protein fractions were pooled and subsequently applied to an amylose resin that binds MBP-fusion proteins with high affinity. The column was washed with lysis buffer, and the protein was eluted with lysis buffer containing 25 mM maltose. Eluted protein fractions were pooled, and buffer was exchanged to desalting buffer [50 mM HEPES, pH 8.0, 300 mM NaCl and 0.5 mM TCEP] using Zeba desalting columns.

For the purification of LC3b and GST-4xUb, the cell pellets were resuspended in lysis buffer [50 mM HEPES, pH 7.4, 300 mM NaCl, 1 mM DTT, and 1 tablet EDTA-free protease inhibitor cocktail] and lysed using a cell disruptor at 1.9 bar. The cleared lysate was applied to Glutathione Sepharose 4 Fast Flow GST-tagged protein purification resin (Cytiva, Cat No 17513201), washed with 10 column volume lysis buffer, and eluted in lysis buffer containing 10 mM reduced glutathione. Elution fractions containing the protein of interest were pooled, further purified by size exclusion chromatography via SD200 16/600 column (Cytiva) [in 50 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM DTT], and frozen at −80°C for storage.

p62 filament formation and labeling
Protein was concentrated to approximately 2 mg/mL using a 30 kDa Amicon concentrator and subsequently incubated with 1:2.5 mass ratio of GST-HRV-3C protease for 3 h at room temperature and overnight at 4°C to remove the MBP and His-tag, allowing p62 filament formation. Filaments were isolated by centrifugation at 20,000 × g for 30 min and resuspended in desalting buffer. Isolated filaments were labeled with lysine residues by incubating with 1:1 molar ratio of Cy5-NHS for 1 h at room temperature and overnight at 4°C. Excess dye was removed by centrifugation, labeled filaments were resuspended in storage buffer [50 mM HEPES, pH 8.0, 50 mM NaCl and 0.5 mM TCEP] and stored at 4°C until further use.

Cryo-EM structure determination of p62 filaments
Quantifoil Cu 200 mesh R1.2/R1.3 grids were glow-discharged, and 2–5 µL of the protein sample was applied in a Vitrobot Mark IV (ThermoFischer Scientific). The sample chamber was maintained at 70–100 % humidity and 4–10°C, with a 2–6 s blotting time before plunging into an ethane/propane mixture. Data collection was performed using SerialEM94 on a Talos Arctica 200 KeV G2 transmission electron microscope (ThermoFischer Scientific), equipped with a K3 direct electron detector camera and Quantum GIF filter (Gatan, Inc). Approximately 4000 micrographs were collected in counted super-resolution mode at 100,000x magnification with an underfocus range of −0.5 µm to −3 µm. The micrographs were motion corrected in WARP95, and particles were picked using the deep-learning-based object detection software crYOLO96. Initially, the picked particle locations and micrographs were imported into RELION and SPRING46,48, which used CTFFIND4 for CTF estimation97. Later, CTF estimated micrographs were imported into cryoSPARC47 and particle locations were extracted with a 90% overlap, yielding approximately 1,000,000 segments of a box size of 503 Å. Two rounds of 2D classification were performed in order to remove heterogeneous particles. Classes with clearly defined PB1 domains were then selected, leading to a total of 600,000 segments. The helical parameters as determined by Jakobi et al.22 of truncated p62 helical filaments were taken as starting conditions for helical refinement in cryoSPARC, where the symmetry parameters converged (Supplementary Fig. 11). The final resolution was estimated by FSC using the 0.143 cutoff98 and FDR-FSC criterion99. Maps were rendered in UCSF ChimeraX100.

AlphaFold3 predictions and modeling of a p62 filament
To model the p62 filament structure, we used AlphaFold3 (AF3) multimer predictions51: first, we tested p62 full-length sequences arranged in dimers, trimers, tetramers, up to decamers. AF3 consistently favored closed ring-like assemblies, even for small oligomers (≥3 subunits), while for larger rings the resulting p62 rings exhibited close-to-rotational symmetry. A single p62 subunit was excised from an AF3-generated p62 closed ring-like decamer with a diameter of 145 Å, approaching the diameter of the filament. To relate these predictions to the p62 filament, we fitted the PB1 domain in the corresponding well-resolved density of one strand of the filament map while including the full-length p62 model and repeated the operation for the opposite PB1 strand. Using the determined helical symmetry parameters of 11.0 Å helical rise and a −26.1° helical rotation, we propagated the two subunits along the helical axis, generating a double-helical filament model composed of repeating p62 units. This helical assembly yielded a plausible model of the p62 filament, with subunits well fitting the experimental cryo-EM density while maintaining interaction geometries close to the AF3-predicted oligomers.

Single-particle fluorescence imaging in TIRF mode
Surface imaging was performed with an inverted microscope (Olympus IX-81, Olympus Corp., Shinjuku, Japan) in total internal reflection fluorescence (TIRF) mode. Molecules were excited using lasers at 488 and 639 nm (Sapphire 488–200 and Obis 637–140, Coherent Inc.), focused off-axis into the back-focal plane of an oil-immersion objective (Olympus UApoN, 100x, NA=1.49, Olympus Corp). The excitation power and corresponding intensity at the sample stage were 3.25 mW and 10.2 W/cm2 for the blue laser, and 2.2 mW and 6.8 W/cm2 for the red laser. The imaging was performed on an EMCCD camera (Andor iXon Ultra 888, Andor Technology) with 50, 100, 40 and 100 ms exposure time for Supplementary Videos 1–4, corresponding to 20, 10, 25 and 10 fps. Midway through acquiring Supplementary Video 2, the illumination was switched from blue to red laser to observe the same p62 filament with LC3b bound to it. Each video consists of 200 frames. Image analysis and video processing were carried out using Fiji (ImageJ, version 2.14.0)101. The emitted fluorescence was collected with the same objective (objective-based TIRF) and passed through a dual-band dichroic mirror as well as a dual-band emission filter (ZT488/640rpc and ZET488/640m, Chroma Technology Corp). p62 filaments-Cy5 and CFP-LC3b were diluted to concentrations of a few nM, and mixed in a 1:1 molar ratio. GST-4xUb was added at a concentration of 5 µM.

Negative staining electron microscopy
For negative staining, 3.5 µL of the sample was applied onto glow-discharged carbon-formvar-coated 300-mesh copper grids and allowed to absorb for 2 min before being blotted according to the side-blotting method. The sample was subsequently stained for 30 s with 6 µL 2% uranyl acetate, blotted and allowed to dry. Negatively stained samples were examined on a Talos L120C G2 transmission electron microscope (ThermoFisher Scientific), operated at 120 kV. Micrographs at high magnification (45,000x and 92,000x) were collected on a 4k x 4k Ceta 16M CEMOS camera using TEM Imaging & Analysis Software (TIA, ThermoFisher Scientific).

Statistical analysis of filament length distribution
We used Origin 2021b to perform the Kolmogorov-Smirnov (K-S) test for the two datasets of filament lengths (p62 filaments without and with LC3b present). We sorted the two datasets in ascending filament length order and computed the cumulative distribution function (CDF) for both datasets. CDF increases from 0 to 1 from the smallest to the largest value in the distribution. The CDFs from both datasets were overlaid in a plot and the largest absolute difference between them was computed (Supplementary Fig. 12).

D = max |F1(x) − F2(x)| (1)

where F1(x) and F2(x) are the CDFs of each dataset. Then we computed the K–S test statistic K

K = D × sqrt((n1 * n2) / (n1 + n2)) (2)

where n1 and n2 are the sizes of each dataset. The approximate p-value was calculated as

p ≈ e−2K2 (3)

Cryo-electron tomography of reconstituted p62 filaments
p62 filament cryo-EM grids were prepared as described above. Tilt series were recorded on a 200 kV Talos Arctica G2 transmission electron microscope (ThermoFisher Scientific), equipped with a Bioquantum GIF (Gatan, Inc), a Volta Phase Plate and a K3 direct electron detector (Gatan, Inc) using SerialEM (Mastronarde, 2005). Each tilt was recorded as a movie at 63,000x magnification with a 0.681 Å/pixel size in super resolution mode, corresponding to a dose of approximately 2.0 e/Å2 at 0° tilt, 10 frames per tilt, and a total dose per tilt series of approximately 130 e/Å2. All tilt series were recorded from −54° to 54° with 3° increments, using a dose-symmetric bidirectional acquisition scheme including ‘the varied intensity’ option (i.e. higher tilt angles received higher doses, as 1/cosine of the tilt angle) in SerialEM and a nominal defocus of −1.2 µm. The raw data were motion corrected and CTF corrected using WARP (version 1.0.9)95 and the tilt series were reconstructed using IMOD102.

GST-4x-linear-Ubiquitin and p62 filaments were prepared as described above. 15 µM of GST-4x-linear-Ubiquitin were mixed with 1 µM p62 filaments. After 4 min incubation time at 4°C, the formed phase separation droplets were negatively stained as described above or plunge frozen with a Vitrobot Mark IV (ThermoFisher Scientific). 50 nm SUVs were prepared containing 5% DSPE-PEG (2000) maleimide (Avanti Polar lipids, Cat No 880126) in DOPS (Avanti Polar lipids, Cat No 840035). The liposomes were incubated with 100 µM LC3b-Cys for 16 h at 4°C before removing unbound LC3b-Cys by size exclusion chromatography on Sephacryl S-500 at Äkta Micro (Cytiva). A total of 2 mg/mL of liposomes was mixed with 1 µM p62 filaments and incubated for 5 min at 4°C. The sample was plunge frozen using a Leica GM2 plunge freezer and imaged in a 300 kV Titan Krios G4 equipped with a BioContinuum energy filter and K3 camera at 64,000x using Tomo5 software (ThermoFisher Scientific). The tilt series were recorded from −60° to 60° with a 3° increment in a dose-symmetric bidirectional acquisition scheme. An electron dose of 4.0 e−/Å2/tilt (at 0° tilt) with 6 frames per tilt resulted in a total dose per tilt series of approximately 160 e−/Å2. The defocus was varied between −2.0 to −4.0 µm. The images were motion corrected in WARP95 and tomograms reconstructed with AreTomo103. Overall binning was 16x and resulted in a pixel size of 11.2 Å. Tomograms were denoised and missing wedge corrected with IsoNet104.

Cell culture and transfection
Cell culture experiments were carried out in human RPE1 cells (hTERT RPE1, CRL-4000, ATCC) cultured at 37°C and 5% CO2 in DMEM GlutaMAX media (Gibco, Cat No 31331028) supplemented with 10% FCS (Sigma-Aldrich, Cat No F7524) and Pen/Strep (Gibco, Cat No 15140122). For confocal microscopy experiments prior to plunge-freezing, the culturing media were substituted with media of low fluorescence background: Fluorobrite DMEM (Gibco, Cat No A1896701). Additional experiments were performed on RPE1 cells stably expressing human mCherry-p62. Transient expression of mCherry-p62 was executed by transfection with Fugene HD (Promega, Cat No E2311) and a modified pDEST vector containing the human WT p62 gene fused with an N-terminal mCherry sequence (Pankiv et al., 2007). Knockdown experiments were carried out by transfection with RNAiMAX (ThermoFisher, Cat No 13778075) and ON-TARGETplus SMARTpool siRNA against ATG5 (Dharmcon Horizon Discovery, Cat No L-004374-00-0005), GAPDH (Dharmcon Horizon Discovery, Cat No D-001830-10-05) and a non-targeting control (Dharmcon Horizon Discovery, Cat No D-001810-10-05). Follow-up experiments were performed 72 h after a reverse transfection with 75 nm siRNA. Lipid droplets were stained with 1:500 Lipi-Blue (Dojindo, Cat No LD01-10) for 24 h. Lysosomes were stained with the CytoPainter Lysosomal staining kit (Abcam, Cat No ab112136).

Preparation of primary postnatal cortical culture and Immunocytochemistry
All animal experiments were approved and performed according to the regulations of the LANUV, NRW, Germany (AZ: 81-02.04.2020.A418, 81-02.05.40.20.075, 81-02.04.2021.A132, Anzeige §4.20.021) guidelines. Mice were maintained in a pathogen-free environment in ventilated polycarbonate cages and housed in groups of five animals per cage with constant temperature and humidity at 12-h/12-h light–dark cycles. Food and water were provided ad libitum. Atg5flox:Slc32a1-Cre:tdTomato (ATG5 cKO) mice were described previously56,74. The following genotyping primers were used to genotype the animals: ATG5 forward 1 (GAATATGAAGCCACACCCCTGAAATG), ATG5 forward 2 (ACAACGTCGAGCACGCTGGCGAAGG), ATG5 reverse (GTACTGCATAATGGTTTAACTCTTGC), Cre_1 (GAACCTGATGGACATGTTCAGG), Cre_2 (AGTGCGTTCGAACGCTAGAGCCTGT), Cre_3 (TTACGTCCATCGTGGACA) and Cre_4 (TGGGCTGGGTGTTAGCC). In accordance with the SAGER guidelines, we have ensured that both male and female mice were included in all experimental groups. Cortical and hippocampal neurons from postnatal pups (P1–5) were isolated as previously described55 and plated on PDL-coated coverslips or dishes. Neurons were grown in a MEM (Gibco) medium, containing 0.5% Glucose (Sigma), 0.02% NaHCO3 (Roth) and 0.01% Transferrin (Merck), which was further supplemented with 5% FBS (Sigma), 0.25% L-GlutMax (100x, Gibco), 2% B27 (50x, Gibco), 1% Pen/Strep (10000 Units/ml Penicillin, 10,000 µg/ml Streptomycin, Gibco). After two days in vitro 2 mM AraC was added to the culture medium to limit glial proliferation. To initiate homologous recombination, primary neurons isolated from floxed animals expressing a tamoxifen-inducible Cre recombinase were treated with 0.4 µM (Z)−4-hydroxytamoxifen (Sigma) immediately after plating. After 24 and 48 h, cells were treated with 0.2 µM and 0.4 µM of tamoxifen, respectively, during medium renewal. Ethanol was added to control neurons (WT) in an amount equal to the tamoxifen. The DIV 14–16 postnatal neurons were fixed in 4% PFA/sucrose in phosphate-buffered saline (PBS) for 15 min at RT. Cells are then washed 3x with PBS and then blocked in 5% NGS in 0.3% Triton X-100 for 1 h at RT. Primary antibodies diluted in 5% NGS and 0.3% Triton X-100. Coverslips were rinsed 3x PBS and incubated with corresponding secondary antibodies diluted in 5% NGS and 0.3% Triton X-100, and incubated for 30 min at RT. The following antibodies were used: polyclonal guinea pig anti-p62 (1:1000, Progen, cat no GP62-C), Goat anti-Guinea Pig Alexa Fluor 568 (1:1000, Invitrogen, cat no A-11075), and Donkey anti-Rabbit Alexa Fluor 647 (1:1000, Invitrogen, cat no A-31573). Cells were washed 3x in PBS and then incubated in 1:1000 Lipidspot 488 (Biotium, cat no #70065-T) for 15 min at RT. Following incubation, cells were directly mounted on glass coverslips. Post-natal neurons were imaged using with Leica SP8 confocal microscope (Leica Microsystems) equipped with a 63x/1.32 oil DIC objective and a pulsed excitation white light laser (WLL; ∼80-ps pulse width, 80-MHz repetition rate; NKT Photonics) at a resolution of 1024x1024 pixels in sequential scanning frame-by-frame mode. A single Z plane was yielded. For quantitative analysis of fluorescent puncta, each channel of interest was processed by subtracting background and the area of the neuron and/or cell body was manually selected using ImageJ (Version 1.54p) selection tools (ROI). Fluorescent puncta were counted with the ImageJ Cell Counter Plugin. Fluorescent puncta size of the lipid droplets (Lipidspot 488) was determined by applying the autothreshold “Huang” algorithm in ImageJ (Version 1.54p) and analyzed using the “Analyze particles” ImageJ (Version 1.54p) plugin to get average puncta size per cell in µM2.

Immunohistochemistry on perfused brain sections
All animal experiments were approved and performed according to the regulations of the LANUV, NRW, Germany (AZ: 81-02.04.2020.A418, 81-02.05.40.20.075, 81-02.04.2021.A132, Anzeige §4.20.021) guidelines. Mice were maintained in a pathogen-free environment in ventilated polycarbonate cages and housed in groups of five animals per cage with constant temperature and humidity at 12-h/12-h light–dark cycles. Food and water were provided ad libitum. Atg5flox:Slc32a1-Cre:tdTomato (ATG5 cKO) mice were described previously56,74. The following genotyping primers were used to genotype the animals: ATG5 forward 1 (GAATATGAAGCCACACCCCTGAAATG), ATG5 forward 2 (ACAACGTCGAGCACGCTGGCGAAGG), ATG5 reverse (GTACTGCATAATGGTTTAACTCTTGC), Cre_1 (GAACCTGATGGACATGTTCAGG), Cre_2 (AGTGCGTTCGAACGCTAGAGCCTGT), Cre_3 (TTACGTCCATCGTGGACA) and Cre_4 (TGGGCTGGGTGTTAGCC). In accordance with the SAGER guidelines, we have ensured that both male and female mice were included in all experimental groups. Mice at 12–15-week were euthanized by an overdose of 1.2% ketamine, and 0.16% xylazine in PBS (i.p., 10 µl per 10 g body weight) and transcardial perfusion was performed as previously described105. Brains were carefully dissected and postfixed in 4% PFA overnight before being processed for immunohistochemistry as previously described105. Corresponding horizontal 40 µm sections from WT and KO littermates were washed 3x in PBS for 5 min each. Sections were blocked with 10% NGS/NDS in 0.5% PBS-T for 1 h at RT. Primary antibodies were incubated in sections in 3% NGS/NDS and 0.3% PBS-T overnight at 4°C. Sections were washed 3x 10 min in 0.3% PBS-T before incubation in fluorescence-labeled secondary antibodies in 3% NGS/NDS and 0.3% PBS-T for 1 h at RT, protected from light. The following antibodies were used: polyclonal guinea pig anti-p62 (1:1000, Progen, cat no GP62-C), recombinant rabbit anti-perlipin 1 (1:500, Proteintech, cat no 83905-4-RR), Donkey anti-Rabbit Alexa Fluor 647 (1:1000, Invitrogen, cat no A-31573) and Goat anti-Guinea Pig Alexa Fluor 488 (1:1000, Invitrogen, cat no A-11073). Sections were imaged at a Leica SP8 confocal microscope (Leica Microsystems) equipped with a 63x/1.32 oil DIC objective and a pulsed excitation white light laser (WLL; ∼80-ps pulse width, 80-MHz repetition rate; NKT Photonics) at a resolution of 1024x1024 pixels in sequential scanning frame-by-frame mode. Stacks of 30 optical sections were acquired, with a fixed stack size of 0.25 µm. For quantitative analysis of fluorescent puncta, each channel of interest was processed by subtracting background and the area of the neuron and/or cell body was manually selected using ImageJ (Version 1.54p) selection tools (ROI). Fluorescent puncta were counted with the ImageJ Cell Counter Plugin. Fluorescent puncta size of the lipid droplets (Perlipin 1) was determined by applying the autothreshold “Huang” algorithm in ImageJ (Version 1.54p) and analyzed using the “Analyze particles” ImageJ (Version 1.54p) plugin to get the average puncta size per cell in µM2.

Western Blot
Cells were lysed by a quick wash with ice-cold PBS (Gibco, Cat No 10010056) and a subsequent 15 min incubation in ice-cold RIPA buffer (Sigma-Aldrich, Cat No R0278-50 mL) supplemented with a protease inhibitor cocktail (Roche, Cat No 11836170001). Cell lysate was collected with a cell scraper before removing the cell debris by centrifugation. Total protein content was quantified and corrected using a Pierce 660 nm assay (ThermoFisher, Cat No 22662) before boiling the sampling in a reducing sample buffer and separation on a 4–15% mini-PROTEAN Precast SDS-PAGE (Bio-Rad, Cat No 4561085). Transfer to PVDF membrane was performed using a Trans-Blot pack (Bio-Rad, Cat No 1704156) and the membrane was blocked for 2 h in 2% BSA in TBST at 4°C. The membrane was incubated overnight with primary antibody in 1% BSA in TBST at 4°C, washed 3 times in TBST before the secondary antibody was added for 2 h in 1% BSA in TBST at 4°C. Prior to imaging on the Azure Sapphire RGB Biomolecular Imager (Azure Biosystems), the membrane was washed 3 times in TBST and incubated in Azure Radiance Q HRP (Biozym, Cat No 512101). The following antibodies were used: polyclonal rabbit anti-p62 (MBL International, Cat No PM045), monoclonal rabbit anti-ATG5 (Abcam, Cat No ab108327), monoclonal mouse anti-GAPDH (Invitrogen, Cat No MA1-16757), polyclonal rabbit anti-LC3 (Abcam, Cat No ab48394), goat anti-mouse HRP conjugate (ThermoFisher, Cat No 32230), and goat anti-rabbit HRP conjugate (ThermoFisher, Cat No 31460). Band intensity quantifications were performed using Image Lab (BioRad, version 6.1.0).

Cellular tomography grid preparation
RPE1 cells were seeded on micropatterned grids before plunge freezing using recently described protocols106,107. Briefly, 200 mesh gold Quantifoil R2/1 grids with a SiO2 support layer (Quantifoil, Cat No N1-S15nAu20-01) were glow discharged and treated with PLL-g-PEG (SuSoS, PLL(20)-g[3.5]-PEG(5)) overnight. Next, the grids were washed in PBS and placed in drops of 14.5 mg/mL PLPP (Alvéole, Cat No PLPP), and 20 µm circles in the center of 10x10 grid squares were micro-patterned with a PRIMO photopatterning device (Alvéole, Photopatterning Device consisting of a DMD + laser 375 nm, 75 mW module, and driving electronics). The grids were then washed thoroughly in PBS and incubated in human fibronectin immediately before use (Gibco, Cat No PHE0023). The cells were trypsinized (Gibco, Cat No 25300054), passed through a 40 µm cell strainer (PluriSelect, Cat No 43-10040-40) and counted before seeding ~200,000 cells in a 30 mm glass-bottom dish (Greiner Bio-One, Cat No 627860) containing the micropatterned grids in a small amount of DMEM. The cells were allowed to attach to the grids for 4–5 h before vitrification in liquid ethane using a Vitrobot Mark IV (ThermoFisher Scientific). The following settings were used; a chamber temperature of 37°C, a chamber humidity of 85% humidity, backside blotting with an 8.5 s blot time and blot force of −10 and a drain time of 1 s.

Confocal microscopy
RPE1 cell phenotypes were studied using a Zeiss LSM710 laser scanning confocal microscope (Carl Zeiss GmbH). The cells were grown in 30 mm glass-bottom dishes with 4 compartments (Greiner Bio-One, Cat No 627870) and imaged at 37°C using a Zeiss 63x oil immersion objective (Plan-Apochromat, DIC, 1.4 NA). Lipi-Blue was excited with a diode laser at 405 nm, and fluorescent emission was detected after passing a suitable emission filter (410–500 nm) using a Multi Alkali photomultiplier tube. The lysosomal stain was excited using an argon ion laser at 488 nm, and emission was detected using an emission filter (499–541 nm). MCherry-p62 was detected using a 543 HeNe 543 laser and an emission filter set to 579–696 nm using a GaAsP photomultiplier tube. Image analysis and quantification were done using Fiji (ImageJ, version 2.14.0)101 and a self-written routine in CellProfiler (version 4.2.6)108. Measurements were exported as .csv files and analyzed in OriginPro 2023 (OriginLab Corporation, version 10.0.0.154). Vitrified grids and thinned lamellae were studied using a Zeiss LSM800 upright laser scanning confocal microscope with a cryo stage (Linkam CMS196). Super-resolution images were acquired using a Zeiss 100x air immersion objective (EC Epiplan NeoFluar, DIC, NA=0.75) and an AiryScan Detector. MCherry-p62 was detected using a 561 nm diode. Image processing was done using Zen Blue (Carl Zeiss GmbH, version 3.3.89).

Correlative light and electron microscopy
Cryo-lamellae were made in a correlative manner using the in-chamber METEOR fluorescence microscope (Delmic, Cat No 2707-999-0014-2) equipped with a LMPLFLN 50x, NA=0.5, WD=10.6 mm objective lens in the Aquilos2 cryo-FIB-SEM system (ThermoFisher Scientific). In short, micropatterned grids with RPE1 cells were loaded under cryogenic conditions, taking special care to avoid contamination build-up from air humidity. Cells were located using the electron beam, and the sample was placed at eucentric height using MAPS software (ThermoFisher Scientific, version 3.19). Cell phenotype was confirmed by imaging each cell in the METEOR fluorescence microscope. Each cell was imaged using the focused ion beam, and the location of the final 150 nm lamella was marked in AutoTEM (ThermoFisher Scientific, version 2.4.1). The lamellae were generated using a step-wise process; the cell was thinned to 1 µm with a 0.5 nA milling current, then thinned to 600 nm with a 0.3 nA current, next to 300 nm with a 0.1 nA current and finally thinned to 200 nm with a 50 pA ion beam. The final polishing step, in which the lamella was thinned to 120-180 nm, took place with a 30 pA ion beam. The final lamella was once again imaged using the METEOR fluorescence microscope, and these images were used to determine where tomograms would be recorded. The lamella was coated with a thin platinum layer; sputtered for 7 s at 7 mA, to prevent sample charging as previously described (Berkamp et al., 2023). Next, the samples were transferred under cryo-conditions to a 200 kV Talos Arctica G2 transmission electron microscope (ThermoFisher Scientific), equipped with a Bioquantum GIF (Gatan, Inc) and a K3 direct electron detector (Gatan, Inc). Tilt series were recorded on p62 positive areas of the lamellae, using SerialEM (version 4.2.0)94 as movies at 24,000x magnification with a 1.7435 Å/pixel size in super resolution mode and a dose of approximately 2.0 e/Å2 at 0° tilt, 10 frames per tilt, and a total dose per tilt series of approximately 130 e/Å2. All tilt series were recorded from −54° to 54° with 3° increments starting at the pre-tilt of the lamella, using a dose-symmetric bidirectional acquisition scheme with weighted dose (i.e. higher tilt angles received higher doses, as 1/cosine of the tilt angle), tilt angle and a nominal defocus of −7 µm. The raw data were motion corrected and CTF corrected using WARP (version 1.0.9)95, and the tilt series were reconstructed using Aretomo2 (SBGrid)103,109. To aid the cryotomogram visualization, they were deconvolved, denoised, and the missing wedge compensated using IsoNet104 and cryoCARE110. To aid in segmentation a pre-trained U-Net model for non-denoised data in MemBrain-Seg (version 9b)111 was used and the processed tomograms were then loaded into Amira 3D (ThermoFisher Scientific, version 2022.2) for semi-manual segmentation. Tomograms were visualized using the IMOD software package (version 4.11.20)102. To aid in targeting the EDX spectra, high dose, single tilt micrographs were recorded on p62 positive areas of the lamellae at 24,000x and a dose of approximately 40 e−/Å2/micrograph and 180 frames per tilt, with each a dose of approximately 0.24 e−/Å2. Motion correction for these micrographs was done in Relion (SBGrid, version 4.0.1)112

Energy-dispersive X-ray (EDX) spectroscopy
The in situ EDX experiments were carried out on a probe-corrected 200 kV FEI Titan microscope (ThermoFisher Scientific) fitted with a Super-X G2 quadrant detector EDX system (Bruker) and a Simple Origin dual grid cryo transfer holder (Simple Origin, Inc). The microscope was operated under cryo-conditions in STEM mode with a 1.5 mrad convergence angle, a 1 nm probe size at a magnification of 55,000, resulting in a 2.9 nm pixel size. EDX spectra were recorded with a 50 µs dwell time and 50 frames, resulting in a total dose of 500 e/Å2, a camera length of 3.5 m and a detector dead time of approximately 70%. We found 500 e−/Å2 to be a good trade-off between a high signal-to-noise ratio in the EDX spectra while still maintaining the ultrastructural integrity of the sample. EDX spectra processing was done in Velox (ThermoFisher Scientific, version 3.14). Atomic map representations were rendered with a 12 px pre-filter average and a 12 px post-filter average for the p62 structures and a 25 px pre-filter average and a 9 px post-filter average for the lipid droplet and lysosome control experiments. The spectra were generated by segmenting the p62 coat in the HAADF-STEM micrograph and segmenting the same number of pixels in the lipid droplet core and a random area of the cytosol. This same process was followed for the control experiments.

Proximity proteomics
Lentivirus was ordered with a vector corresponding to V5-miniTurbo-mCherry-p62-IRES-puro (Sirion Biotech GmbH). The virus was added to WT RPE1 cells, clones were puromycin selected, and transduction confirmed via fluorescence microscopy. The cells were seeded in T75 flasks, and ATG5 was knocked down using siRNAs (ON-TARGETplus SMARTpool siRNA against ATG5, Dharmacon Horizon Discovery), and the cells were incubated for 72 h. Next, 500 µM biotin was added (Sigma-Aldrich, Cat No B4501-1G,) and 5 mM ATP (AppliChem, Cat No A1348, 0100) and the samples were incubated for 2 h at 37°C. The cells were placed on ice, washed in ice-cold PBS (Gibco, Cat No 10010056) and harvested with a cell scraper and RIPA buffer (Sigma-Aldrich, Cat No R0278-50 mL) supplemented with protease inhibitor cocktail (Roche, Cat No 11836170001) and 1 mM PMSF. Cell debris was removed via centrifugation. All experiments were done in quadruplicates, on mCherry-p62 RPE1 cells, miniTurbo-mCherry-p62 RPE1 cells and ATG5 KD + miniTurbo-mCherry-p62 RPE1 cells. The lysates were incubated with 200 µL washed Pierce Streptavidin Magnetic Beads (Thermo Fisher Scientific, Cat No 88817) overnight at 4°C. The beads were then washed with 3x 500 µL 100 mM Hepes, pH=7.4, 1 mL 1M KCl, 1 mL 0.1 M freshly made Na2CO3, 3x 1 mL 3 M urea and finally the beads were suspended in 100 µL 3 M urea in 10 mM Tris, pH=8. DTT was added to the final concentration of 10 mM, and the sample was incubated for 30 min at 56°C with 1000 rpm shaking. Next, the sample was alkylated with 50 mM CAA (Sigma-Aldrich, Cat No 22790-250G) for 30 min in the dark. The reaction was quenched with 50 mM DTT for 20 min, and the beads washed twice with 100 uL 2 M urea. On-bead digestion was performed overnight at 37°C in 50 µL 100 mM HEPES pH=7.4 using a 1:1 trypsin to proteome ratio (Promega, Cat no V5111). The supernatant was collected and pooled with 2x 100 µL 2 M urea wash steps. The samples were acidified with 2% TFA (Sigma Aldrich, Cat no 1082180050), to pH < 3. StageTips (Affinisep, Cat No Tips-RPS-M.T1.200.96) were equilibrated as follows: they were washed with pure methanol, with 0.1% formic acid in 80% acetonitrile and then twice with 0.1% formic acid. Each sample was split over 6 StageTips and centrifuged at 500 x g for 2 min, and then washed twice in 30 uL 0.1% formic acid and centrifuged at 500 x g for 2 min. Peptides were eluted with 60 µL of 60% ACN, 5% NH3 and vacuum-concentrated with the concentrator plus (Eppendorf). The samples were stored at −20°C, and right before mass spectrometry measurement, reconstituted in 0.1% TFA. The concentration was determined with a Nanodrop before injecting the peptides on a Q Exactive Orbitrap (Thermo Fisher Scientific) coupled to an UltiMate® 3000 UHPLC-System (Thermo Fisher Scientific) ran in positive mode utilizing a µPAC™ C18 pillar array column (200 cm bed length, Thermo Fisher Scientific) heated to 40°C. The 140 min gradient was applied in the following solvent B concentration: 6% to 5 min, 6–10% (5–10 min), 10–45% until 110 min, 45–99% until 118 min, 99% for 7.9 min, 99–6% in 0.1 min, and 6% for 14 min. Mobile phase A consisted of 0.1% formic acid (LC-MS grade) in water, and mobile phase B consisted of 0.1% formic acid in 86% acetonitrile (LC-MS grade). After peptide separation, electrospray ionization (ESI) was used as the ionization method. Therefore, a Nanospray Flex ion source (Thermo Fisher Scientific), liquid Junction™ PST-HV-NFU (MS Wil) and a fused silica emitter (20 µm inner diameter, 360 µm outer diameter, MicroOmics Technologies LLC) were used. A spray voltage of 1.8 kV and a capillary temperature of 250°C were applied. Full or overview MS scans were acquired in the m/z range of 385–1050 with a resolution of 70000 (at m/z 200) and an automatic gain control (AGC) target of 3x 106 ions with a maximum injection time of 60 ms. Each DIA cycle consisted of one full MS scan followed by 50 DIA windows with 12.5 m/z isolation width overlapping by 0.25 m/z covering the mass range of 400-1000. Each DIA window had a resolution of 35000, an AGC target of 1x 106 ions, and a maximum injection time of 60 ms. The peptides were fragmented by higher-energy collision-induced dissociation (HCD) with a normalized collision energy (NCE) of 26. Raw data was first converted by MSConvert GUI (64-bit, version 3.0.21229-9668f52) to the mzML format files using Peak Picking filtering. FragPipe (version 22.0) was used for database search by matching DIA spectra to the FASTA file path according to Proteome (i.e. UniProt H. sapiens canonical and isoform protein sequences, downloaded in May 2023), added with decoys and contaminants. MSFragger settings included peptide digestion with trypsin; for a peptide length from 7 to 45, a number of missed cleavages was set to 2, a precursor mass range from 300 to 5000 and the mass tolerance for precursor and fragment ions was set to 20 ppm. Commonly, methionine oxidation (M) and N-terminal acetylation ([^) were set as variable modifications and cysteine alkylation as a fixed modification. Validation settings of DIA_speclib_Quant were used, including MSBooster for deep learning prediction Peptide Spectrum Match (PSM) Validation with Percolator to increase confident identifications, and Protein Interference with ProteinProphet to generate the protein list with a 1% false discovery rate (FDR). The report.pg_matrix.tsv file from the FragPipe output was processed with RStudio (R version 4.4.0 (2024-04-24) x86_64-apple-darwin20) using the packages limma and dplyr. A log2 transformation was applied to the data to stabilize variance and normalize the data distribution. Next, a threshold was defined to filter rows based on the presence of valid (non-missing) values. Only rows where at least 75% of the values in each specified condition are valid were retained. The linear model was fitted to the data using the lmFit function from the limma package. An empirical Bayes moderation was performed to obtain more stable estimates of the variance. Moderated t-statistics and p-values for the comparison between two conditions were extracted, and the p-values were also adjusted for multiple hypothesis testing using the FDR method113. Finally, the log fold change between the two conditions was calculated for each comparison. All proteins found in control samples from cells expressing mCherry-p62 (mCh-p62) were considered non-specific contaminants and removed from the dataset before comparison of protein abundance in cells expressing T-mCh-p62.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript the Sachse laboratory is building on their previous seminal work identifying the formation of p62 filaments by cryo-EM. The improvements in the purification of the full length p62 (which is challenging to say the least) allowed the authors to generate robust filamentous structures and investigate their interactions with LC3B and poly-ubiquitin producing significant impact on the filament length and branching. Most interestingly, the authors also show the formation of a calcium, phosphate, and magnesium (as well as oxygen?)-rich p62 coat enwrapping lipid droplets in cellulo by oligomers, potentially shedding light on the mechanism of action of p62 in the early stages of lipophagy. The study is technologically exciting as the robust filament assembly allowed the authors to visualise how these structures can interact with membrane-conjugated LC3 and ubiquitin, shedding light on their possible behaviour during autophagy initiation. The in situ imaging of p62 and EDX imaging of lipid droplets is intriguing and certainly provides a lot of food for thought, with potentially important (albeit still to be determined) functional consequences.\nMajor comments:\n1. For Figure 2G/H, is there a statistically significant decrease in filament length with LC3b? Although it can be seen that that the modal filament length has shifted from around 200um to 100um, the overall distribution looks fairly similar, so it would be useful to include some statistical analysis to see whether the change is significant.\n2. If, as shown in supplementary Figure 1A, the p62 filaments seen at pH 6 are largely unwound at pH 8, what is the physiological relevance of the filament structure described, given that intracellular pH is typically around 7-7.5? Would filament structures at neutral pH interact with LC3/liposomes/ubiquitin in the similar manner to the lower pH?\n3. On lines 442-443, the authors state that the control focussing on lipid droplets showing no p62 colocalisation (shown in supplementary Figure 7) obtained a very similar EDX signal. This reads as though the signal is similar to that of the p62-encapsulated lipid droplets, when in fact the droplets without p62 do not show additional Mg, Ca and P signals seen in the p62 coat in Figure 6E. This should be rewritten to clarify that without the p62 coat, the atoms are not detected. More importantly, the mechanism by which p62 drives the formation of the calcium and phosphate coat around LDs is currently not explored. The authors should attempt to address this, either experimentally (can the EDX spectroscopy be applied to the p62 filament preparations in vitro?) or by providing additional discussion exploring possible pathways, including, but not limited to, p62-mediated recruitment of mineralisation factors.\n4. Similarly, what is the rationale for attributing the detection of the atoms by EDX spectroscopy to inorganic molecules such as calcium phosphate as opposed to calcium, phosphorus and magnesium being incorporated into proteins within the LD coat? What about calcium-binding and phosphorylated proteins? Is it similar to the oxygen, which is also clearly present within the coat, and which would be assumed to be incorporated into proteins and other biomolecules in the cytoplasm but also on the surface of LDs? Would calcium chelators or phosphatases disrupt the association of respective atoms with LDs? Fully answering these questions might not be possible within this study but the authors should provide a balanced interpretation of their data.\n5. What is the connection between the filaments detected in vitro with the p62 coat on LDs? There is no evidence that p62 in situ is polymerised, and beyond the effect of calcium on the filament bundling in vitro there is no support for the filament organisation of p62 on the surface of the LD in the diagram 6I. Will the in situ cryo-EM be able to clarify this issue now or in the future?\n6. Likewise, the rationale for including ubiquitinated proteins on LD surfaces in Figure 6I requires clarification. Do the authors have direct evidence that p62 recruitment is driven by ubiquitination? Are LDs displaying p62 ubiquitination-enriched, while those lacking p62 are not? The authors should discuss known ubiquitination machinery implicated in lipid droplet ubiquitination (doi: 10.1016/j.bbalip.2017.06.006), and assess whether these might mediate p62 recruitment. This contextualisation would provide a more comprehensive mechanistic perspective.\nMinor comments:\n7. In line 73 of introduction, the authors refer to the characterisation of other selective autophagy receptors beyond p62 in mitophagy. Various SARs have been identified as cargo receptors across a wide range of autophagy subtypes, so it might be worth referencing a review that provides an overview of this (e.g., Farre & Subramani 2016, doi: 10.1038/nrm.2016.74), rather than just referring to mitophagy alone.\n8. When describing domains of p62 in lines 87-89, it is worth noting the disordered linker region between the PB1 and ZZ domains, and its possible role in p62 disulphide-linked conjugate formation (e.g. doi: 10.1038/s41467-017-02746-z).\n9. In the seconds paragraph of the discussion, the authors describe the unwinding and structural opening up of the double-helical filament architecture to form a p62 “bubble” structure, allowing access to binding sites. A diagrammatic figure may be useful here to help the reader visualise what is meant by this.", "reviewer_2": "The manuscript is very comprehensive but a bit difficult to read because of many abbreviations and different techniques that are presented within the experimental analysis. It also contains a lot of data, probably enough for two papers. Overall, all experiments seem technically sound and according to current standards. The method section is sufficient, only a description for AlphaFold3 usage (especially for creating filaments) is missing.", "reviewer_3": "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_4": "This manuscript “NCOMMS-25-03646” presents a comprehensive structural analysis of full-length p62/SQSTM1, combining state-of-the-art techniques such as high-resolution cryo-electron microscopy single particle analysis (SPA), in situ cryo-electron tomography (cryo-ET), complementary biochemical methods, correlative light and electron microscopy (CLEM), STEM, and EDX. The authors describe a double-helical filament architecture built upon a PB1 domain scaffold with a flexible C-terminal region. They further investigate how binding partners (LC3b and polyubiquitin) and divalent cations influence filament organization. Additionally, the in situ imaging of p62-enwrapped lipid droplets with a calcium phosphate-rich coat offers novel insights into early autophagy stages. Overall, the study is robust and provides valuable structural insights linking molecular architecture to cellular function in autophagy. I recommend acceptance following the consideration of one major and several minor points:\nMajor:\nLow-resolution regions:\nAlthough the PB1 domain scaffold is resolved at ~4.5 Å, the flexible C-terminal and ZZ domains remain poorly defined. These lower-resolution regions are crucial for interpreting binding interactions. Although the authors present AlphaFold predictions and some binding experiment using imaging method, I suggest including additional biochemical characterization to further strengthen these conclusions.\nMinor suggestions:\n1) It would be beneficial if the authors could explain why the resolution for the full-length structure does not match that previously achieved for the isolated PB1 domain in 2020.\n2) An explanation of the differences observed at varying concentrations of p62 filament and GST-4xUbiquitin between Figure 2C (condensates) and Figure 3B (bundles) would be appreciated.\n3) Figures 6D and E (right EDX figures) should include scale bars.\n4) In Figure S1A, it would be helpful to present the 2D classification results at the same scale across different pH conditions.\n5) Typo error: line 1169 (methods, Cryo-electron tomography of reconstituted p62 filaments, line 5) should read “63,000x magnification” instead of “63.000x magnification.”", "reviewer_5": "The authors present cryo-electron microscopy imaging of the selective autophagy receptor p62, revealing that full-length p62 forms regular double-helix filaments in vitro. AlphaFold3 was used to predict the interactions of full-length p62 filaments with LC3b and ubiquitin. The interaction of p62 with soluble LC3b, LC3B-conjugated liposome membranes and poly-ubiquitin was studied. These three interactions led to structural disassembly, lateral association and bundling, respectively, providing insights into how p62 filaments respond to autophagy-related factors.\nIn vivo experiments showed that p62 puncta colocalized with lipid droplets in cells, with p62 forming a discontinuous coat around the droplets. Elemental analysis of the p62 coat revealed an enrichment of calcium and phosphorus, and the effects of other divalent cations were also examined. The authors argue that these findings support the hypothesis that calcium ions may be a key regulator in autophagy.\nWhile this manuscript presents an improved structural model of p62 filaments, the biological significance of these findings is unclear, and the possible significance for autophagy mechanisms in cells was not clearly stated by the authors. Judging the significance is also made harder by the fact that the context of the present work within the history of this subfield is not clearly spelled out - a general reader would find it difficult to know what precisely has been established in this field, and what has not been established. Further, the in vitro and in vivo experiments are somewhat disconnected.\nMajor comments:\n1. The findings presented in this manuscript are close to previously published findings by the same authors in [Ciuffa et al., 2015] and [Jakobi et al., 2020]. The present study is a worthy extension of these earlier studies; however, the present manuscript’s additional impact on the community’s understanding of autophagy appears somewhat limited.\n2. The authors state that “in contrast to the previously determined PB1 assemblies [Jakobi et al., 2020], the here determined full-length p62 filament structure formed a double helical filament architecture”. However, their previous study, [Ciuffa et al., 2015], reported that filaments of PB1-ZZ (a truncated version of full length p62) are organized in a double helix, while full-length p62 filaments consist only of a single strand [Ciuffa et al., 2015]. Thus, the authors own earlier findings appear to contradict their findings in the present work. This is not discussed in the manuscript.\nAdditionally, Ciuffa et al. described that the C-terminal part of the protein forms a solvent-accessible stretch, a feature that is also highlighted in this manuscript. Furthermore, Ciuffa et al. found that octa-ubiquitin but not LC3 provokes filament shortening, which seems to significantly overlap with the work presented in this manuscript (see section “P62 filament interactions with LC3b and ubiquitin”). The authors should clarify how their findings differ from or extend these previous studies.\n3. It would really help if the context of the work presented in this manuscript were clearly described. The manuscript would greatly benefit from a clear exposition, including an explanatory figure, of the current understanding of autophagy as it involves p62, LC3, ubiquitin, Atg8 family proteins, membrane compartments etc etc. For a general reader it is very difficult to comprehend from the present Introduction and other parts of the manuscript.\n4. The biological significance of this study is not stated, and no “model” figure is presented that visually announces the authors’ hypotheses. What do these findings tell us about the mechanisms of p62-mediated autophagy?\nFor example, the influence of LC3b and poly-ubiquitin on p62 is studied – but how is this related to autophagy? Another example: in the section “p62 filament interactions with LC3b and ubiquitin”, p62 filaments form condensates in the presence of ubiquitin, whereas, in “p62 filament membrane interactions”, ubiquitin instead induces parallel filaments bundles. Which of these two behaviors better reflects in vivo conditions? Again, what do these two different outcomes tell us about autophagy?\nAnother example: Ca and p62 are reported to colocalize on lipid droplets in cells – what mechanism involving Ca does this suggest to the authors?\n5. The authors emphasize that most p62 filaments have regular helical structure at pH=6, while a fraction unwind at pH=8. However, in the section “p62 filament interactions with LC3b and ubiquitin”, the authors polymerized p62 filaments at pH=8. Why is this pH used, when this apparently disrupts filaments?\n6. In the section “p62 filament membrane interactions”, the authors characterized the ultrastructure of p62 filaments at a high concentration and at pH=6. However, in subsequent experiments, they used a 10-fold lower concentration and pH=7.4. How does this impact the ultrastructure?\n7. The authors revealed the structural organization of p62 in human RPE1 cells. However, they did not establish a clear connection between the in vivo and in vitro experiments. Specifically, the physiological concentration of p62 in cells was not reported. Was it comparable to the in vitro conditions? Providing this information would help interpret the relevance of the in vitro findings in the cellular context.\nMinor comments:\n1. Fig. 2G and 2H show the distribution of p62 filament length in the absence and presence of LC3b, suggesting that LC3b induces depolymerization. For a clearer comparison, plotting both distributions within the same graph would be more effective.\n2. In Fig. S7F, the label for the peak at around e = 500 eV is missing.\n3. The authors compare the p62 structure to the DNA double-helix model (Line 494). However, given the subunit composition and the pitch of the p62 double helix filaments, perhaps a comparison to actin filaments or microtubules might be more appropriate."}, "author_response": "Preliminary Response to Reviewer Comments\nWe thank the reviewers for their detailed comments on the manuscript. In this point-by-point\nresponse, we addressed the remaining raised concerns.\nIn summary, we added two major experiments in this revised version of the manuscript:\n1. Proteomic characterization of ATG5 phenotype by p62 proximity labeling\n2. Extend biological significance of ATG5 knockdown phenotype to primary cortical\nneurons and brain cells by fluorescence characterization\nThe included experiments extend the scope of the conclusions. The remaining questions, e.g.\nregarding the experimental pHs, were succinctly addressed by amending the presentation in\nthe figures as well as adding introductory and discussion items. Noteworthy, we added a clear\ngraphical model in Figure 7E relevant for the context of the autophagy process.\nReviewer #1 (Remarks to the Author):\nIn this manuscript the Sachse laboratory is building on their previous seminal work identifying\nthe formation of p62 filaments by cryo-EM. The improvements in the purification of the full\nlength p62 (which is challenging to say the least) allowed the authors to generate robust\nfilamentous structures and investigate their interactions with LC3B and poly-ubiquitin\nproducing significant impact on the filament length and branching. Most interestingly, the\nauthors also show the formation of a calcium, phosphate, and magnesium (as well as\noxygen?)-rich p62 coat enwrapping lipid droplets in cellulo by oligomers, potentially shedding\nlight on the mechanism of action of p62 in the early stages of lipophagy. The study is\ntechnologically exciting as the robust filament assembly allowed the authors to visualise how\nthese structures can interact with membrane-conjugated LC3 and ubiquitin, shedding light on\ntheir possible behaviour during autophagy initiation. The in situ imaging of p62 and EDX\nimaging of lipid droplets is intriguing and certainly provides a lot of food for thought, with\npotentially important (albeit still to be determined) functional consequences.\nThank you for the overall positive assessment of our manuscript.\nMajor comments:\n1. For Figure 2G/H, is there a statistically significant decrease in filament length with LC3b?\nAlthough it can be seen that that the modal filament length has shifted from around 200um to\n100um, the overall distribution looks fairly similar, so it would be useful to include some\nstatistical analysis to see whether the change is significant.\nStimulated by the reviewer’s comments, we added an extended statistical treatment on the\nfilament length. Interestingly, the filament length distribution does not follow a Gaussian\ndistribution. Therefore, we performed the analysis using an empirical cumulative density\nfunction: Details have been added to the legend of new Supplementary Fig. 11B and the\nMethods section of the manuscript (page 40 (Supplementary Fig. 11B), 58):\nThe cumulative density distribution (CDF) was calculated for p62 filaments without\nLC3b (blue line) and p62 filaments in the presence of LC3b (orange line). The dashed\nred line gives the maximum absolute difference between the two CDFs, D=0.17787.\nThe Kolmogorov-Smirnov test statistic was calculated K= 2.10023 and the approximate\np-value was derived p= 0.00014746. Since p<0.05 the observed shift of the distribution\nin the presence of LC3b towards shorter filament lengths is statistically significant.\n2a. If, as shown in supplementary Figure 1A, the p62 filaments seen at pH 6 are largely\nunwound at pH 8, what is the physiological relevance of the filament structure described, given\nthat intracellular pH is typically around 7-7.5?\nWe thank the reviewer for asking this question. We address this point in a dedicated section\n1\nin the discussion (page 26):\nIn the cryo-EM images at physiological pH 7-8, we also found an unwinding and\nstructural opening up of the double-helical filament architecture located on the\nmembrane surface. In contrast, our full length p62 filament structure was solved at pH\n6 in order to obtain a more homogeneous closed filament conformation following the\nhelical symmetry. At a cytosolic pH of approximately 7.4, the p62 filaments reveal the\nability to adopt an open “assembly configuration”, in which previously inaccessible\navailable binding sites in the interior will become accessible in an opened p62 “bubble”\nstructure while still maintaining functional polymers and oligomers. At the slightly lower\npH values present in the fully assembled autophagosome, estimated to be closer to pH\n6, p62 will assemble in more rigid filaments. In future work, it will be interesting to\ncharacterize LC3 and poly-ubiquitin binding at various pH values to better understand\nthis regulatory mechanism, similar to the mechanism described for arginylated\nsubstrate45.\nb. Would filament structures at neutral pH interact with LC3/liposomes/ubiquitin in the similar\nmanner to the lower pH?\nDuring the restricted revision period, we attempted to perform quantitative biochemical binding\nstudies at different pHs. Unfortunately, the experiments have not been conclusive. Please find\nthe comment on biochemical bulk characterization of p62 filaments requested by reviewer #4,\npoint 2.\n3. On lines 442-443, the authors state that the control focussing on lipid droplets showing no\np62 colocalisation (shown in supplementary Figure 7) obtained a very similar EDX signal. This\nreads as though the signal is similar to that of the p62-encapsulated lipid droplets, when in fact\nthe droplets without p62 do not show additional Mg, Ca and P signals seen in the p62 coat in\nFigure 6E. This should be rewritten to clarify that without the p62 coat, the atoms are not\ndetected.\nWe apologize for the misunderstanding and added a clarifying statement:\nAs a control, we focused on lipid droplets showing no p62 co-localization and obtained\na very similar EDX signal of the core lipid droplet region while lacking the high levels of\nCa and P in their surrounding (Supplementary Figure 8).\n4. More importantly, the mechanism by which p62 drives the formation of the calcium and\nphosphate coat around LDs is currently not explored. The authors should attempt to address\nthis, either experimentally (can the EDX spectroscopy be applied to the p62 filament\npreparations in vitro?) or by providing additional discussion exploring possible pathways,\nincluding, but not limited to, p62-mediated recruitment of mineralisation factors.\nThe reviewer asks an intriguing question. To address the raised point, we turned to Turbo-ID\nproximity labeling followed by subsequent proteomic mass spectrometry to identify the\nmolecular protein composition and differences in p62 proximity with regards to wildtype and\nATG5 K.D. lipid droplets (page 22, new Figure 7A-D):\nIn order to provide further molecular insights in the observed p62-encapsulated lipid\ndroplets, we turned to a proximity proteomics experiment61. In short, the miniTurbo\nenzyme was fused to mCherry-p62 and a stable RPE1 cell line was generated.\nSubsequently, proteins in the immediate proximity of p62 were biotinylated, pulled down\nand identified using mass spectrometry (Figure 7A/B). The abundance of biotinylated\nproteins was estimated for the 72-hour ATG5 knockdown (including mCherry-p62 and\nminiTurbo), i.e. T-mCh-p62 ATG5 KD, and compared with a control of endogenously\nexpressed ATG5 (including mCherry-p62 and miniTurbo), i.e. T-mCh-p62, as well as a\nsecond control devoid of the miniTurbo enzyme (including mCherry-p62), i.e. mCh-p62.\nWestern blot confirmed the absence of ATG5 in the T-mCh-p62 ATG5 KD sample while\nConsistently, we found neither ATG5 nor ATG16 in the proteomic data of the T-mCh-\np62 ATG5 KD sample (ProteomeXchange-ID PXD066163). We detected several\nknown interactors of p62 and other early autophagy proteins in both the T-mCh-p62\nand T-mCh-p62 ATG5 KD samples: NBR1, ATG13, ZFYVE1/DFCP1 and TRAF6, while\nthey were not present in the mCh-p62 control. Statistical analysis of relative peptide\nabundance between the T-mCh-p62 ATG5 KD and the T-mCh-p62 endogeneous ATG5\nsample identified several other proteins related to autophagy that were only present in\nthe absence of ATG5 (Figure 7D). Most interestingly is the presence of SIRT1 and\nalpha-synuclein as these proteins have been described to be present on lipid droplets\ninvolved in lipophagy62,63. Analysis of protein enrichment and depletion between\nendogeneous ATG5 and ATG5 knockdown also identified proteins related to lipid\ndroplets or lipid and steroid metabolism: TPRG1L64, SQLE and PIP4P1.\nFinally, we inspected proteins that were possibly related to the observed calcium\naccumulation around the p62-lipid droplet structures. Intracellular calcium chelators,\nsuch as calmodulin or calcineurin, were neither enriched nor depleted upon ATG5\nknockdown. However, we identified a calcium channel and a regulator of calcium\nhomeostasis in the ATG5 knockdown but not in the endogeneous ATG5 sample: First,\nITPR2, an ER-resident, inositol 1,4,5-trisphosphate-gated calcium channel and,\nsecond, MCUb known as a negative regulator for the mitochondrial calcium uniporter.\nThe former proteins, i.e. ITPR channels, release calcium from the ER and have been\nshown affect autophagy65.The latter protein, i.e. MCUb, has been described to block\ncalcium overload of nearby mitochondria, and also been associated with the early\nstages of autophagy and mitophagy66–68. Together, our proximity proteomics\nexperiments on the ATG5 knockdown induced p62-encapsulated lipid droplets revealed\nproteins such as SIRT1 and alpha-synuclein, linking lipid droplets to autophagy.\nMoreover, upon ATG5 knockdown proteins involved in calcium transport from the ER\nsuch as ITPR2 and MCUb were found in close proximity to p62 supporting the notion\nthat the observed calcium layer and nearby single phospholipid bilayer may represent\ncalcium released from the ER.\nWe also highlight the role of identified ER calcium transporters in the discussion on page 30:\nThe fact that we identified calcium transporters in our proteomics studies, such as\nITPR2, further lends support to this hypothesis. ITPR2, an inositol 1,4,5-trisphosphate-\ngated calcium channel found in the ER membrane, has been shown to be involved in\nautophagy initiation and senescence66. Additional reports demonstrated that\nknockdown of its paralogue, ITPR1, led to autophagosome accumulation92, and\nITPR2/ITPR3 knockdown reduced FIP200 punctae, WIPI2 punctae and LC3 punctae\nunder starvation conditions90.\n5. Similarly, what is the rationale for attributing the detection of the atoms by EDX spectroscopy\nto inorganic molecules such as calcium phosphate as opposed to calcium, phosphorus and\nmagnesium being incorporated into proteins within the LD coat? What about calcium-binding\nand phosphorylated proteins? Is it similar to the oxygen, which is also clearly present within\nthe coat, and which would be assumed to be incorporated into proteins and other biomolecules\nin the cytoplasm but also on the surface of LDs? Would calcium chelators or phosphatases\ndisrupt the association of respective atoms with LDs? Fully answering these questions might\nnot be possible within this study but the authors should provide a balanced interpretation of\ntheir data.\nFor the response, please refer to point 4 above. The performed proximity labeling addresses\nthe question.\n3\n6. What is the connection between the filaments detected in vitro with the p62 coat on LDs?\nThere is no evidence that p62 in situ is polymerised, and beyond the effect of calcium on the\nfilament bundling in vitro there is no support for the filament organisation of p62 on the surface\nof the LD in the diagram 6I.\nWe thank the reviewer for pointing out this question and added our state of understanding to\nthe discussion (page 29):\nAn intriguing question arises whether the here observed double-helical filaments from\npurified material are compatible with our cryo-ET observations in cells. Given the dense\ncellular environment and limited resolution of the lamellae we could not find direct visual\nevidence of the helically assembled filaments. Nevertheless, we located p62\nsurrounding the lipid droplet as part of a calcium-rich layer and believe it to be present\nas structurally modified polymeric assemblies like the ones visualized on the liposome\nsurface or our in vitro reconstituted calcium-induced filament bundles (see Figure 3D\nand 6H).\n7. Will the in situ cryo-EM be able to clarify this issue now or in the future?\nAs actin filaments (7 nm width) are routinely visualized in vitrified cells by cryo-ET, the currently\nachievable resolution is sufficient to locate such structures in cells. Therefore, at this moment\nit appears that the chosen ATG5 knockdown conditions do not favor the double-helical filament\nassembly of p62 while they are compatible with polymeric assemblies as part of the calcium-\nrich layer (refer to point 6 above).\n8. Likewise, the rationale for including ubiquitinated proteins on LD surfaces in Figure 6I\nrequires clarification. Do the authors have direct evidence that p62 recruitment is driven by\nubiquitination? Are LDs displaying p62 ubiquitination-enriched, while those lacking p62 are\nnot? The authors should discuss known ubiquitination machinery implicated in lipid droplet\nubiquitination (doi: 10.1016/j.bbalip.2017.06.006), and assess whether these might mediate\np62 recruitment. This contextualisation would provide a more comprehensive mechanistic\nperspective.\nWe attempted to address this contextualization question and discuss our proteomic results in\nthe manuscript (page 27):\nHowever, without an additional ubiquitin enrichment step we were unable to\nexperimentally confirm that PLIN ubiquitination recruited p62 to these lipid droplets,\nand\nwe did not identify other proteins of the lipid droplet ubiquitination machinery76 in our\nproteomic mass spectrometry experiments.\nMinor comments:\n9. In line 73 of introduction, the authors refer to the characterisation of other selective\nautophagy receptors beyond p62 in mitophagy. Various SARs have been identified as cargo\nreceptors across a wide range of autophagy subtypes, so it might be worth referencing a\nreview that provides an overview of this (e.g., Farre & Subramani 2016, doi:\n10.1038/nrm.2016.74), rather than just referring to mitophagy alone.\nWe added the review reference as requested.\n8. When describing domains of p62 in lines 87-89, it is worth noting the disordered linker region\nbetween the PB1 and ZZ domains, and its possible role in p62 disulphide-linked conjugate\nformation (e.g. doi: 10.1038/s41467-017-02746-z).\nWe added the reference and mentioned the disulphide-linked conjugate as requested.\n9. In the seconds paragraph of the discussion, the authors describe the unwinding and\nstructural opening up of the double-helical filament architecture to form a p62 “bubble”\nstructure, allowing access to binding sites. A diagrammatic figure may be useful here to help\n4\nthe reader visualise what is meant by this.\nStimulated by the reviewer’s comment, in order to help the discussion on this item, we included\na little cartoon of this bubble (Supplementary Fig. 11A).\nReviewer #2 (Remarks to the Author):\n1. The manuscript is very comprehensive but a bit difficult to read because of many\nabbreviations and different techniques that are presented within the experimental analysis. It\nalso contains a lot of data, probably enough for two papers. Overall, all experiments seem\ntechnically sound and according to current standards.\nThank you for the overall positive assessment of the manuscript. We firmly believe that\ndifferent techniques are essential to comprehensively characterize the structure and function\nof this important autophagy receptor p62/SQSTM1. We made sure that the used abbreviations\nare not excessive while respecting the standards of the respective fields. We will keep them\nrestricted to ascertain readability while they are transparently introduced at the beginning of\nthe manuscript.\n2. The method section is sufficient, only a description for AlphaFold3 usage (especially for\ncreating filaments) is missing.\nAccording to the request, we added a dedicated methods section (page 43) in addition to the\npreviously presented supplementary Figure S2:\nTo model the p62 filament structure, we used AlphaFold3 (AF3) multimer predictions\n(Abramson et al., 2024): first we tested p62 full-length sequences arranged in dimers,\ntrimers, tetramers up to decamers. AF3 consistently favored closed ring-like\nassemblies, even for small oligomers (≥3 subunits) while for larger rings the resulting\np62 rings exhibited close-to-rotational symmetry. A single p62 subunit was excised from\na AF3-generated p62 closed ring-like decamer with a diameter of 145 Å approaching\nthe diameter of the filament. To relate these predictions to the p62 filament, we fitted\nthe PB1 domain in the corresponding well-resolved density of one strand of the filament\nmap while including the full-length p62 model and repeated the operation for the\nopposite PB1 strand. Using the determined helical symmetry parameters of 11.0 Å\nhelical rise and a -26.1° helical rotation, we propagated the two subunits along the\nhelical axis, generating a double-helical filament model composed of repeating p62\nunits. This helical assembly yielded a plausible model of the p62 filament, with subunits\nwell fitting the experimental cryo-EM density while maintaining interaction geometries\nclose to the AF3-predicted oligomers.\nReviewer #4 (Remarks to the Author):\nThis manuscript “NCOMMS-25-03646” presents a comprehensive structural analysis of full-\nlength p62/SQSTM1, combining state-of-the-art techniques such as high-resolution cryo-\nelectron microscopy single particle analysis (SPA), in situ cryo-electron tomography (cryo-ET),\ncomplementary biochemical methods, correlative light and electron microscopy (CLEM),\nSTEM, and EDX. The authors describe a double-helical filament architecture built upon a PB1\ndomain scaffold with a flexible C-terminal region. They further investigate how binding partners\n(LC3b and polyubiquitin) and divalent cations influence filament organization. Additionally, the\nin situ imaging of p62-enwrapped lipid droplets with a calcium phosphate-rich coat offers novel\ninsights into early autophagy stages. Overall, the study is robust and provides valuable\nstructural insights linking molecular architecture to cellular function in autophagy. I recommend\nacceptance following the consideration of one major and several minor points:\nWe thank the reviewer for the overall positive assessment of the manuscript.\nMajor:\n5\n1. Low-resolution regions:\nAlthough the PB1 domain scaffold is resolved at ~4.5 Å, the flexible C-terminal and ZZ domains\nremain poorly defined. These lower-resolution regions are crucial for interpreting binding\ninteractions.\nFor clarification: the limited resolution of the densities C-terminal to the ZZ-domain is due to\nmolecular flexibility (complete or partial structural disorder). Such structures cannot be\nresolved by typical averaging based structural methods such as cryo-EM in particular when\nthey do not follow the imposed helical symmetry. We added a clarifying sentence in the results\nsection (page 6):\nThe densities of the associated C-terminal domains were weak and poorly resolved as\nthey did not follow helical symmetry presumably due to molecular flexibility with respect\nto the PB1 strands, which is in agreement with the assigned structurally disordered\nregion in residues 169-33821.\nWe added another sentence to the discussion (page 25):\nThe density located C-terminally to the ZZ-domain could only be resolved at lower\nresolution as it does not strictly follow the helical symmetry of the PB1 scaffold. An\nadditional reason for the limited resolution is likely the at least partial structural disorder\nof the region 168 – 338 in agreement with previous disorder predictions21 also reflected\nby the lower confidence score of the AF3 predictions included in our modeled p62\nfilament.\n2. Although the authors present AlphaFold predictions and some binding experiment using\nimaging method, I suggest including additional biochemical characterization to further\nstrengthen these conclusions.\nWe thank the reviewer for the suggestion. In the light of the reviewer’s comment, we wanted\nto share that we performed proposed biochemical characterization for full-length p62 with LC3\nand ubiquitin using surface plasmon resonance (SPR) and bilayer interferometry (BLI),\nhowever, without success. We believe that the results were not conclusive due to the\naggregation-prone nature of full-length p62. Once full-length p62 is freed from its MPB-tag,\np62 maintains an immediate assembly/disassembly equilibrium. In this context, we emphasize\nthis point in the discussion section of the manuscript (page 25):\nPrevious studies have successfully employed bulk structural and biochemical\ncharacterization methods to characterize p62 interactions but only for relevant p62\nfragments, e.g. with the isolated PB1 domain, LIR motif peptide and UBA domain12,70,71\nbut not for full-length p62 investigated here. When working with full-length p62, bulk\ncharacterization methods are often not conclusive due to the contributions of p62 self-\ninteractions and associated heterogeneity. Therefore, we turned to single-molecule\nstructure characterization methods such as negative staining and cryo-EM as well as\nsingle-particle fluorescence microscopy that allow targeted identification of the relevant\nsubpopulations for the interactions.\nMinor suggestions:\n3. It would be beneficial if the authors could explain why the resolution for the full-length\nstructure does not match that previously achieved for the isolated PB1 domain in 2020.\nRegarding the resolution, please refer to the response of point 1.\nWe highlight the structural differences between the isolated PB1 and full-length p62\nassemblies more clearly in the discussion (page 25):\nIn contrast, the more recently determined PB1 (1-102) assemblies22 acquired on direct\nelectron detectors allowed reliable atomic model building revealing triple or quadruple\nlength structure revealed only two well-resolved antiparallel PB1 scaffold strands\nresulting in an open major groove while the previously determined PB1 domain-only\nassemblies had one or two additional PB1 strands inserted into the closed helical\nassembly. In the full-length structure, the space required by the additional 340 C-\nterminal amino acids restricts the assembly to two antiparallel PB1 strands leaving\nspace for a major groove.\n4. An explanation of the differences observed at varying concentrations of p62 filament and\nGST-4xUbiquitin between Figure 2C (condensates) and Figure 3B (bundles) would be\nappreciated.\nThe differences arose primarily from different experimental requirements in the single-\nmolecule TIRF microscopy and the cryo-EM studies. We added a clarifying discussion on page\n25:\nAs a consequence of the chosen single-molecule characterization methods, for\nsuccessful detection of the relevant interactions we also had to adjust the p62 working\nconcentrations to nM and μM for single-particle fluorescence microscopy/negative\nstaining EM and cryo-EM, respectively. These concentration differences also explain\nthe differences in the observed fine p62 structures after incubation with GST-4xUb, e.g.\nbetween negative staining EM and cryo-EM (see Figure 2C and 3B). The formation of\nlong filaments is favored at μM over the much shorter p62 fragments at nM\nconcentration while both morphologies are consistent with the formation of cross-linked\nlarger p62 assemblies.\n3) Figures 6D and E (right EDX figures) should include scale bars.\nWe added scale bars as requested.\n4) In Figure S1A, it would be helpful to present the 2D classification results at the same scale\nacross different pH conditions.\nIn order to address this point, we updated the class averages at the same scale.\n5) Typo error: line 1169 (methods, Cryo-electron tomography of reconstituted p62 filaments,\nline 5) should read “63,000x magnification” instead of “63.000x magnification.”\nThank you for spotting the typo.\nReviewer #5 (Remarks to the Author):\nThe authors present cryo-electron microscopy imaging of the selective autophagy receptor\np62, revealing that full-length p62 forms regular double-helix filaments in vitro. AlphaFold3 was\nused to predict the interactions of full-length p62 filaments with LC3b and ubiquitin. The\ninteraction of p62 with soluble LC3b, LC3B-conjugated liposome membranes and poly-\nubiquitin was studied. These three interactions led to structural disassembly, lateral\nassociation and bundling, respectively, providing insights into how p62 filaments respond to\nautophagy-related factors.\nIn vivo experiments showed that p62 puncta colocalized with lipid droplets in cells, with p62\nforming a discontinuous coat around the droplets. Elemental analysis of the p62 coat revealed\nan enrichment of calcium and phosphorus, and the effects of other divalent cations were also\nexamined. The authors argue that these findings support the hypothesis that calcium ions may\nbe a key regulator in autophagy.\nWhile this manuscript presents an improved structural model of p62 filaments, the biological\nsignificance of these findings is unclear, and the possible significance for autophagy\nmechanisms in cells was not clearly stated by the authors. Judging the significance is also\nmade harder by the fact that the context of the present work within the history of this subfield\nis not clearly spelled out - a general reader would find it difficult to know what precisely has\nbeen established in this field, and what has not been established. Further, the in vitro and in\nvivo experiments are somewhat disconnected.\nWe thank the reviewer for taking the time to review the manuscript in detail.\nMajor comments:\n1. The findings presented in this manuscript are close to previously published findings by the\nsame authors in [Ciuffa et al., 2015] and [Jakobi et al., 2020]. The present study is a worthy\nextension of these earlier studies; however, the present manuscript’s additional impact on the\ncommunity’s understanding of autophagy appears somewhat limited.\nWe respectfully disagree with the conclusion of this reviewer. Ciuffa et al. established that\nhelical filaments of p62 are formed in vitro by low-resolution cryo-EM (see more details on\npoint 2 below) while Jakobi et al. focused the structural studies on the close-to-atomic\nresolution structure of the p62-PB1 domain and its requirements in cells. The current study\nreveals state-of-the-art structural results and addresses, first, the full-length p62 three-\ndimensional double-helical organization (that was not studied previously) and second, for the\nfirst time, by high-resolution cryo-EM in cellulo of how p62 encapsulates lipid droplets during\nlipophagy and discovers an unexpected role in divalent cation p62 binding. In point 3, this\nreviewer requests a model for better understanding of the autophagy process. We are happy\nto provide and highlight these novel details in the revised version of the manuscript.\n2. The authors state that “in contrast to the previously determined PB1 assemblies [Jakobi et\nal., 2020], the here determined full-length p62 filament structure formed a double helical\nfilament architecture”. However, their previous study, [Ciuffa et al., 2015], reported that\nfilaments of PB1-ZZ (a truncated version of full length p62) are organized in a double helix,\nwhile full-length p62 filaments consist only of a single strand [Ciuffa et al., 2015]. Thus, the\nauthors own earlier findings appear to contradict their findings in the present work. This is not\ndiscussed in the manuscript.\nWe believe that this point (as well as related point 1) most likely arose from a lack of technical\nunderstanding of the associated structural EM data quality in our previous Ciuffa et al., Jakobi\net al. and the current manuscripts. Therefore, we explain the different structural findings in the\ndiscussion in comparison (page 25):\nOur own earlier work of full-length p62 revealed the ability to form filamentous\nassemblies while the cryo-EM images were acquired on a CCD camera and single as\nwell as double-helical filaments could not be discerned unambiguously at the limited\nresolution21. In contrast, the more recently determined PB1 (1-102) assemblies22\nacquired on direct electron detectors allowed reliable atomic model building revealing\ntriple or quadruple PB1 domain strands leading to a closed tube. In comparison, the\nhere determined full-length structure revealed only two well-resolved antiparallel PB1\nscaffold strands resulting in an open major groove while the previously determined PB1\ndomain-only assemblies had one or two additional PB1 strands inserted into the closed\nhelical assembly.\nIn this manuscript, we unambiguously determined the double-helical architecture of full-length\np62 at 4.5 Å resolution and deposited the PDB coordinates in the protein data bank fully\ntransparently representing state-of-the-art cryo-EM standards.\n8\n3. Additionally, Ciuffa et al. described that the C-terminal part of the protein forms a solvent-\naccessible stretch, a feature that is also highlighted in this manuscript. Furthermore, Ciuffa et\nal. found that octa-ubiquitin but not LC3 provokes filament shortening, which seems to\nsignificantly overlap with the work presented in this manuscript (see section “P62 filament\ninteractions with LC3b and ubiquitin”). The authors should clarify how their findings differ from\nor extend these previous studies.\nWe understand the confusion that may have arisen from comparing the two studies. We wish\nto clarify this confusion briefly for the reviewer:\nIn Cuiffa et al., we included two basic binding experiments simply mixing p62 filaments with\nLC3 and octa-ubiquitin separately and reported co-sedimentation and negatively stained\nmicrographs. Here, we significantly go beyond the earlier work, perform single-particle\nfluorescence microscopy to record the association over time and show a comprehensive\nanalysis of the interactors including their mixtures (ubiquitin + LC3) mimicking condensate-\nforming conditions and perform experiments in the presence of membrane-conjugated LC3.\nMoreover, we explore the importance of different concentrations highly relevant for these\ninteractions (refer to point reviewer #4, point 4). Furthermore, we spent significant efforts to\nanalyze the associated fine structures of p62 assemblies by cryo-electron tomography at\nunprecedented detail including image analysis of segmentation to characterize the molecular\nmorphologies of the observed condensates and filament bundles. Given this comprehensive\nset of new experimental setups, we cannot fully understand the conclusion of this reviewer.\n3. It would really help if the context of the work presented in this manuscript were clearly\ndescribed. The manuscript would greatly benefit from a clear exposition, including an\nexplanatory figure, of the current understanding of autophagy as it involves p62, LC3,\nubiquitin, Atg8 family proteins, membrane compartments etc etc. For a general reader it is very\ndifficult to comprehend from the present Introduction and other parts of the manuscript.\nWe understand the reviewer’s concern. While we cannot dedicate an entire Figure 1 just\nsummarizing previous results, we extended the introduction text with regards to that comment\n(page 3): “At the early stages of autophagy, selective autophagy receptors like p62 thereby\nbridge ubiquitinated cargo via conjugated Atg8 to the forming autophagosome.” We also added\na minimal panel to Figure 1A including the primary structure and binding partners.\n4. The biological significance of this study is not stated, and no “model” figure is presented that\nvisually announces the authors’ hypotheses. What do these findings tell us about the\nmechanisms of p62-mediated autophagy?\nWe thank the reviewer for pointing this out. We addressed the wider biological mechanism by\ntwo far-reaching experiments: First, we extended the analysis of ATG5 knockout with respect\nto p62 to primary neurons and brain cells. Second, we initiated a proteomic analysis of the p62\nproximity labeled cells (refer to response of reviewer #1, point 4).\nFirst, we initiated a study in an alternative primary neuron cell line as well as cortical neurons\nwith ATG5 knocked out and confirmed the wider presence of the described p62 positive lipid\ndroplet structures in cells using fluorescence microscopy (page 14):\nIn order to verify that our observations were not limited to adherent RPE1 cells, we\nextended our experiments to mouse primary neurons and brain cortical tissue. First, we\nisolated primary neuronal cells from conditional Atg5flox:CAG-CreTmx newborn mice, a\nmodel previously shown to exhibit robust and complete knockout (KO) of ATG555. After\nfixation, we labeled WT and KO astrocytes and neurons with an antibody against p62,\nand stained lipid droplets with Lipidspot488 (Figure 4D-G). Second, we analyzed\ncortical brain sections from three-month-old WT and conditional ATG5 (cKO) mice, in\nwhich ATG5 deletion is driven by the neuronal-specific Scl32a1 promoter56. These\nsections were stained with antibodies against p62 and Perilipin1, a protein associated\n9\nwith the surface of lipid droplets (Figure 4H-I). We used confocal microscopy to image\nthese samples and quantified the amount of lipid droplets per cell, the amount of p62\npunctae per cell, the average size of the lipid droplets and the percentage of lipid\ndroplets that co-localized with p62 signal in each cell. Both in human and mouse cells,\nwe observed an increase in the amount of lipid droplets, while this effect was most\npronounced in mouse primary KO astrocytes and cortical 3-month-old KO neurons\n(Figure 4J). Additionally, as expected, we found a significant increase in the number of\np62 punctae upon ATG5 KD or KO in the analyzed cells (Figure 4K). In the absence of\nATG5, the average size of the lipid droplets increased from 0.3 to 0.4 μm2, from 0.3 to\n0.6 μm2, from 0.7 to 0.8 μm2, for mCherry-p62 RPE1, mouse astrocytes and neurons,\nrespectively (Figure 4L). The mouse cortical neurons cells were harder to quantify as\nthey, on average, contained few, very large lipid droplets. Finally, we also detected a\nsignificant increase in the number of lipid droplets that co-localized with p62 punctae in\nthose samples lacking ATG5 (Figure 4M).\nIn the discussion on page 27, we highlight:\nWe observed more and larger lipid droplets in our ATG5 knockdown cells, as well as a\nhigher percentage of lipid droplets co-localized with p62. We confirmed that this is not\na unique phenotype limited to human RPE1 cells, as we observed the same trends in\nmouse ATG5 KO primary astrocytes, mouse primary neurons and mouse cortex (see\nFigure 4).\n5a. For example, the influence of LC3b and poly-ubiquitin on p62 is studied – but how is this\nrelated to autophagy?\nIn the paragraphs on page 3 and 4 of the manuscript, we openly introduced how LC3b and\npoly-ubiquitin binding are relevant to autophagy progression and point to the critical\nreferences.\nb. Another example: in the section “p62 filament interactions with LC3b and ubiquitin”, p62\nfilaments form condensates in the presence of ubiquitin, whereas, in “p62 filament membrane\ninteractions”, ubiquitin instead induces parallel filaments bundles. Which of these two\nbehaviors better reflects in vivo conditions? Again, what do these two different outcomes tell\nus about autophagy?\nThe observed differences are related to the chosen different experimental conditions. It was\nour aim to recapitulate the key binding events of selective autophagy in the test tube – a\ncommon strategy of biochemical studies also known as bottom-up. First, we mixed p62\nfilaments and LC3 and GST-4xUb in solution concluding correctly: “filaments form\ncondensates in the presence of ubiquitin”. Second, we mixed p62 filaments with LC3-\nconjugated liposomes and GST-4xUb and conclude the formation of filament bundles. In the\nsecond experiment, we are obviously closer to the cellular situation as LC3 is conjugated to\nlipid bilayer membrane. All of the studies aim to study the fine structure of p62 assemblies\nupon addition of the relevant molecular components involved in the early events of selective\nautophagy. While both outcomes are somewhat different, both have common features: the\nrecruitment of key binding partners of LC3 and Ubiquitin to polymeric p62 assemblies.\nc. Another example: Ca and p62 are reported to colocalize on lipid droplets in cells – what\nmechanism involving Ca does this suggest to the authors?\nWe laid out our findings and showed for the first time the detailed characterization of the ATG5\nknockdown condition. Surprisingly, we discover a role for calcium together with p62 in a layer\nsurrounding lipid droplet cargo. We realize that we have not concluded so clearly on a role in\nautophagy. We now present a model of how the calcium-rich p62 layer surrounding a lipid\ndroplet may be involved in the early stages of selective autophagy (Figure 7E, page 30):\nGiven the multitude of observations in a series of biochemical and cellular experiments,\nwe summarize our findings in a model of the early stages of selective autophagy (Figure\n10\n7E): p62 polymers recognize the ubiquitinated surface of lipid droplets destined for\ndegradation in proximity to the ER. In parallel, an unknown signal leads to calcium efflux\nfrom the ER that is collected by the p62 polymers surrounding the cargo with a calcium-\nrich layer. Additional autophagy factors are recruited leading to the progression of\nautophagy, e.g. the ATG5/12/16 complex conjugating LC3b to the phosphatidyl\nethanolamine (PE) membrane and thereby mediating phagophore formation and\nextension up to an autophagosome. In the absence of ATG5 membrane extension,\nLC3b is not conjugated to PE leading to an accumulation of the calcium-rich p62\npositive lipid droplet cargo structures.\n6. The authors emphasize that most p62 filaments have regular helical structure at pH=6, while\na fraction unwind at pH=8. However, in the section “p62 filament interactions with LC3b and\nubiquitin”, the authors polymerized p62 filaments at pH=8. Why is this pH used, when this\napparently disrupts filaments?\nThis point was also independently raised by Reviewer #1, point 2. Find the response above.\nWe clarified our rationale in the respective results sections:\na) On page 5 rationale for cryo-EM structure determination including new Supplementary\nFigure 1A: “As previous studies suggested an effect of pH on the assembly conditions45,\nwe compared the preparations at different pHs ranging from 6 to 8 (Supplementary\nFigure 1A).”\nb) On page 8 rationale for binding studies: “We used the above-characterized preparations\nof p62 filaments at pH=8 as they were closer to physiological pH than the stable pH 6\nfilaments formed for structural analysis.”\n7. In the section “p62 filament membrane interactions”, the authors characterized the\nultrastructure of p62 filaments at a high concentration and at pH=6. However, in subsequent\nexperiments, they used a 10-fold lower concentration and pH=7.4. How does this impact the\nultrastructure?\nThis point on different experimental concentrations was independently raised by Reviewer #4,\npoint 4. Primarily, they are restricted by the employed experimental characterization method.\nTherefore, we took care to report them transparently. Refer to the response above.\n8. The authors revealed the structural organization of p62 in human RPE1 cells. However, they\ndid not establish a clear connection between the in vivo and in vitro experiments. Specifically,\nthe physiological concentration of p62 in cells was not reported. Was it comparable to the in\nvitro conditions? Providing this information would help interpret the relevance of the in vitro\nfindings in the cellular context.\nRelating the in vivo concentrations with the in vitro experiments is an intriguing question that\nwe included to address in the discussion on page 28:\nAs we learnt from our in vitro experiments, the tendency of p62 filament\nassembly/disassembly and structural properties such as filament length is dependent\non the concentrations present in the test tube. In this context, it is an important question\nwhat the cellular concentration of p62 is. Previous reports estimated for mouse\nfibroblast cells and HEK293T cells the average p62 copy number to be 25,000 and\n97,000, respectively82,83. Assuming an average cytoplasmic volume of 1500 μm3 of an\nRPE1 cell84, the resulting average concentration is between 0.1 μM/100 nM and 0.4/400\nnM. This concentration is about three orders of magnitude lower than the 2 mg/ml (41.9\nμM) used in this study to generate filaments for structural analysis. While the 100 nM\nconcentrations are of the same order of magnitude as used in the nM binding studies\nexamined by single-particle fluorescence microscopy and negative staining EM.\nNevertheless, in the cell we expect significant variation in local concentrations, e.g.\nwhen p62 forms a condensate or a dense layer around a lipid droplet cargo, making\n11\nsignificant local structural enrichment and effective higher concentrations possible.\nMinor comments:\n1. Fig. 2G and 2H show the distribution of p62 filament length in the absence and presence of\nLC3b, suggesting that LC3b induces depolymerization. For a clearer comparison, plotting both\ndistributions within the same graph would be more effective.\nWe agree with the reviewer and made the two graphs more comparable including the statistical\nsignificance tests that were requested by point1 of reviewer #1.\n2. In Fig. S7F, the label for the peak at around e = 500 eV is missing.\nAdded as requested.\n3. The authors compare the p62 structure to the DNA double-helix model (Line 494). However,\ngiven the subunit composition and the pitch of the p62 double helix filaments, perhaps a\ncomparison to actin filaments or microtubules might be more appropriate.\nThe comparison to the DNA double helix is intriguing in one aspect as a pH dependent opening\nmay affect the accessibility. Based on the suggestion of reviewer #1, point 9, we included an\nexplanatory figure (Supplementary Fig. 11A) in to make this point clearer. We do not believe\nthat actin filaments or microtubules are necessarily a good comparison as they do not share\nthe basic antiparallel architecture and they tend to be more stable polymers easily discernible\nin electron microscopy images as opposed to the more flexible nature of the p62 filaments.\nReviewer #1 (Remarks to the Author):\nThe authors performed extensive revisions which significantly improved the manuscript. The new\nproteomics data adds to the proposed working model of the p62/Ca2+ coat formation. Other concerns\nfrom this reviewers were also constructively addressed. As such, I would recommend the manuscript for\npublication in the current form.\nI would still recommend spell check of the text and figures, e.g. Holliday junctions in one of the figures is\nspelled with one L.\nCorrected as requested.\n\"In cellulo\" should be spelled \"in cellula\" according to Latin grammar.\nThe term “in cellulo” is not genuine Latin, but has been coined in recent biology to mean experiments\nconducted within live cells (often in tissue culture or isolated single cells but not a whole organism)\n(https://doi.org/10.1016/j.sbi.2024.102843). To avoid any confusion, we revert to the more commonly\nused term “in situ” instead.\nI’ve read the Reviewer 5 comments and responses by the authors and I think they are satisfactory. It is\nimpossible for me to know if the Reviewer would accept them, but from the extensive experimental and\ntextual revisions I would say most or all of the criticisms have been addressed. The authors did a very\nnice job in providing clarifications requested by the reviewer and formalising their model further.\nUndoubtedly, there are many uncertainties about the role of p62 and inorganic molecules at LDs but the\ndata is robust, technology is cutting edge, and scientific advance and significance for the autophagy field\nand cell biology in general is obvious to me.\nReviewer #2 (Remarks to the Author):\nThe revised manuscript is fundamentally o.k.. It is in my opinion too long, however. But this is an editorial\ndecision.\nNo changes required.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the\nNature Communications initiative to facilitate training in peer review and to provide appropriate\nrecognition for Early Career Researchers who co-review manuscripts.\nNo changes required.\nReviewer #4 (Remarks to the Author):\nThe revision substantially strengthens the manuscript and addresses most of my previous comments. I\nrecommend acceptance pending minor revisions and one concern.\nConcern:\n1) The authors report a total dose of 500 e/Å^2 for the EDX measurements. I am concerned that the dose\nmight be too high, which caused too much damage to the sample.\nThank you for raising the concern. The reviewer may have compared the typical doses applied in cryo-ET\nwith the reported total dose of 500 e-/Å2 used for the EDX analysis. As reported in the manuscript (page\n52): “EDX spectra were recorded with a 50 μs dwell time and 50 frames resulting in a total dose of 500 e-\n/Å2”. Therefore, we were able to examine intermediate stages of the recording. Between 300 and 400 e-\n/Å2, we observed initial sample bubbling and the resulting damage most likely compromised highest\nspatial resolution while the integrity of the ultrastructure (lipid droplet and electron-dense ring) was not\naffected. When we analyzed EDX spectra of the relevant areas from a total of 300 e-/Å2, we qualitatively\nidentified the same elements as presented in Figure 6 and Supplementary Figures 7-9. Given this\nobservation, we have no evidence for any dose-induced excessive mass loss that may affect the element\nidentification. As EDX requires significant doses to detect element-specific signal, and therefore, add the\nfollowing sentence to the Methods:\nWe found 500 e-/Å2, to be a good trade-off between high signal-to-noise ratio in the EDX spectra\nwhile still maintaining the ultrastructural integrity of the sample.\nMinor revisions\n2) Total tomography dose: Please recalculate and confirm the cumulative dose; the per-tilt values\nreported in Methods (lines 1030–1031 and 1233–1235) imply a total dose of ~80 e⁻ /Å².\nThis was not an error. We added clarifying statements to both parts: the basic dose per tilt estimate is\nonly valid for 0º tilt while it is increased for higher tilts as part of a SerialEM setting:\nEach tilt was recorded as movie … corresponding to a dose of approximately 2.0 e/Å2 at 0º tilt…\nAll tilt series were recorded from -54° to 54° with 3° increments, using a dose-symmetric\nbidirectional acquisition scheme including ‘the varied intensity’ option (i.e. higher tilt angles\nreceived higher doses, as 1/cosine of the tilt angle) in SerialEM.\n3) Typo: Methods, “Cryo-electron tomography of reconstituted p62 filaments,” line 5 (around line 1030)\nshould read “63,000× magnification” (not “63.000×”).\nChanged as requested.\n4) Supplementary figure quality: Supplementary Figures 7–9 are more blurred in the current merged file\nthan in the previous version. Please check the file format/resolution and restore the prior quality.\nThank you for pointing out this quality deterioration. We made sure to import high-resolution/quality\nimages in the provided Supplementary information file."}]

Decision letter