Journal of Cell Biology

While the physical interactions between the Golgi apparatus (Golgi) and lipid droplets (LDs) have been suggested through system-level imaging, the bona fide and functional Golgi-LD membrane contact sites (MCSs) remain largely uncharacterized. Here, we demonstrate that vacuolar protein sorting-associated protein 13B (VPS13B) mediates trans-Golgi network (TGN)-LD interactions. VPS13B is specifically accumulated at TGN-LD MCSs with its C-terminal region targeting LDs via an amphipathic helix while a putative WD40 module and a C-terminal Pleckstrin homology (PH) domain independently recognizing TGN via directly binding to Rab6. A putative lipid transfer domain (LTD) at the N-terminal portion of VPS13B binds glycerophospholipids in vitro. VPS13B suppression results in severe fragmentation of the TGN, an effect that can be almost completely rescued by the expression of VPS13B-LTD. Collectively, our findings demonstrate that VPS13B mediates lipid transfer at TGN-LD MCSs to maintain TGN integrity and function.

The Golgi-localized, {gamma}-ear containing, ADP-ribosylation factor binding proteins (GGAs) are a family of adaptor proteins that regulate transport of specific cargo receptors from the Golgi to endosomes. For many years it was assumed that GGAs transport cargo via interaction with the adaptor complex AP-1. However, recent findings suggest that GGA and AP-1 may have opposing roles, with GGAs facilitating forward transport between Golgi and endosomes, and AP-1 mediating the opposite trafficking step. To shed light on the functional connection of GGAs with AP-1, we combined CRISPR-Cas9 gene editing with live-cell imaging and TurboID-based proximity labelling. We find that GGAs localize not only to the Golgi apparatus but also, to a greater extent, to peripheral ARF1-positive compartments responsible for secretory trafficking and endocytic recycling. At both, the Golgi and peripheral sites, we observe distinct sorting domains containing either AP-1 or GGAs alone, as well as domains in which both adaptors are present. Interestingly, GGAs can recruit clathrin lattices independently of AP-1. Proximome mapping shows that AP-1 specific cargoes only localize to AP-1 domains in the absence of GGAs. These findings point to a regulatory role of GGAs in AP-1 transport. We speculate that GGAs prevent binding of AP-1 to its cargo clients to avoid premature retrieval and to modulate bi-directional trafficking between the Golgi and endosomes.

The eukaryotic p24 family, consisting of -, {beta}-, {gamma}- and {delta}-p24 subfamilies, has long been known to be involved in regulating secretion. Despite increasing interest in these proteins, fundamental questions remain about their role. Here, we systematically investigated Drosophila p24 proteins. We discovered that members of all four p24 subfamilies are required for general secretion, and that their localizations between ER exit site (ERES) and Golgi are interdependent in an [->]{beta}{delta}[->]{gamma} sequence. We also found that localization of p24 proteins and ERES determinant Tango1 requires interaction through their respective GOLD and SH3 lumenal domains, with Tango1 loss sending p24 proteins to the plasma membrane and vice versa. Finally, we show that p24 loss expands the COPII zone at ERES and increases the number of ER-Golgi vesicles, supporting a restrictive role of p24 proteins on vesicle budding for efficient transport. Our results reveal Tango1-p24 interplay as central to the generation of a stable ER-Golgi interface. SummaryYang et al. systematically analyze in Drosophila the function of the four p24 protein subfamilies and discover that interaction with Tango1 is essential for their concentration between ER and Golgi and for efficiency of COPII-mediated general secretory transport.

Macroautophagy (autophagy) is a fundamental catabolic process requiring the biogenesis of the autophagosome to support cell survival during stress. While the roles of F-actin and microtubule cytoskeleton in autophagy are well established, the contribution of intermediate filaments (IFs) remains poorly understood. Here, we investigated the role of the type III IF vimentin in supporting the early steps of starvation-induced autophagy. We demonstrate that starvation triggers a rapid, perinuclear compaction of vimentin IFs, correlating with transient phosphorylation at serine 56 and enhanced overlap with the endoplasmic reticulum (ER). We reveal that autophagic proteins accumulate at the vimentin/ER interface, physically connecting the autophagosome biogenesis machinery to the vimentin IF network. Knock-out or pharmacological perturbation of vimentin-IFs dynamics using Withaferin-A significantly impairs starvation-induced autophagic flux. Mechanistically, we reveal that vimentin IFs are essential coordinators for the mobilization of endosome-ER-membrane contact sites (EERCS), a critical hub for autophagosome nucleation. Together, our findings uncover a novel role for vimentin IFs as a dynamic cytoskeletal coordinator that spatially organizes membrane contact sites to promote the efficient initiation of autophagosome biogenesis in response to nutrient stress. Summary statementThis study reveals that vimentin intermediate filaments rapidly reorganize to mobilize ER-endosome contact sites, establishing a critical spatial platform for starvation-induced autophagy initiation.

Constitutive secretion from the trans-Golgi network (TGN) to the cell surface proceeds via carriers thought to form without a canonical cytoplasmic coat, yet how these carriers are generated remains poorly understood. Here, we identify a distinct population of TGN-to-cell surface carriers transporting influenza hemagglutinin (HA) and uncover a coat-like mechanism underlying their formation. HA carrier biogenesis requires non-vesicular lipid transfer at endoplasmic reticulum (ER)-Golgi membrane contact sites (MCSs) and protein kinase D (PKD) activity. We show that caveolin promotes membrane budding by assembling into cholesterol- and PKD-associated oligomers that act as a membrane-embedded, coat-like scaffold at lipid nanodomain-enriched TGN subdomains. These findings establish caveolin as a structural and regulatory component of TGN export and support a model in which a PKD-caveolin axis couples ER-Golgi lipid transfer to cargo sorting, membrane remodeling and fission during secretory carrier biogenesis.

Golgi fragmentation and ER exit site dissociation are considered as the leading causes of mitotic block of secretion from the ER. Although the mechanisms of Golgi fragmentation have been extensively characterized, ER exit block early in mitosis is not well-understood. We previously found that TANGO1 organizes ER exit sites by directly interacting with Sec16. Here, we showed that TANGO1 is phosphorylated by casein kinase 1 (CK1) during mitosis. Interestingly, the interaction with Sec16 was abrogated by phosphorylation of TANGO1, leading to dissociation of the ER exit sites. Moreover, a TANGO1 mutant deficient in phosphorylation inhibited the mitotic dissociation of ER exit sites. In contrast, a TANGO1 mutant mimicking CK1-mediated phosphorylation dissociated ER exit sites in interphase cells. Although CK1 activity remains constant throughout the cell cycle, PP1, a phosphatase for which activity decreases during mitosis, participates in the regulation of TANGO1 phosphorylation. This is the first report demonstrating the mechanisms of ER exit site dissociation during mitosis.

Tepsin is an established accessory protein found in Adaptor Protein 4 (AP-4) coated vesicles, but the biological role of tepsin remains unknown. AP-4 vesicles originate at the trans-Golgi network (TGN) and target the delivery of ATG9A, a scramblase required for autophagosome biogenesis, to the cell periphery. Using in silico methods, we identified a putative LC3-Interacting Region (LIR) motif in tepsin. Biochemical experiments using purified recombinant proteins indicate tepsin directly binds LC3B, but not other members, of the mammalian ATG8 family. Calorimetry and structural modeling data indicate this interaction occurs with micromolar affinity using the established LC3B LIR docking site. Loss of tepsin in cultured cells dysregulates ATG9A export from the TGN as well as ATG9A distribution at the cell periphery. Tepsin depletion in a mRFP-GFP-LC3B HeLa reporter cell line using siRNA knockdown increases autophagosome volume and number, but does not appear to affect flux through the autophagic pathway. Re-introduction of wild-type tepsin partially rescues ATG9A cargo trafficking defects. In contrast, re-introducing tepsin with a mutated LIR motif or missing N-terminus does not fully rescue altered ATG9A subcellular distribution. Together, these data suggest roles for tepsin in cargo export from the TGN; delivery of ATG9A-positive vesicles at the cell periphery; and in overall maintenance of autophagosome structure.

Vimentin intermediate filaments (VIFs) form complex, tight-packed networks; due to this density, traditional ensemble labeling and imaging approaches cannot accurately discern single filament behavior. To address this, we introduce a sparse vimentin-SunTag labeling strategy to unambiguously visualize individual filament dynamics. This technique confirmed known long-range dynein and kinesin transport of peripheral VIFs and uncovered extensive bidirectional VIF motion within the perinuclear vimentin network, a region we had thought too densely bundled to permit such motility. To examine the nanoscale organization of perinuclear vimentin, we acquired high-resolution electron microscopy volumes of a vitreously frozen cell and reconstructed VIFs and microtubules within a [~]50 {micro}m3 window. Of 583 VIFs identified, most were integrated into long, semi-coherent bundles that fluctuated in width and filament packing density. Unexpectedly, VIFs displayed minimal local co-alignment with microtubules, save for sporadic cross-over sites that we predict facilitate cytoskeletal crosstalk. Overall, this work demonstrates single VIF dynamics and organization in the cellular milieu for the first time SummarySingle-particle tracking demonstrates that individual filaments in bundles of vimentin intermediate filaments are transported in the cytoplasm by motor proteins along microtubules. Furthermore, using 3D FIB-SEM the authors showed that vimentin filament bundles are loosely packed and co-aligned with microtubules.

Human cells face a wide range of external mechanical stimuli that vary with cell type, state, and pathological conditions. The rapidly growing field of mechanobiology investigates how cells sense and respond to these forces. While most work has focused on focal adhesions (FAs), plasma membrane, and nucleus as primary mechanosensors, how reciprocal inside-out signals adapt intracellular organelles to extracellular mechanics has remained largely unexplored. Here, we show that extracellular mechanical signals influence the secretory function of the Golgi apparatus. By subjecting adherent cells to various mechanical challenges -cell spreading on surfaces coated with different ligands, altering substrate stiffness, or applying equibiaxial strains to the cells-, we reveal that extracellular forces modulate Golgi transport carrier biogenesis, thereby regulating exocytosis. Together with modulation of Golgi membrane tension, we identify molecular determinants of the underlying mechanotransduction pathway, including microtubule acetylation, diacylglycerol (DAG) production, and protein kinase D (PKD) activity. These findings uncover a bidirectional mechanotransduction axis in which extracellular mechanics tune Golgi secretory output, providing a framework for investigating organelle-based mechanoadaptation in physiology and disease, particularly in cancer and fibrosis where secretion is critical.

The endoplasmic reticulum (ER) is the master regulator of various cellular processes. To achieve its diverse functions, the ER interacts with other organelles at membrane contact sites, regions where organelles are brought into proximity. Most ER membrane contact sites are facilitated by the ER-resident VAP proteins. To address the role of VAP proteins in regulating cellular lipid homeostasis, we performed a targeted lipidomic screen after silencing individual VAPs. The loss of VAPB increases cellular levels of neutral lipids stored in lipid droplets (LDs). The increase in neutral lipids is reflected in the size, number and motility of LDs, and is due to the impaired degradation of these organelles. VAPB requires its contact site forming ability to regulate LDs, prompting the identification of protein kinase A (PKA) anchor AKAP11 as a VAPB interaction partner in regulating LD degradation and dynamics. Collectively, our findings identify a role for the ER-resident VAPB-AKAP11 interaction and PKA activity in regulating LD homeostasis. SummaryThe ER-resident membrane contact site protein VAPB and its interaction partner AKAP11 regulate lipid droplet size and motility by mediating neutral lipid degradation. VAPB requires its ability to form membrane contact sites and interact with AKAP11, protein kinase A anchor protein, for mediating lipid droplet homeostasis. This paper uncovers a role for VAPB-AKAP11-PKA axis in regulating the homeostasis of lipid droplets.

Mitochondrial fission is required for proper segregation during cell division, quality control, and cellular homeostasis (metabolism and energy production). Despite its importance, models of the process remain speculative. Here we apply cryogenic electron tomography to image the nanoscale architecture of mitochondrial fission in mammalian cells. We find that constriction of the inner and outer membranes is coordinated, suggesting that force on both membranes is applied externally. While we observe ER at constriction sites, it did not encircle constrictions. Instead, we find long bundles of both unbranched actin and septin filaments enriched at constrictions. Actin bundles align with the central region of division bridges and septin bundles with the necks on either side. Septin bundles appear to guide microtubules to constriction sites, suggesting, along with autolysosomes observed in the vicinity, a pathway for mitophagy. Together, our results rule out several existing models for mitochondrial fission and provide empirical parameters to inform the development of realistic coarse-grained models in the future.

The autophagy core machinery mediates the enclosure of cytosolic cargo destined for degradation in the lysosome. The Atg9-Atg2-Atg18 complex coordinates phagophore expansion via directed lipid transfer until closure of the phagophore rim. Using an Atg2 variant (Atg2-PM4) as a model of decelerated autophagosome biogenesis, we visualized the morphological states prior to autophagosome closure by cryogenic correlative light and electron microscopy in S. cerevisiae. Using in situ cryo-electron tomography, we find an enlarged rim morphology of an expanding phagophore in Atg2-PM4 cells in comparison with Atg2 wildtype condition. Analysis of segmented rim membrane features as well as surrounding and attached vesicles suggest that the enlarged rims are a result of cytosolic vesicles fusing with the growing phagophore. High-resolution imaging in this study shows that, apart from the initial nucleation phase, vesicle fusion can also contribute to phagophore expansion during later stages of autophagosome biogenesis.

Autophagy is an essential cellular recycling process that maintains protein and organelle homeostasis. ATG9A vesicle recruitment is a critical early step in autophagy to initiate autophagosome biogenesis. The mechanisms of ATG9A vesicle recruitment are best understood in the context of starvation-induced non-selective autophagy, whereas less is known about the signals driving ATG9A vesicle recruitment to autophagy initiation sites in the absence of nutrient stress. Here we demonstrate that loss of ATG9A or the lipid transfer protein ATG2 leads to the accumulation of phosphorylated p62 aggregates in the context of basal autophagy. Furthermore, we show that p62 degradation requires the lipid scramblase activity of ATG9A. Lastly, we present evidence that poly-ubiquitin is an essential signal that recruits ATG9A and mediates autophagy foci assembly in nutrient replete cells. Together, our data support a ubiquitin-driven model of ATG9A recruitment and autophagosome formation during basal autophagy.

Secretory proteins are sorted at the trans-Golgi network (TGN) for export into specific transport carriers. However, the molecular players involved in this fundamental process remain largely elusive. Here, we identified the human transmembrane protein TGN46 as a receptor for the export of secretory cargo protein PAUF in CARTS - a class of protein kinase D-dependent TGN-to-plasma membrane carriers. We show that TGN46 is necessary for cargo sorting and loading into nascent carriers at the TGN. By combining quantitative fluorescence microscopy and mutagenesis approaches, we further discovered that the lumenal domain of TGN46 encodes for its cargo sorting function. In summary, our results define a cellular function of TGN46 in sorting secretory proteins for export from the TGN.

At the early endosome, cargos are sorted into subdomains; receptors destined for recycling to the plasma membrane are sorted into tubulovesicular structures that undergo fission and release cargo-laden vesicles that traffic along microtubules. Although branched actin has been implicated in the establishment/maintenance of endosomal membrane subdomains, its role in cargo segregation, fission, and recycling has not been extensively studied. Using inhibitors of formin-and ARP2/3-mediated actin assembly, we show that branched actin, but not linear actin, is required for endosome fission and receptor recycling. To examine the spatial relationship between actin and cargo, we transfected cells with constitutively active RAB5 Q79L to generate enlarged endosomes and demonstrated that internalized transferrin localized to discrete endosomal regions adjacent to branched actin. ARP2/3 inhibition disrupted this organization, resulting in broader cargo distribution on the endosomal membrane and coalescence of degradative and retrieval subdomains. Consistent with impaired endosomal sorting and fission, branched actin inhibition led to cargo accumulation. Our findings identify ARP2/3-mediated branched actin as a key regulator of cargo segregation, subdomain maintenance, and fission at the early endosome.

Retrograde protein transport from the cell surface and endosomes to the trans-Golgi network (TGN) is essential for membrane homeostasis in general and for the recycling of mannose-6-phosphate receptors (MPRs) for sorting of lysosomal hydrolases in particular. Several different sorting machineries have been implicated in retrieval from early or late endosomes to the TGN, mostly for the cation-independent MPR (CIMPR), mainly by analysis of steady-state localization and by interaction studies. We employed a nanobody-based sulfation tool to more directly determine transport kinetics from the plasma membrane to the TGN - the site of sulfation - for the cation-dependent MPR (CDMPR) with and without silencing of candidate machinery proteins. The clathrin adaptor AP-1 that operates bidirectionally at the TGN-to-endosome interface, which had been shown to cause reduced sulfation when rapidly depleted, produced hypersulfation of nanobodies internalized by CDMPR upon long-term silencing, reflecting accumulation in the TGN. In contrast, knockdown of retromer (Vps26), epsinR, or Rab9 reduced CDMPR arrival to the TGN. No effect was observed upon silencing of TIP47. Most surprisingly, depletion of the GGA (Golgi-localized, {gamma}-adaptin ear-containing, Arf-binding) proteins inhibited retrograde transport rather than TGN exit. This study illustrates the usefulness of derivatized, sulfation-competent nanobodies to analyze retrograde protein transport to identify the contributions of different machineries.

Phosphatidic acid (PA) is a central metabolic intermediate that can fuel triacylglycerol (TAG) synthesis in lipid droplets (LDs) or cardiolipin production in mitochondria, but how cells partition PA between these competing fates has remained a fundamental unresolved question in lipid cell biology. We identify the lipid transfer protein ORP5 as a key regulator of PA partitioning at Mitochondria-Associated endoplasmic reticulum Membranes (MAM) that contact lipid droplets (LD), referred to as MAM-LD junctions. Cell imaging analysis shows that ORP5 stabilizes PA levels at MAM to promote TAG synthesis. On the other hand, loss of ORP5 causes PA accumulation on mitochondrial membranes, leading to excess cardiolipin synthesis and mitochondrial hyperfusion, while impairing triacylglycerol (TAG) synthesis and LD formation. Finally, reconstitution assays using liposomes or giant organelles further demonstrate that ORP5 can transfer PA from mitochondria to the ER via its ORD domain. Together, these findings reveal that ORP5 functions as a PA lipid transfer protein at tripartite MAM-LD contacts, where it balances LD formation with mitochondrial lipid metabolism, protecting mitochondria from cardiolipin overload.

During autophagosome biogenesis, the incorporation of transmembrane proteins into the expanding phagophore is not readily observed. In addition, the membrane surface area of the organelle expands rapidly, while the volume of the autophagosome is kept low. Several recent studies have suggested a model of membrane expansion that explains how these attributes are maintained. The autophagosome expands predominantly through the direct protein-mediated transfer of lipids through the lipid transfer protein ATG2. As these lipids are only introduced into the cytoplasmic-facing leaflet of the expanding phagophore, full membrane growth also requires lipid scramblase activity. ATG9 has been demonstrated to harbor scramblase activity and is essential to autophagosome formation, however if and when it is integrated into mammalian autophagosomes remains unclear. Here we show that in the absence of lipid transport, ATG9 vesicles are already fully competent to collect proteins normally found on mature autophagosomes, including LC3-II. Further, through the novel use of styrene-maleic acid lipid particles as a nanoscale interrogation of protein organization on intact membranes, we show that ATG9 is fully integrated in the same membranes as LC3-II, even on maturing autophagosomes. The ratios of these two proteins at different stages of maturation demonstrate that ATG9 proteins are not continuously integrated, but rather are present on the seed vesicles only and become diluted in the rapidly expanding autophagosome membrane. Thus, ATG9 vesicles are the seed membrane from which mammalian autophagosomes form.

Cell adhesion molecules of the immunoglobulin superfamily (IgCAMs) coordinate adhesive interactions with intracellular organization during tissue morphogenesis. In the Drosophila follicular epithelium, epithelial maintenance depends on reintegration, a process in which mitotically displaced cells reincorporate into the epithelial monolayer. Previous work identified the IgCAMs Fasciclin 2 (Fas2) and Neuroglian (Nrg) as parallel, partially redundant regulators of reintegration, but the intracellular mechanisms linking adhesion to reintegration remained unclear. Here, we show that Fas2 supports reintegration through two mechanistically distinct modes: a transmembrane mode and a GPI-linked mode. Although both contribute to reintegration, the transmembrane mechanism is more effective and depends on stabilization of a cortical Fas2 pool through intracellular coupling. Using yeast two-hybrid screening, genetics, and fluorescence recovery after photobleaching (FRAP), we identify the scaffold protein Discs large (Dlg1) as a functional intracellular partner of transmembrane Fas2. Partial disruption of Dlg1 preferentially sensitizes epithelia in which the parallel Nrg-dependent reintegration mechanism is compromised, consistent with Dlg1 functioning primarily within the Fas2-dependent reintegration arm. While Dlg1 is not required for Fas2 membrane localization, Dlg1 disruption increases the mobile fraction of transmembrane Fas2, indicating that Dlg1 promotes retention of a stabilized cortical Fas2 pool. Together, these findings support a model in which epithelial reintegration depends on coordinated adhesion-scaffold coupling and reveal mechanistic parallels between epithelial reintegration and IgCAM-dependent processes in the developing nervous system.

Neurons are polarized cells that require accurate membrane trafficking to maintain distinct protein complements at dendritic and axonal membranes. The Kinesin-3 family members KIF13A and KIF13B are thought to mediate dendrite-selective transport, but the mechanism by which they are recruited to polarized vesicles and the differences in the specific trafficking role of each KIF13 have not been defined. We performed live-cell imaging in cultured hippocampal neurons and found that KIF13A is a dedicated dendrite-selective kinesin. KIF13B confers two different transport modes, both dendrite- and axon-selective transport. Both KIF13s are maintained at the trans-Golgi network by interactions with the heterotetrameric adaptor protein complex AP-1. Interference with KIF13 binding to AP-1 resulted in disruptions to both dendrite- and axon- selective trafficking. We conclude that AP-1 is the molecular link between the sorting of polarized cargoes into vesicles and the recruitment of kinesins that confer polarized transport.