Journal of Cell Biology

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.

Microtubules in cells consist of functionally diverse subpopulations carrying distinct post-translational modifications (PTMs). Akin to the histone code, the tubulin code regulates a myriad of microtubule functions ranging from intracellular transport to chromosome segregation. Yet, how individual PTMs only occur on subsets of microtubules to contribute to microtubule specialization is not well understood. In particular, microtubule detyrosination, which is the removal of the C-terminal tyrosine on -tubulin subunits, marks the stable population of microtubules and modifies how microtubules interact with other microtubule-associated proteins to regulate a wide range of cellular processes. Previously, we found that, in certain cell types, only a small subpopulation of microtubules is highly enriched with the detyrosination mark ([~]30%) and that detyrosination spans most of the length of a microtubule, often adjacent to a completely tyrosinated microtubule. How the activity of a cytosolic detyrosinase, Vasohibin (VASH) leads to only a small subpopulation of highly detyrosinated microtubules is unclear. Here, using quantitative super-resolution microscopy, we visualized nascent microtubule detyrosination events in cells consisting of 1-3 detyrosinated -tubulin subunits after Nocodazole washout. Microtubule detyrosination accumulates slowly and in a disperse pattern across the microtubule length. By visualizing single molecules of VASH in live cells, we found that VASH engages with microtubules stochastically on a short time scale suggesting limited removal of tyrosine per interaction, consistent with the super-resolution results. Combining these quantitative imaging results with simulations incorporating parameters from our experiments, we propose a stochastic model for cells to establish a subset of detyrosinated microtubules via a detyrosination-stabilization feedback mechanism.

Membrane contact sites (MCSs) are areas of close proximity between organelles that allow the exchange of material, among other roles. The endoplasmic reticulum (ER) has MCSs with a variety of organelles in the cell. MCSs are dynamic, responding to changes in cell state, and are therefore best visualized through inducible labeling methods. However, existing methods typically distort ER-MCSs, by expanding contacts or creating artificial ones. Here we describe a new method for inducible labeling of ER-MCSs using the Lamin B receptor (LBR) and a generic anchor protein on the partner organelle. Termed LaBeRling, this versatile, one-to-many approach allows labeling of different types of ER-MCSs (mitochondria, plasma membrane, lysosomes, early endosomes, lipid droplets and Golgi), on-demand, in interphase or mitotic cells. LaBeRling is non-disruptive and does not change ER-MCSs in terms of the contact number, extent or distance measured; as determined by light microscopy or a deep-learning volume electron microscopy approach. We applied this method to study the changes in ER-MCSs during mitosis and to label novel ER-Golgi contact sites at different mitotic stages in live cells.

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.

The sub-cellular positioning of endolysosomes is crucial for regulating their function. Particularly, the positioning of endolysosomes between the cell periphery versus the peri-nuclear region impacts autophagy, mTOR (mechanistic target of rapamycin) signaling and other processes. The mechanisms that regulate the positioning of endolysosomes at these two locations are still being uncovered. Here, using super-resolution microscopy, we show that the retrograde motor dynein forms nano-clusters on endolysosomal membranes containing 1-2 dyneins, with an average of ~3 nanoclusters per endolysosome. These data suggest that a very small number of dynein motors (1-6) drive endolysosome motility. Surprisingly, dynein nano-clusters are slightly larger on peripheral endolysosomes having higher cholesterol levels compared to peri-nuclear ones. By perturbing endolysosomal membrane cholesterol levels, we show that dynein copy number within nano-clusters is influenced by the amount of endolysosomal cholesterol while the total number of nano-clusters per endolysosome is independent of cholesterol. Finally, we show that the dynein adapter protein ORP1L (Oxysterol Binding Protein Homologue) regulates the number of dynein motors within nano-clusters in response to cholesterol levels. We propose a new model by which endolysosomal transport and positioning is influenced by the cholesterol sensing adapter protein ORP1L, which influences dyneins copy number within nano-clusters.

The yeast vacuole membrane forms ordered microdomains that facilitate micro-lipophagy under nutrient limitation. We previously found that this process involves the intracellular sorting of sphingolipids to the vacuole. While multiple vacuole protein pathways have been identified, corresponding mechanisms for lipid sorting remain undefined. Here we use a range of approaches to identify how endocytic sorting and intraluminal transport of sphingolipids contribute to the formation of vacuole domains. To visualize sphingolipid trafficking, we employed the ceramide analogue BODIPY C12-ceramide (BODIPY-Cer), which is internalized by cells and stains the vacuole. We observed that cells lacking Vps29 and Vps30, proteins involved in endosomal sorting, show altered vacuole domains and accumulate BODIPY-Cer at sites proximal to the plasma membrane. Subsequent incorporation of endocytic-derived ceramide into the vacuole is dependent on the Niemann-Pick Type C 2 protein (Npc2). Loss of Npc2 reduces domain formation and causes BODIPY-Cer to accumulate within the vacuole lumen. Both intra-vacuole trafficking of BODIPY-Cer and membrane phase separation were not dependent on Npc2s canonical receptor, Ncr1. Lipidomics of isolated vacuoles confirmed that Npc2 independently mediates sphingolipid sorting under micro-lipophagy conditions. In liposome assays, Npc2 robustly transports analogues of ceramide and inositol phosphorylceramide, a complex sphingolipid that is enriched in phase-separated vacuoles. We propose that the enlarged binding cavity of yeast Npc2 is specialized for the incorporation of sphingolipids into the vacuole membrane.

AO_SCPLOWBSTRACTC_SCPLOWRab5 is required for macropinosome formation, but its site and mode of action remain unknown. We report that Rab5 acts at the plasma membrane, downstream of ruffling, to promote macropinosome sealing and scission. Dominant-negative Rab5, which obliterates macropinocytosis, had no effect on the development of membrane ruffles. However, Rab5-containing vesicles were recruited to circular membrane ruffles, and SNARE-dependent endomembrane fusion was necessary for completion of macropinocytosis. This fusion event coincided with the disappearance of PtdIns(4,5)P2 that accompanies macropinosome closure. Counteracting the depletion of PtdIns(4,5)P2 by expression of phosphatidylinositol-4-phosphate 5-kinase impaired macropinosome formation. Importantly, we found that removal of PtdIns(4,5)P2 is dependent on Rab5, through the Rab5-mediated recruitment of the inositol 5-phosphatases OCRL and Inpp5b, via APPL1. Knockdown of OCRL and Inpp5b, or APPL1 prevented macropinosome closure, without affecting ruffling. We therefore propose that Rab5 is essential for the clearance of PtdIns(4,5)P2 needed to complete macropinosome scission from the plasmalemma.

Complex sphingolipids form in the Golgi apparatus and require transport by vesicular carriers to reach the plasma membrane (PM) where they assemble with cholesterol to affect membrane function. The caveolin proteins have been implicated in sphingolipid trafficking but by mechanisms unknown. Here, we found that cells lacking caveolin-1 (Cav1) distributed the complex sphingolipids to the lysosome rather than to the PM. This was not seen in Cavin-1 KO cells, implicating a function for Cav1 independent of caveolae. The defect in trafficking localized to the sorting endosome where the complex sphingolipids failed to enter recycling tubules serving the PM. Sphingolipid trafficking was rescued by over-expression of Cav1, but not by a Cav1 mutant that lacked the S-palmitoylation sites. Thus, noncaveolar and palmitoylated Cav1 appears to act as a chaperone, or selectivity filter, enabling entry of the complex sphingolipids into endocytic recycling tubules to shape the composition of the PM.

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.

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.

Due to the local enrichment of factors that influence its formation, dynamics, and organization, the actin cytoskeleton displays different shapes and functions within the same cell. In yeast cells post-Golgi vesicles ride on long actin cables to the bud tip. The scaffold proteins Boi1 and Boi2 participate in tethering and docking these vesicles to the plasma membrane. Here we show that Boi1/2 also recruit nucleation and elongation factors to form actin filaments at sites of exocytosis. Disrupting the physical connection between Boi1/2 and the nucleation factor Bud6 impairs filament formation in the bud, reduces the directed movement of the vesicles to the tip, and shortens their tethering time at the cortex. Artificially transplanting Boi1 from the bud tip to the peroxisomal membrane partially redirects the actin cytoskeleton and the vesicular flow towards the peroxisome, and creates an alternative, rudimentary vesicle-docking zone. We conclude that Boi1/2 is sufficient to induce the formation of a cortical actin structure that receives and aligns incoming vesicles before fusing with the membrane.

How neurons accomplish the growth of an axon, and several dendrites sequentially is a cellular process not well understood. Here, we show that preferential somatic F-actin delivery to neurites inhibits neurite growth during axon formation. Thus, the neurite receiving less somatic F-actin is growing as an axon. During dendrites outgrowth, radial somatic F-actin translocation into all neurites is drastically diminished suggesting that cellular growth is restricted by the somatic F-actin flow into the growing neurites. Mechanistically, we report that radial somatic F-actin translocation is mediated via the Myosin II motor. Pharmacological inhibition of these molecular motors promotes neurite elongation and precludes F-actin translocation into neurites. Moreover, we show that actin phosphorylation at Y53, which promotes F-actin instability, is enriched selectively in some neurite shafts to exclude/minimize translocation of somatic F-actin only into those neurites. Therefore, enrichment of actin pY53 in the neurite shaft correlates with the longer neurite during axon formation. Accordingly, the Kinesin-1 motor domain, which accumulates transiently in neurites of stage 2 neurons and only in the emerging axon of stage 3 neurons, localizes preferentially in the neurite with the increased actin pY53 signal in the neurite shaft. Finally, we demonstrate that microtubule acetylation promotes actin phosphorylation, and these cytoskeleton modifications coexist in the longest neurite that grows as an axon. Collectively, our data support a model in which somatic F-actin translocation into undifferentiated neurites impedes growth counteracted by actin pY53, consequently, supporting neuronal polarization during axon formation.

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.

It is well-known that in addition to its classical role in protein turnover, ubiquitination is required for a variety of membrane protein sorting events. However, and despite substantial progress in the field, a long-standing question remains: given that all ubiquitin (Ub) units are identical, how do different elements of the sorting machinery recognize their specific cargoes? Here we provide an answer to this question as we discovered a mechanism based on the coincidence detection of lysine ubiquitination and Ser/Thr phosphorylation for the endocytic adaptor epsin to mediate the internalization of the yeast Na+ pump Ena1. Internalization of Ena1-GFP was abolished in double epsin knock-out in S. cerevisiae and was rescued by re-introducing either one of the 2 yeast epsins, Ent1 or Ent2 in an UIM (Ub Interacting Motif)-dependent manner. Further, our results indicate that ubiquitination of its C-terminal Lys1090 is needed for internalization of Ena1 and requires the arrestin-related-trafficking adaptor, Art3. We determined that in addition to ubiquitination of K1090, the presence of a Ser/Thr-rich patch (S1076TST1079) within Ena1 was also essential for its internalization. Our results suggest that this ST motif is targeted for phosphorylation by casein kinases. Nevertheless, phosphorylation of this S/T patch was not required for ubiquitination. Instead, ubiquitination of K1090 and phosphorylation of the ST motif were independently needed for epsin-mediated internalization of Ena1. We propose a model in which a dual detection mechanism is used by Ub-binding elements of the sorting machinery to differentiate among multiple Ub-cargoes.

Autophagy involves the rapid growth of phagophores through membrane addition. This growth is triggered by vesicles containing the Atg9A protein. However, Atg9A is not incorporated into mature autophagosomes. We now demonstrate that Dynamin-2 (Dnm2) colocalizes with the BAR domain protein Endophilin-B1 (EndoB1/Bif-1/SH3GLB1) and other autophagy proteins when autophagy is induced. Our data suggest that Atg9A is retrieved from phagophores via fission, with Dnm2 acting as the membrane scission protein. Blocking Atg9A recycling, either by mutating Dnm2, using RNA interference, or applying chemical inhibitors, results in Atg9A remaining in autophagosomes and being degraded during autophagy. Overall, these findings provide new insights into the roles of membrane-scission proteins in autophagy.

Microtubules assemble from tubulin heterodimers composed of conserved - and {beta}- tubulins. In many species, including humans, tubulins are expressed from multiple genes. While the resulting tubulin isotypes show only subtle sequence differences, microtubules made of distinct isotypes can differ in their dynamic behaviour, as well as in their structure, for example in the number of their protofilaments. In cells, tubulin isotypes co-polymerize into mixed-isotype microtubules. How the mixing of tubulin isotypes affects microtubule functionality is unknown. Here we show that co-polymerization of recombinant tubulin dimers containing two different human {beta}-tubulin isotypes, 1{beta}3 and 1{beta}4, generates sectioned microtubules in which tubulin content and protofilament number differ from one section to the next. We demonstrate that two microtubule-associated proteins (MAPs), TPPP1 and optineurin (OPTN), bind differentially to these sections. Microtubules grown from natively mixed-isotype sources, either HeLa cells or mammalian brain tissue, also consist of sections that are differentially recognized by TPPP1 and OPTN, suggestive of isotype sorting. Our results demonstrate that co-polymerization of multiple tubulin isotypes can drive microtubules to assemble into distinct sections that locally regulate the interactions of MAPs with the microtubule lattice.

Membrane remodeling leading to fragmentation is crucial for autophagy programs that control capture by phagophores or endolysosomes of portions of organelles to be removed from cells. It is driven by membrane-bound autophagy receptors that display cytoplasmic intrinsically disordered modules (IDRs) engaging Atg8/LC3/GABARAP (LC3). Studies on endoplasmic reticulum (ER)-phagy receptors of the FAM134 family revealed the importance of sequential FAM134 proteins phosphorylation, ubiquitylation and clustering for execution of the ER-phagy programs. In this model, ER fragmentation is promoted/facilitated by the membrane-remodeling function of FAM134 reticulon homology domains (RHDs). However, RHDs are not conserved in ER-phagy receptors. The question that we tackle in this work is if activation of ER-phagy receptors anchored at the ER membrane with conventional membrane spanning domains, i.e., most of the ER-phagy receptors known to date, eventually trigger ER remodeling and fragmentation, and how. Here, we show that the membrane-tethering modules of ER-phagy receptors (RHDs for FAM134B, single/multi spanning transmembrane domains for TEX264 and SEC62) determine the sub-compartmental distribution of the receptors but are dispensable for ER fragmentation, regardless of their propensity to remodel the ER membrane. Rather, ER fragmentation is promoted by the ER-phagy receptors intrinsically disordered region (IDR) modules that are a conserved feature of all ER-phagy receptors exposed at the cytoplasmic face of the ER membrane. Since cytoplasmic IDRs with net negative charge are conserved in autophagy receptors at the limiting membrane of other organelles, we anticipate that conserved mechanisms of organelle fragmentaVon driven by cytoplasmic exposed IDRs could operate in eukaryoVc cells.

Myosin-X (MYO10), a molecular motor localizing to filopodia, is thought to transport various cargo to filopodia tips, modulating filopodia function. Yet, only a few MYO10 cargoes have been described. Here, using GFP-Trap and BioID approaches combined with mass spectrometry, we identified lamellipodin (RAPH1) as a novel MYO10 cargo. We report that the FERM domain of MYO10 is required for RAPH1 localization and accumulation at filopodia tips. Previous studies have mapped RAPH1 interaction with adhesome components to its talinbinding and Ras-association domains. Surprisingly, we find that the RAPH1 MYO10-binding site is not within these domains. Instead, it comprises an area with previously unknown functions. Functionally, RAPH1 supports MYO10 filopodia formation and stability but is not involved in regulating integrin activity in filopodia tips. Taken together, our data indicate a feed-forward mechanism whereby MYO10 filopodia are positively regulated by MYO10-mediated transport of RAPH1 to the filopodium tip.

Peroxisomes play a central role in fatty acid metabolism. To correctly target to peroxisomes, proteins require specialized targeting signals. One mystery in the field is sorting of proteins that carry both a targeting signal for peroxisomes as well as for other organelles such as mitochondria or the endoplasmic reticulum (ER). Exploring several of these dually localized proteins in Saccharomyces cerevisiae, we observed that they can act as dynamic tethers bridging organelles together through an affinity for organelle-destined targeting factors. We show that this mode of tethering involves the peroxisome import machinery, the ER- mitochondria encounter structure (ERMES) in the case of mitochondria and the GET complex in the case of the ER. Depletion of each of the targeting factors resulted in the accumulation of smaller peroxisomes. We propose that dual targeting of proteins occurs at contact sites and that protein import per se contributes to the maintenance of these membrane proximities. This introduces a previously unexplored concept of how targeting of dual affinity proteins can support organelle attachment, growth and communication.