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.

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 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.

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.

Phagocytosis requires coordinated remodeling of the actin cytoskeleton to generate protrusive and contractile forces that drive target engulfment. Class I myosins Myo1e and Myo1f (Myo1e/f) have been implicated in linking the plasma membrane to the actin network, but their specific roles during Fc-receptor-mediated phagocytosis remain unclear. Using CRISPR-edited RAW 264.7 macrophages lacking Myo1e and Myo1f, we show that double knockout (dKO) cells exhibit markedly reduced uptake of IgG-coated beads, a phenotype that is partially rescued by re-expression of either myosin. Lattice-light-sheet and confocal imaging revealed distinct F-actin architectures corresponding to the various stages of cup progression, including basal podosome-like adhesions, individual phagocytic podosomes (actin teeth) along the rim of the cup, and a contractile phagocytic ring formed by the reorganization of podosomes into a higher-order network. In Myo1e/f- deficient cells, podosome formation was diminished, actin teeth were largely absent, and the phagocytic ring formed prematurely, which was often accompanied by stalled cup progression and repeated engulfment attempts. Myo1e/f localized both to podosomes and to the inner surface of the phagocytic ring, non-muscle myosin II (NM2) localized to the outer surface, and the absence of Myo1e/f correlated with the diffuse distribution of NM2. In addition, Myo1e/f-deficient macrophages exhibited increased trogocytosis of antibody-opsonized HL-60 cells, indicating a shift from whole-target engulfment toward partial target ingestion. These results suggest that Myo1e/f coordinate spatial and temporal transitions between protrusive and contractile actin networks, thereby ensuring efficient phagocytic cup progression. Our findings highlight a dual role for Myo1e/f in adhesion regulation and force balance during macrophage phagocytosis.

Transport and regulation of messenger ribonucleoprotein complexes (mRNPs) is critical for spatial control of gene expression within cells. How mRNPs are trafficked in hyphae of different filamentous fungi remains poorly understood. Here we investigate the transport of SsdA, the Aspergillus nidulans ortholog of budding yeast RNA-binding protein Ssd1, which is thought to function in translational repression. SsdA forms cytoplasmic puncta that colocalize with the poly(A)-binding protein FabM, marking them as mRNPs, and punctum formation depends on conserved RNA-binding residues of SsdA. SsdA puncta move bidirectionally along microtubules by hitchhiking on early endosomes, and this requires the adaptor proteins PxdA and DipA--machinery previously associated exclusively with peroxisome transport. SsdA puncta are largely depleted from hyphal tip-proximal regions, despite an abundance of early endosomes near hyphal tips, and the length of SsdA puncta depletion zones correlates with hyphal growth rate. We show that acute inhibition of Nuclear Dbf2-related (NDR)-family protein kinase CotA causes rapid accumulation of SsdA puncta near hyphal tips, indicating that puncta depletion depends on CotA activity. Mutation of predicted CotA phosphorylation sites within SsdA to nonphosphorylatable residues also leads to accumulation of SsdA puncta near tips, while mutation to phosphomimetic residues disrupts puncta entirely. Together, our findings establish SsdA-containing mRNPs as a new endosomal hitchhiking cargo in A. nidulans and reveal that NDR kinase signaling spatially regulates SsdA-mRNP distribution during normal polarized growth.

The class III phosphatidylinositol 3-kinase complex (PI3K(III)) generates phosphatidylinositol-3-phosphate (PI(3)P), a lipid that defines endosomal membrane identity. Two PI3K(III) complexes share core subunits but differ in their fourth component: the Atg14-containing complex I functions in autophagy, whereas the Uvrag-containing complex II is required for endosomal maturation. Despite this, the mechanism by which complex II promotes lysosomal function remains unclear. Using Drosophila nephrocytes, we show that PI(3)P is enriched on Rab7-positive late endosomes and that the Hsp70 chaperone Hsc70-4 binds phosphoinositides. Loss of PI3K complex II disrupts endolysosomal organization and phenocopies Hsc70-4 inhibition. In both cases, clathrin accumulates on intracellular, often endosomal membranes, Rab7 compartments are disorganized, and abnormal endolysosomal structures form. These defects are accompanied by impaired HOPS recruitment, lysosomal dysfunction, and secretion of endolysosomal content. Importantly, clathrin depletion partially rescues these defects. Together, our findings identify a role for PI3K complex II in promoting clathrin removal from endosomal membranes and link PI(3)P and Hsc70-4 activity to lysosomal maturation.

The ULK1 complex (ULK1C) and the class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) act together to initiate autophagy. Human ULK1C consists of ULK1 itself, FIP200, and the HORMA domain heterodimer ATG13:ATG101. PI3P generated by PI3KC3-C1 is essential to recruit and stabilize ULK1C on membranes for ULK1 to phosphorylate its membrane-associated substrates in autophagy induction, even though ULK1C subunits do not contain any PI3P-binding domains. Here we show that the ATG13:ATG101 dimer forms a tight complex with the PI3P-binding protein WIPI3, as well as with WIPI2. Bound to WIPI2-3, ATG13:ATG101 aligns with the membrane to insert its Trp-Phe (WF) finger into the membrane. Molecular dynamics simulations show that alignment of WIPIs and the ATG101 WF finger cooperatively stabilizes the complex on membranes, explaining the essential role of the WF residues in autophagy. Biochemical reconstitution and a cell-based assay show that WIPI3:ATG13 engagement is required for ATG16L1 phosphorylation by ULK1, ATG13 puncta formation, and bulk autophagic flux. We further showed that a kinase domain (KD)-proximal PVP motif within the ULK1 IDR docks onto the surface of the ATG13:ATG101 HORMA dimer and used molecular modeling to show how the ULK1 KD is brought close to the membrane surface. Biochemical reconstitution and cell-based assays show that the PVP motif is essential for in vitro ULK1 phosphorylation of ATG16L1 and important for starvation-induced autophagy and BNIP3/NIX-dependent mitophagy. These data establish a stepwise pathway for recruitment of the ULK1 KD to the vicinity of the membrane surface downstream of PI3KC3-C1.

The spindle checkpoint ensures accurate chromosome segregation by monitoring whether chromosomes, via kinetochores, are properly attached to the spindle. If chromosomes fail to establish bipolar attachment, the checkpoint delays the cell cycle to enable error correction. In C. elegans early embryos, activation of the spindle checkpoint produces a longer mitotic delay in primordial germ cells than somatic cells. We show that the conserved nucleoporin and spindle matrix component, NPP-21/TPR, is required for the stronger spindle checkpoint in germline cells. A checkpoint-proficient NPP-21::GFP transgene localizes to a spindle-like structure during mitosis and is enriched in germline cells, consistent with a cell-fate specific function for this protein. Finally, NPP-21 controls spindle checkpoint strength in germline cells via two, potentially linked, mechanisms: concentrating PCH-2 around mitotic chromosomes and promoting the localization of the checkpoint effector, Mad2, to unattached kinetochores. These experiments demonstrate a developmental role for NPP-21, and the spindle matrix, in controlling spindle checkpoint strength in immortal germline cells in C. elegans.

Microtubules attach to kinetochores to facilitate chromosome movement to opposite spindle poles. Defective kinetochore-microtubule attachments lead to phosphoryation of the outer kinetochore protein KNL1 at conserved MELT motifs, which triggers spindle assembly checkpoint activation and recruitment of the fibrous corona. To identify additional phosphorylation sites that regulate kinetochores, we treated HEK 293T/17 cells with nocodazole, paclitaxel, or STLC to create defective kinetochore-microtubule attachment states. We then purified KNL1 and performed proteomics and identified 111 phosphorylation sites on KNL1, including several that may be attachment-state specific. These data demonstrate that KNL1 is extensively phosphoregulated in response to treatment with microtubule-disrupting compounds.

Telomere-led rapid prophase chromosome movements (RPMs) during meiotic prophase are critical for homologous chromosome pairing and proper meiotic progression. These movements are generated by the cytoskeleton and are transmitted to the telomeres via the LINC complex, yet the cytoplasmic components that generate these forces remain poorly defined. Among candidates of microtubule-associated motor proteins in mouse primary spermatocytes, we confirmed KIF5B as a specific interactor of the KASH5-LINC complex. Total internal reflection fluorescence microscopy and microtubule sedimentation assays performed with purified recombinant proteins suggest a direct interaction between KASH5 and KIF5B on microtubules, enhanced by MAP7, a known KIF5B-recruiting and activating cofactor. Mapping the KIF5B-binding surface of KASH5 revealed that KASH5 N-terminal EF-hand domains mediate the interaction. Further, in vivo KIF5B-KASH5 interaction and KIF5B role in RPMs are evidenced as (1) KIF5B is recruited by KASH5-SUN1 to the nuclear envelope in two different cultured somatic cell models, (2) KIF5B is telomere-associated and colocalizes with KASH5, and microtubules associated with the nuclear envelope in mouse spermatocytes, and (3) chemical inhibition of KIF5B reduces telomere-led chromosome motions. Altogether, our findings identify the KIF5B kinesin as a previously unrecognized component of the force-generating machinery that drives chromosome movement during meiotic prophase I, acting through KASH5 as a specific nuclear membrane adaptor.

While the role of autophagy-related (ATG) proteins in microautophagy remains unclear, their absence in budding yeast has been reported to impair stationary-phase microlipophagy. Here, we show that this defect in ATG-deficient (atg{Delta}) cells arises not from a direct requirement of ATG proteins for the execution of microlipophagy but from accumulation of acetic acid (AA) in the medium. High concentrations of AA in the medium of atg{Delta} cells trigger the clustering of Niemann-Pick type C (NPC) proteins, causing impairment of raft-like vacuolar microdomain formation and suppression of microlipophagy. Lowering extracellular AA rapidly dissolves NPC protein clusters, restores vacuolar microdomains, and rescues microlipophagy in atg{Delta} cells. Conversely, elevating AA concentrations in the medium of wild-type cells induces NPC protein clusters and microlipophagy defects. These findings demonstrate that stationary-phase microlipophagy can proceed independently of ATG proteins and that the defect in atg{Delta} cells can be rescued by normalizing extracellular AA levels.

Synaptic vesicle proteins (SVPs) are synthesised in the neuronal soma trafficked as precursor synaptic vesicles (pre-SVs) on route to synapses. While pre-SVs are known to have heterogeneous protein composition and can co-traffic with lysosomal proteins. In this study, we assess the trafficking routes and kinetics of Synatobrevin-1 (SNB-1) released from the ER using the RUSH system in vivo in C. elegans touch receptor neurons. We showed that ER-released SNB-1 follows at least two temporally distinct trafficking routes. A predominantly anterogradely moving population of SNB-1 carrying vesicles appeared early, within 20 minutes of ER release in the axon without overlap with lysosomal proteins. Another SNB-1 population at 45 minutes post-ER release overlapped with endolysosomal compartments in both the cell body and the axon. Early SNB-1 carrying vesicles co-migrate with a transmembrane synaptic vesicle protein Synaptogyrin (SNG-1) and RAB-27 but fewer with RAB-3, suggesting that SVPs can be co-sorted into the same carriers prior to overlap with lysosomal proteins. The SV-lysosomal protein overlap occurs even when SNB-1 endocytosis on the plasma membrane is reduced in unc-11/ap180 mutants. Finally, we identified SAM-4/Myrlysin, a subunit of the BORC complex, as a regulator of both the trafficking kinetics of Synaptobrevin-1 intermediates and the cargo composition of pre-SVs. Loss of SAM-4 accelerated SV-lysosomal protein overlap and reduced co-transport of SNG-1 with SNB-1 in early pre-SVs in the axon. Together, these findings reveal heterogeneity in pre-SV biogenesis routes and identify SAM-4 as a key regulator of both the kinetics and cargo composition of synaptic vesicle precursors.

In metazoans, mitochondria optimally distribute to sites of need through long-range transport events on microtubules. The prevailing model for this trafficking mechanism is that the tail-anchored calcium-binding GTPase, Miro, recruits cytosolic TRAK and associated molecular motors to the outer mitochondrial membrane. Therefore, Miro is proposed to be an obligate adaptor for TRAK required for bulk mitochondrial transport, a process that is considered particularly important for long-range trafficking in neurons, and thus, for viability. Here, we impaired Miro-TRAK interaction in vivo by introducing a point mutation into the Drosophila TRAK orthologue Milton, that impairs its interaction with Miro, based on recent structural evidence. Flies harbouring this point mutation are viable to adulthood. Moreover, neurons carrying this mutation exhibit little to no observable reduction in axonal mitochondria. Mutant flies, however, display progressive loss of motor function with age and reduced lifespan. We therefore call into question the long-standing view that Miro plays an obligatory role in mitochondrial trafficking and challenge the canonical model for mitochondrial transport.

The distinct compositions of the two mitochondrial membranes are generated through a combination of phospholipids that mitochondria can make and those they take; both processes depend on a series of distinct lipid trafficking steps. Mitochondria make phosphatidylethanolamine (PE) through the action of the phosphatidylserine decarboxylase Psd1, an intermembrane space (IMS)-facing integral inner membrane (IM) protein. Psd1 has been proposed to act on its endoplasmic reticulum-derived substrate, phosphatidylserine (PS), after its transport to the mitochondrial outer membrane (OM) and either following its Ups2/Mdm35-mediated transport across the IMS to the IM or instead, on the IMS-side of the OM in a process enabled by the mitochondrial contact site and cristae organizing system (MICOS). Here, we implement a two-pronged Psd1 rewiring-based strategy predicted to either 1) circumvent the need for Ups2/Mdm35 and/or MICOS; or 2) selectively ablate the ability of Psd1 to work in trans. Our results with yeast harboring Psd1 targeted to the OM demonstrate that, with respect to mitochondrial PE production, Ups2/Mdm35 and MICOS indeed function within the IMS. Using yeast expressing a topologically inverted Psd1 chimera that faces the matrix, we identify previously unappreciated transbilayer lipid trafficking steps within the IM and show that Psd1 does not operate via a MICOS-organized in trans mechanism. Further, retained flux through inverted Psd1 when both Ups2/Mdm35 and MICOS are absent strongly implicates the existence of a major, yet presently unknown, mediator(s) of lipid movement across the IMS. Collectively, these data suggest a new model of how mitochondrial membrane diversity is established and maintained.

During maturation of a Golgi cisterna, multiple vesicular transport pathways recycle resident Golgi proteins. Recycling vesicles are captured by Golgi-associated tethers. To assign individual tethers to specific recycling pathways in Saccharomyces cerevisiae, we examined tether arrival and departure using kinetic mapping, and we examined tether function using an ectopic tether localization assay. Those approaches yielded mutually consistent results. Our analysis focused on two coiled coil golgin tethers and the multi-subunit tether GARP. At an intermediate stage of cisternal maturation, the golgin Sgm1 tethers proteins that follow an intra-Golgi recycling pathway dependent on COPI. At a late stage of cisternal maturation, GARP and the golgin Imh1 tether trans- Golgi network (TGN) proteins that follow an intra-Golgi recycling pathway dependent on the AP-1 and Ent5 clathrin adaptors. This involvement of GARP in intra-Golgi recycling had not previously been documented. Imh1 also tethers proteins that recycle from prevacuolar endosome compartments to the TGN. Our findings contribute to an integrated model of Golgi membrane traffic.

Membrane contact sites (MCSs) enable communication between organelles and play central roles in lipid metabolism. In budding yeast, the nucleus-vacuole junction (NVJ) functions as a dynamic platform that integrates lipid metabolism and stress responses. However, it remains unclear whether NVJ structure and function are broadly conserved across eukaryotes, particularly because Nvj1, the key membrane tethering factor that mediates NVJ formation in budding yeast, is absent in higher eukaryotes. Here, we investigated whether an MCS analogous to the NVJ in budding yeast exists in fission yeast (Schizosaccharomyces pombe), which lacks Nvj1. We show that an NVJ is present in fission yeast and serves as a platform for the accumulation of sterol synthesis factors, including the HMG-CoA reductase Hmg1 and the INSIG homolog Ins1. We further demonstrate that the localization of these factors depends on the membrane protein insertase Snd302 and is dynamically regulated by nutrient conditions. Our findings reveal that, despite the absence of Nvj1, the NVJ is functionally conserved as a site for sterol synthesis in fission yeast, suggesting a conserved role of spatial organization in lipid metabolism.

The nuclear pore complex (NPC) is the only gateway that connects the nucleus with the cytoplasm in eukaryotic cells. Its nucleoplasmic face is decorated by the nuclear basket, a filamentous structure with important roles in mRNA export and chromatin organization. In contrast to major parts of the nuclear pore scaffold, the architecture and organization of the nuclear basket remain poorly defined. In this study, we investigate the interaction network required for formation of the nuclear basket in vivo using budding yeast. We demonstrate that the filamentous Mlp1 protein relies on coiled-coil segments outside its previously characterized NPC-binding region to stably interact with the NPC. Furthermore, our results reveal that Mlp1s paralogue, Mlp2, plays a central role in nuclear basket architecture. Specifically, Mlp2 associates with the NPC independently of Mlp1 and together with Mlp1 is essential for the efficient recruitment of Pml39 and additional Mlp1 subunits. Our findings allow us to propose a refined model of nuclear pore basket architecture and organization.

The tight junction (TJ) protein cingulin binds directly to nonmuscle myosin 2B (NM2B) through sequences in its C-terminal rod-tail region and recruits it to tight junctions (TJ) to control membrane cortex mechanics, epithelial morphogenesis and cingulin conformation. However, the minimal sequence required for cingulin-NM2B interaction and how this interaction is regulated is not known. Here we identify a 19-aminoacid sequence at the hinge between the cingulin rod and tail that is required for cingulin-NM2B interaction, and we investigate the role of phosphorylation of Ser residues within this region in regulating this interaction. Immunofluorescence microscopy localization of NM2B in cingulin-KO cells rescued with mutant cingulin constructs shows that phospho-mimetic but not dephospho-mimetic cingulin mutants inhibit NM2B recruitment to junctions and downstream regulation of cingulin conformation and TJ tortuosity, correlating with cingulin-NM2B interaction, as determined by GST pulldown analysis. In contrast, either phospo-or dephospho-mimetic mutants of Ser residues within the cingulin head domain do not affect either NM2B recruitment to TJ, or cingulin conformation and localization in cells, or TJ membrane tortuosity. Finally, Ser residues within the hinge display the consensus sequence for protein kinases CK1 and CK2, and, through in vitro phosphorylation, site mutation analysis and use of inhibitors, we identify a complex interplay between CGN phospho-sites, with a prominent negative role of Ser1162 phosphorylation in the regulation of cingulin-NM2B interaction. In summary, we show that cingulin-NM2B interaction is regulated by cingulin phosphorylation within the hinge and identify a potential role for CK1 and CK2 kinases in cingulin phosphorylation.

Actin-based membrane protrusions such as filopodia, microvilli, and stereocilia support a range of cell functions, from nutrient absorption to mechanosensation. In each case, membrane deformation is supported by a core bundle of actin filaments, organized in a unipolar barbed-end out manner. Although their structures and proteomes are well characterized, mechanisms governing the growth and stability of these protrusions remain less clear. Factors that localize to the distal tips of these structures are of particular interest, as they are well positioned to control actin assembly at filament barbed ends. One such factor, EPS8, localizes to distal tip puncta in multiple protrusion types. While early biochemical studies suggested a role in filament capping, loss of EPS8 in multiple models shortened microvilli and stereocilia, suggesting roles in elongation. More recent studies in differentiating epithelial cells suggested that EPS8 promotes protrusion growth and stability. To clarify EPS8s function in the distal tip compartment, we leveraged acute loss-of-function experiments and titrated gain-of-function approaches in combination with live imaging. Acute sequestration of EPS8 led to rapid depletion of filopodia. Conversely, increasing cellular EPS8 levels elevated EPS8 per distal tip punctum, increased F-actin content within individual filopodia, reduced filopodia elongation rates, increased protrusion lifetimes, and protected filopodia against cytochalasin D-induced collapse. These findings suggest that EPS8 binds filament barbed ends as a leaky capper, slowing monomer addition while stabilizing bundles and preventing collapse. These activities are likely critical for building and maintaining the large arrays of protrusions that are assembled by diverse epithelial cell types.