Journal of Cell Biology

Animal microtubule-organizing centers, including the centrosome and the Golgi apparatus, regulate microtubule nucleation and anchoring through the {gamma}-tubulin ring complex ({gamma}-TuRC) and CAMSAP-mediated minus-end stabilization. However, functional redundancy between these pathways has impeded dissection of their contributions to controlling microtubule organization and density. Here, we addressed this problem using combinatorial gene knockouts, protein depletions and Expansion Microscopy. By simultaneously eliminating CAMSAP2 and the {gamma}-TuRC-targeting proteins AKAP450, pericentrin, CDK5RAP2, myomegalin, ninein and AKNA, we generated viable RPE1 cells that lack both Golgi-derived microtubules and {gamma}-TuRC localization within the pericentriolar material and at subdistal appendages. Despite the disruption of these major microtubule-organizing pathways, overall microtubule density was only partially reduced. The remaining microtubules depended on CEP192 and NEDD1, which, together with ch-TOG, can activate {gamma}-TuRC at the centriole wall, in acentriolar cells, and in biochemical reconstitution assays. Our results demonstrate that in the absence of CAMSAP-mediated stabilization, interphase microtubule formation strongly relies on {gamma}-TuRC activation, which occurs through several redundant pathways.

Chromosome-bound RCC1 generates Ran-GTP signals to organize functional spindles for faithful chromosome segregation during mitosis and meiosis. RCC1 is the sole guanine nucleotide exchange factor (GEF) for Ran and is essential for spindle assembly during early, but not late, embryonic divisions. However, how RCC1 organizes the specialized embryonic spindle and when its function changes during early embryogenesis remain unclear. Here, using time-resolved RCC1 depletion and depletion-rescue experiments in medaka embryos, we show that RCC1 GEF activity is specifically required before the blastula stage to organize a specialized metaphase spindle mid-plane that ensures faithful chromosome segregation. Mechanistically, RCC1 promotes the accumulation of the canonical Ran effectors HURP and KIFC1/HSET, and unexpectedly, the microtubule motor dynein at the spindle midplane during early embryonic divisions. Intriguingly, a five-fold increase in RCC1 expression phenocopies RCC1 depletion, disrupting spindle-midplane organization and the accumulation of KIFC1 and dynein in a GEF activity-dependent manner. Together, our findings demonstrate that both insufficient and excessive RCC1 GEF activity compromise embryonic spindle assembly, revealing that balanced Ran activation is required to organize the specialized spindle midplane during vertebrate cleavage divisions. HighlightsRCC1 requirement changes with embryonic spindle remodeling before the blastula stage. RCC1 GEF activity is required to organize the specialized embryonic spindle midplane. RCC1 promotes the accumulation of HURP, KIFC1, and dynein at the spindle midplane. Both insufficient and excessive RCC1 GEF activity disrupt the spindle midplane organization.

Macroautophagy is a conserved catabolic process that facilitates the degradation of cellular material by capturing it in double membrane vesicles termed autophagosomes. In Saccharomyces cerevisiae, selective macroautophagy is initiated by the scaffolding protein Atg11. Atg11 recruits the transmembrane protein Atg9, which resides in small vesicles, to autophagic cargo. Atg9 vesicles then fuse, forming the initial membrane sheet that expands into the autophagosomal membrane. While it is known that Atg9 interacts with Atg11 via a set of hydrophobic amino acids in the disordered N-terminus of Atg9, it is unclear how Atg11 mediates this interaction. To gain insight into this unknown aspect of autophagy initiation we utilized a combination of biochemical, structural, and cellular approaches. We demonstrate that the N-terminal domain (NTD) of Atg11 is the primary interaction site for Atg9, but the NTD requires clustering by the C-terminal region of Atg11 for its complete interaction with Atg9. We investigated the structure of the Atg11-NTD using cryo-EM which, in combination with AlphaFold modeling, revealed a positively charged binding pocket within the Atg11-NTD that is essential for Atg9 binding. Mutation of this conserved binding pocket leads to a loss of Atg9 binding in yeast and a reduction in the selective autophagy of mitochondria. Taken together, our results demonstrate the mechanism by which Atg11 recruits Atg9 to autophagy initiation sites.

Endocytosis internalises nutrients, regulates extracellular signals, and recycles membrane components. Clathrin polymerises into a coat that shapes the endocytic vesicle from the plasma membrane. However, the role of clathrins dynamic assembly in the endocytic process remains unclear. We show, using two-colour fluorescence recovery after photobleaching assays in yeast, that the clathrin coat turns over rapidly in the early phase of endocytosis, dependent on the auxilin Swa2 and its ATPase. In the late phase the turnover is stopped by the coat protein Sla1. Regulated clathrin turnover is critical for the timing of endocytic progression and for controlling coat size. In the absence of this dynamic regulation the endocytic coats become abnormally large, resulting in the failure of the final actin-driven vesicle budding. These findings reveal that, in addition to its classic structural function, the dynamic properties of the clathrin lattice are critical for both the temporal and mechanical aspects of endocytosis.

TMEM165 is a Golgi-resident multi-pass membrane protein involved in divalent cation homeostasis and associated with congenital disorders of glycosylation, yet its N-terminal biogenesis has remained unresolved. Here, we demonstrate that TMEM165 contains a functional cleavable signal peptide required for correct Golgi targeting and membrane topology. Loss of this signal peptide causes protein mislocalization, and altered topology with N-terminal cytosolic exposure, whereas extended N-terminal deletion restores both Golgi localization and overall membrane topology, consistent with insertion mediated by the first transmembrane domain as commonly described for multi-pass membrane proteins. Importantly, this N-terminally truncated form remains responsive to manganese-induced degradation and partially restores glycosylation defects associated with TMEM165 deficiency, indicating that the extended N-terminal region is dispensable for core TMEM165 function. Together, these findings identify the signal peptide as a key determinant of TMEM165 biogenesis and suggest that its conservation may contribute not only to membrane targeting, but also to maintaining the proper luminal environment of the N-terminus during early biogenesis.

Cargo receptors are central organizers of the secretory pathway, yet the mechanisms controlling their abundance remain poorly understood. The endoplasmic reticulum (ER)-resident intramembrane protease RHBDL4 promotes substrate turnover via a non-canonical branch of ER-associated degradation and has recently been implicated in regulating secretory pathway components. We previously identified the p24 cargo receptor TMED7 as an RHBDL4 substrate, suggesting that cargo receptor turnover contributes to secretory pathway regulation. Here, quantitative proteomics identify members of the ER-Golgi intermediate compartment (ERGIC) cargo receptor family as endogenous RHBDL4 substrates, demonstrating that RHBDL4 targets multiple cargo receptor families within the early secretory pathway. Accordingly, RHBDL4 modulates multiple ERGIC-dependent transport pathways. In addition, unbiased secretome analysis reveals increased secretion of lysosomal precursor proteins upon RHBDL4 ablation. Mechanistically, we show that this phenotype is mediated, at least in part, by RHBDL4-dependent cleavage of the lysosomal cargo receptor sortilin/SORT1. Together, these findings identify cargo receptors as a major class of RHBDL4 substrates and establish proteolytic remodeling of cargo receptor networks as a mechanism for regulating secretory pathway flux.

Extracellular vesicles (EVs) comprise molecularly diverse populations generated through multiple membrane-trafficking pathways, yet the intracellular basis of this heterogeneity remains poorly understood. Here, we identify the EV-associated tetraspanin TSPAN3 as a marker of a secretory multivesicular body (MVB) population that is molecularly and functionally distinct from canonical CD63-positive compartments. Using endogenous genome editing, live-cell and super-resolution microscopy, electron microscopy, quantitative EV secretion assays, and complementary proteomic approaches, we show that TSPAN3 localizes to fusion-competent MVBs but exhibits limited overlap with CD63 during secretion. Unlike CD63, which extensively traffics through the plasma membrane and depends on YXX{Phi}-mediated endocytic retrieval, TSPAN3 reaches secretory MVBs predominantly through an intracellular trafficking route that relies on a dileucine-containing sorting region. Orthogonal proximity-labeling and affinity-purification proteomics revealed that TSPAN3-positive compartments are associated with a selective LC3/ATG8-related membrane network, including GABARAPL2 and proteins involved in endosomal membrane remodeling and fusion. Perturbation of residues required for this association impaired localization to LC3-positive compartments and reduced secretory MVB fusion. Consistent with these findings, pharmacological disruption of autophagy- and endolysosomal-associated pathways differentially altered TSPAN3-positive EV secretion. Finally, proximity-labeled EV proteomics demonstrated that TSPAN3-associated EVs possess cargo signatures distinct from CD63-associated EVs, with greater representation of endosomal and endolysosomal proteins suggesting that tetraspanin-associated membrane nanodomains retain molecular signatures consistent with their intracellular trafficking history. Together, our findings identify TSPAN3 as a marker of a previously unrecognized secretory MVB population distinguished by its intracellular trafficking, molecular interactions, and EV composition, supporting a model in which distinct tetraspanin-organized membrane nanodomains are associated with different intracellular trafficking routes and molecularly distinct EV populations.

Eukaryotic cells contain multiple biochemically distinct actin filament networks, which enable versatile functions of actin in a range of cellular processes. Yet, the mechanisms by which specific actin filament networks are assembled in a common cytoplasm remain elusive. Here, we investigated how functionally distinct actin nanoscale layers, specified by -actininand tropomyosin isoforms, Tpm1.6 and Tpm3.2, are assembled at focal adhesions. By combining genetic perturbations with mitochondrial-targeting of actin polymerases, we discovered that DAAM1 formin assembles Tpm3.2-actin filaments, whereas Ena/VASP family proteins polymerize -actinin cross-linked actin filament bundles at focal adhesions. Consequently, loss of DAAM1 dampened Tpm3.2 protein levels and impaired focal adhesion disassembly, thus phenocopying Tpm3.2-deficient cells. In contrast, Ena/VASP depletion led to defective focal adhesion maturation and loss of -actinin from focal adhesions. More broadly, our study highlights specific roles for formin and Ena/VASP family proteins in assembling biochemically and functionally distinct linear actin filament arrays in cells.

Peripheral sensory neurons innervate the skin to detect mechanical, thermal, and noxious stimuli. Within the epidermis, nerve fibers terminate beneath tight junctions, shielding them from environmental exposure. Although epidermal differentiation coordinates tight junction assembly, its role in organizing nerve terminals is poorly understood. Here, we show that activation of Notch, a master regulator of epidermal differentiation, caused near-complete loss of epidermal innervation. This was largely the result of increased contractility rather than impaired differentiation. Inducing epidermal contractility was sufficient to deplete nerve fibers with striking spatial precision, and restoring normal contractility reversed this effect. Actomyosin contractility is highest in the granular layers of the epidermis, where tight junctions form and nerve fibers terminate. Ablation of nonmuscle myosin II allowed nerve fibers to extend beyond their normal termination zone and caused touch hypersensitivity. Together, these findings demonstrate that epidermal contractility positions sensory nerve endings through spatially controlled pruning and defines a mechanical boundary established by epidermal cells that restricts neuronal outgrowth.

Lysosomal ion channels play key roles in regulating membrane trafficking, autophagy, and cell death. RECS1 is a pH-sensitive lysosomal calcium channel previously implicated in lysosome-mediated apoptosis. Here, we identify a novel role for RECS1 in exosome biology. Using immunoprecipitation followed by mass spectrometry, we mapped RECS1 interactors under apoptotic and lysosomal stress conditions. Notably, Syntenin-1, a key scaffolding protein in ESCRT-independent exosome biogenesis, emerged as the top hit. We validated the physical interaction between RECS1-Syntenin-1 using various approaches. RECS1 localized to secreted exosomes, and its overexpression increased exosome production, as measured by nanoparticle tracking analysis. Intriguingly, a channel-dead RECS1 retained both Syntenin-1 interaction and the ability to promote exosome release, suggesting a channel-independent mechanism. Our findings identify RECS1 as a structural component of the exosomal trafficking machinery and a modulator of extracellular vesicle biogenesis. This work connects lysosomal signalling with intercellular communication and suggests a broader role for RECS1 in stress-responsive secretion.

Electron microscopy (EM) is essential for resolving cellular ultrastructure, yet quantitative analysis remains limited by labor-intensive segmentation and the scarcity of generalizable models. Here we present QuantEM, an open-source platform for segmentation and analysis of EM data across imaging modalities, tissues, and species. We assembled the largest curated collection of intracellular EM datasets to date, comprising over 15,000 two-dimensional images and 1,700 three-dimensional acquisitions from more than 600 datasets, including nearly 4,000 newly released acquisitions. Using this resource, we trained an EM-specific vision transformer foundation model and systematically optimized adaptation strategies for organelle segmentation. QuantEM provides pretrained models for mitochondria, endoplasmic reticulum, nuclei, and lipid droplets, integrated with interactive proofreading and downstream quantitative analyses through standalone and napari interfaces. Across diverse naive datasets, QuantEM consistently matches or exceeds existing models on zero-shot segmentation while requiring less data for finetuning. We further demonstrate its utility by revealing previously unrecognized subcellular compartmentation of hepatic glucokinase using immuno-electron microscopy.

Three-dimensional live-cell fluorescence imaging of yeast cells is crucial for studying cell-cycle mechanics and regulation. However, extracting multi-channel phenotypes within dense cell clusters remains an image-processing bottleneck. Standard deep-learning models segment cells but fail to track mother-bud boundaries, mitotic spindle shapes and spindle-localizing proteins. Investigators rely on labour-intensive manual coordinate plotting, introducing observer bias and often exclude clustered cell data due to visual complexity. Here, we present an open-source Fiji pipeline for automated yeast cell image processing and deterministic classification of cell-cycle, spindle and protein dynamics. The workflow utilizes a dual-segmentation architecture via custom Cellpose models to capture the mother-bud cell boundaries. Extracted masks are integrated with multi-channel fluorescence data using a Difference-of-Gaussians framework to resolve SPB coordinates and localized protein kinetics, which a rule-based decision-tree maps to precise mitotic phenotypes. Validation demonstrates a 50-fold acceleration with ~6% deviation from manual analysis. Availability: Zenodo at https://doi.org/10.5281/zenodo.22083016.

Rubicon is a negative regulator of autophagy and the endolysosomal network (ELN) and an antagonist of the class III phosphatidylinositol 3-kinase complex II (PI3KC3-C2). Inhibition of Rubicon is considered a potential means to therapeutically upregulate autophagy and the ELN to treat Parkinsons disease and other conditions characterized by autophagic and ELN dysfunction. Rubicon is specific for the UVRAG-containing PI3KC3-C2 over the purely autophagic ATG14- containing PI3KC3-C1 complex. Here, we determined the high-resolution cryo-electron microscopy structure of PI3KC3-C2 in complex with the PI3KC3-binding domain (PIKBD) of Rubicon and compared it to cryo-EM structures of unbound PI3KC3-C2 and PI3KC3-C1. Rubicon binds directly to PI3KC3-C2 only via the BARA domain of the BECN1 subunit, which is common to both C1 and C2. The selectivity of Rubicon for the PI3KC3-C2 complex over the PI3KC3-C1 complex is attributed to a conformation of the BECN1BARA domain induced by UVRAG, rather than to direct contact with UVRAG or direct antagonism by the ATG14 subunit of PI3KC3-C1. Targeted disruption of the Rubicon:PI3K3-C2 structural interface by site-directed mutations enhances mitophagic activity in human epithelial cells to levels comparable to those observed in Rubicon knockout (KO) cells. Similarly, disruption of the interaction in Rubicon-overexpressing hippocampal neurons restored lysosomal flux to wild-type levels. These data show that suppressing the function of PI3K3- C2 can fully account for the negative regulatory effects of Rubicon in the autophagy and ELN pathways. Significance StatementEndolysosome maturation and autophagosome-lysosome fusion require the production of phosphatidylinositol 3-phosphate (PI(3)P) by the class III phosphatidylinositol 3-kinase complex II (PI3KC3-C2). Rubicon is a key negative regulator of endolysosomes and autophagy that suppresses PI3KC3-C2 activity. Here, we reveal in atomistic detail how Rubicon selectively recognizes PI3KC3-C2. Disrupting the Rubicon-PI3KC3-C2 interaction restores mitophagy and enhances lysosomal activity to the same extent as Rubicon gene deletion, establishing that PI3KC3-C2 inhibition fully accounts for the biological regulatory effects of Rubicon in the autophagy and lysosome pathways.

Increased activity of leucine-rich repeat kinase 2 (LRRK2) confers Parkinson's disease risk. LRRK2 dynamically localizes to lysosomal membranes in response to various stresses, yet the mechanisms by which distinct lysosomal perturbations are communicated to LRRK2 remain unclear. Here, we show that inhibition of the lysosomal lipid kinase PIKfyve promotes LRRK2 recruitment and signaling through a pathway that requires the lysosomal chloride/proton antiporter ClC-7. ClC-7 in turn controls the accumulation of multiple Rab GTPases on lysosomes. LRRK2 signaling under these conditions requires its established Rab-binding surfaces, with Rab12 contributing significantly to this response. This pathway operates independently of CASM. In contrast, lysosomal stresses that induce CASM require both Rab-binding sites on LRRK2 and GABARAP for robust LRRK2 signaling. These findings identify ClC-7-dependent lysosomal remodeling and Rab accumulation as key features linking PIKfyve inhibition to LRRK2 signaling and reveal that distinct lysosomal stresses engage different combinations of Rab and GABARAP inputs to activate LRRK2.

Accurate chromosome segregation requires the spatiotemporally regulated removal of sister chromatid cohesion. Cohesin cleavage by the endopeptidase separase depends on destruction of its inhibitor securin by the ubiquitin-proteasome system (UPS) and on phosphorylation-mediated priming of the cohesin kleisin subunit. Whether the UPS also contributes to cohesin priming has remained unknown. Here, we show that the 26S proteasome mediates cohesin removal during meiosis II through branches of two parallel pathways. Using separation-of-function mutants targeting either the proteasome's core or regulatory particles, we identify a proteasome function that is required specifically for centromeric cohesin removal during meiosis II but dispensable for separase activation. Defects in this proteasome function causes centromeric accumulation of phosphatase anchor shugoshin (Sgo1), impaired cleavage of meiotic kleisin Rec8, and frequent failure of sister chromatid segregation. Bypassing the requirement for Rec8 priming, either through a phosphomimetic rec8 allele or by separase-independent Rec8 cleavage, restores chromosome segregation, demonstrating that the proteasome mediates cohesin removal independently of its established role in activating separase. Consistent with a direct role, proteasomes localize prominently to kinetochores during meiosis II. Together, these findings identify the proteasome as a dual-function regulator that mediates both separase activation and cohesin priming, revealing how a single proteolytic machine coordinates the two molecular pathways underlying stepwise chromosome segregation during meiosis.

During spermiogenesis, early round spermatids differentiate into specialized spermatozoa through an extensive reshaping of the nucleus driven by coordinated cytoskeletal and chromatin-based mechanisms. In mammals, this process critically relies on the transient manchette, a microtubule-based structure that remodels the spermatid nucleus and serves a track to transport material required for flagellum assembly. However, the existence, organization, and molecular composition of such a structure in other vertebrates have remained poorly investigated. Here, we establish that an organized microtubule network is present in Xenopus spermatids and shares key architectural and molecular hallmarks of the mammalian manchette. We further uncover a large structural heterogeneity of spermatid microtubules, with variable protofilament numbers, skew angles, and lattice organizations. We reveal the presence of a Spaca9-Saxo2 internal scaffold in spermatid microtubules suggesting an internal reinforcement mechanism necessary for extensive nuclear reshaping and cytoplasm remodeling.

During eukaryotic cell division, kinetochores couple duplicated sister chromatids to mitotic spindle microtubules to mediate faithful chromosome segregation. Although the main kinetochore attachment sites to centromeric chromatin and microtubules are known, additional factors including microtubule-associated proteins are required for efficient chromosome biorientation and segregation in vivo. However, the roles and mechanisms of these factors in kinetochore function remain to be fully understood. Here, we characterise a previously unrecognised interaction between the microtubule plus-end tracking protein Bim1 and the outer kinetochore Ndc80 complex (Ndc80c) in S. cerevisiae. We show this interaction is mediated by a conserved SxIP motif within the intrinsically disordered Ndc80 N-terminus (Ndc80N), augmented by a secondary binding site containing an -helical segment. This Ndc80 interaction with Bim1 increases the strength of Ndc80c-microtubule attachments. Phosphorylation of the Bim1-binding region of Ndc80N by the error correction Ipl1/Aurora B protein kinase alters its secondary structure and weakens the Bim1-Ndc80c interaction, providing a potential additional regulatory mechanism for how incorrect kinetochore-microtubule attachments are destabilised during error correction.

Macrophages are central to immune homeostasis, clearing both pathogens and apoptotic cells. While these processes share some features, they differ functionally: efferocytosis of apoptotic cells is typically anti-inflammatory, whereas bacterial uptake triggers strong inflammatory responses. During infections, both targets may coexist, yet how macrophages handle such complex particles is poorly understood. Previously, we showed that Pseudomonas aeruginosa adheres to apoptotic cells, forming stable composites that can be jointly phagocytosed. Here, using quantitative confocal and live-cell imaging, we investigate how macrophages engage and internalize these dual targets. We find that macrophages employ distinct strategies depending on target composition. Apoptotic cells alone are engulfed intact within tight, actin-rich membrane cups, whereas bacteria-laden apoptotic cells induce protrusive, actin-driven extensions that navigate along the apoptotic scaffold to access bacteria. Individual bacteria are selectively extracted and internalized, while apoptotic material is internalized in a piecemeal manner. Early phagosome analysis shows that most contain either bacteria or apoptotic material alone, with mixed cargo being less frequent. These findings demonstrate that macrophages can discriminate and sort the components of complex targets during uptake, revealing a previously unrecognized sophistication in phagocytic processing. This work provides a framework for understanding how innate immune cells integrate pathogen clearance while managing apoptotic material.

Biomolecular condensates are membraneless compartments critical for the functional organization of cellular macromolecules in essential processes such as cell division, gene transcription or stress responses. We previously reported that vimentin filaments remodel into phase separated biomolecular condensates upon oxidative stress. This process requires vimentin single cysteine, C328, suggesting the involvement of oxidative modifications of this residue. Here, we aimed to generate vimentin condensates by inserting mutations mimicking posttranslational modifications associated with oxidative stress. In vimentin deficient cells, a cysteine oxidation mimetic mutant, vimentin C328D, formed only elongated particles or short filaments that evolved towards droplets upon serum deprivation or treatment with the oxidant diamide. Among vimentin posttranslational modifications rapidly responding to these stimuli, glycosylation confers filament stability whereas phosphorylation promotes disassembly. We observed that the O-deglycosylation inhibitor thiamet G, and the kinase inhibitors staurosporine and H-89, attenuated diamide-elicited vimentin C328D droplet formation, suggesting a potential glycosylation/phosphorylation interplay in this effect. Indeed, introducing phosphomimetic residues at certain single vimentin glycosylation and/or phosphorylation sites induced the formation of droplets, only if combined with the C328D mutation. In particular, the vimentin S49D,C328D mutant formed condensates that were reversibly dispersed by dilution through hypotonic shock. Therefore, mimicking C328 oxidation and S49 phosphorylation was sufficient to elicit vimentin phase separation. In vitro, purified vimentin S49D,C328D polymerized into a mixture of aberrant filaments and aggregates, which, in the presence of crowders, evolved towards paracrystals or clusters of beaded assemblies depending on pH. These findings highlight the role of C328 perturbations in the formation of biomolecular condensates and suggest a modulatory role of glycosylation/phosphorylation, thus shedding light on the processes regulating vimentin phase separation.

Skin, the bodys largest mechanosensitive organ, relies on a tension gradient across epidermal layers to maintain structure and function, but how mechanical force contributes to epidermal development and disease pathogenesis is poorly understood. By anchoring intermediate filaments (IF) to the plasma membrane, desmosomes, the most abundant intercellular junctions in the epidermis, help create a supracellular scaffolding that provides mechanical resilience to the tissue. However, the contribution of the desmosome-IF network to the epidermal response to mechanical strain remains unknown. Here we show that the desmosome-IF connection is not only required to induce a proper cellular mechano-response but is actively strengthened in response to stretch through the PP2A-mediated phospho-regulation of the cytoskeletal linker protein desmoplakin (DP). Additionally, we show in human skin dephosphorylated DP localizes to high tension layers, suggesting this mechano-response mechanism is coordinated with the epidermal tension gradient. Furthermore, in models of Carvajal syndrome, a cardio-cutaneous disorder caused by truncating DP mutations, cells lose mechano-responsive behavior and exhibit abnormal morphology in high-tension epidermal layers. Together, these findings identify the DP-IF network as a key component of the response to mechanical strain and show that its disruption compromises epidermal homeostasis and contributes to disease pathogenesis.