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.

COPI-mediated retrograde transport is essential for maintaining homeostasis between the endoplasmic reticulum (ER) and the Golgi in eukaryotic cells. However, its spatiotemporal dynamics remain unclear. In budding yeast, ER export occurs through a hug-and-kiss mechanism, wherein mobile cis-Golgi network (CGN) elements transiently approach ER exit sites (ERES) to capture cargo before departing to deliver it to the Golgi. Here, using high-resolution 3D and 4D live imaging, we show that the COPI-mediated recycling of a cargo receptor from the CGN back to the ER occurs during the hug-and-kiss event. Furthermore, this recycling is complete before the CGN detaches from the ERES, suggesting that COPI-mediated retrograde transport is spatiotemporally coupled to ER export during a single and transient CGN-ERES association. Consistent with this model, when COPI function is impaired, the receptor fails to return efficiently to the ER and instead remains in the CGN after separation from the ERES. Additionally, COPI impairment prolonged CGN-ERES association, suggesting that COPI function also facilitates CGN detachment from ERES. Thus, we propose that rapid COPI-mediated recycling drives CGN maturation directly at the ERES interface, enabling the CGN to detach and proceed along the secretory pathway.

During cell migration, branched actin filaments nucleated by the Arp2/3 complex induce leading edge protrusions, whereas directionality of cell migration is controlled by microtubules. We showed previously that Adenomatous Polyposis Coli (APC) initiates branched actin assembly at microtubule tips, which can explain how microtubules control directional protrusion. Here, we investigate a link from APC to Arp2/3 complex activity. We show that protrusion-generating activity of APC resides in its N-terminal region containing the Armadillo Repeat Domain (ARD). Furthermore, Asef1/ARHGEF4, a Cdc42 GEF known to be activated by the ARD of APC, as well as Cdc42 itself and its effector N-WASP, an Arp2/3 complex activator, are all required for the assembly of branched actin filaments in neuronal growth cones and neurite outgrowth. As downregulation of Asef1, Cdc42 or N-WASP produces phenotypes similar to APC knockdown, we propose that microtubules regulate directional cell migration and neuron navigation by inducing local membrane protrusion through the APC - Asef1 - Cdc42 - N-WASP - Arp2/3 pathway. These findings bridge a significant gap in our knowledge of cell migration.

During cytokinesis in amphibian and echinoderm embryos, propagating waves of Rho activity and F-actin assembly - cortical excitability - are amplified and focused at the equatorial cortex by the mitotic spindle. Such cortical waves are proposed to result from fast positive feedback based on Rho and the Rho GEF, Ect2, and delayed negative feedback involving F-actin and the Rho GAP, RGA-3/4. Currently, this model is tenuous, in part because the relationship between F-actin and RGA-3/4 is not well understood. Therefore, we investigated how RGA-3/4 regulates F-actin structure both in vivo and in vitro. Full-length RGA-3/4, when expressed alone in Xenopus oocytes, localizes to and bundles cortical F-actin. In vitro, purified full-length RGA-3/4 protein directly binds to and bundles F-actin. TIRF microscopy revealed that RGA-3/4 binds to F-actin cooperatively and accumulates on the filaments before filament crosslinking events. Structure-function analyses demonstrated both the amino-terminal region and the intrinsically disordered region at the carboxyl-terminal portion of RGA-3/4 are essential for normal F-actin interaction. These results provide the first demonstration that RGA-3/4 is a direct, cooperative F-actin binding and bundling protein. We propose that its special features make RGA-3/4 uniquely suited to provide F-actin-dependent negative feedback during cortical excitability and cytokinesis.

The spindle assembly checkpoint (SAC) prevents anaphase until chromosomes have formed correct bipolar attachments to the mitotic spindle. Spatial separation of pericentromeric Aurora B from outer-kinetochore substrates explains how tension-dependent biorientation stabilises microtubule-kinetochore attachments. How this geometry controls the SAC and is ultimately sensed by the key checkpoint kinase MPS1 has remained unclear. Here, we show that MPS1 recruitment is regulated by the spatial relationship between pericentromeric Aurora B-INCENP and the outer kinetochore. Shortening INCENP reduces MPS1 recruitment, whereas extending INCENP delays MPS1 removal and SAC silencing. Partial NDC80 depletion prevents normal Aurora B-kinetochore separation while retaining MPS1-dependent checkpoint signalling. Conversely, acute recruitment of Aurora B to microtubule-attached outer kinetochores under tension bypasses spatial separation and rapidly restores MPS1 and downstream SAC signalling. We propose that error correction and MPS1-dependent SAC signalling share an upstream spatial-sensing mechanism in which force-dependent separation from Aurora B favours attachment stabilisation while limiting MPS1 recruitment.

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.

Dendrites are vital to neuronal function, and dendrite injury occurs after neurological traumas such as stroke, traumatic brain injury, or neurodegenerative diseases. Despite their importance, the mechanisms underlying dendrite maintenance or regeneration remain poorly understood. The Drosophila gene short stop (shot), orthologous to ACF7/MACF1 in mammals, functions as an actin-microtubule crosslinker during neuronal development. Here, we investigate shot's role in dendrite stability and repair using Drosophila sensory neurons. We find that shot plays opposing, cell type-specific roles in dendrite maintenance: it restricts excessive branch growth in simple, stable neurons, while being required for dendrite coverage in complex, dynamic neurons. These opposing functions are reflected in distinct localization patterns in stable versus dynamic dendrite arbors. Loss of shot destabilizes the microtubule cytoskeleton in stable neurons, and activates Wallenda/DLK signaling in both stable and dynamic neurons. Downstream of Wallenda/DLK, JNK signaling diverges between neuron types, with canonical basket/JNK activation occurring only in neurons with stable dendritic arbors. After injury, shot promotes dendrite regeneration in both neuron types and accumulates in distinct shapes in regenerated dendrites, with specific domains critical for proper Shot accumulation patterns. Collectively, these findings establish shot as a context-dependent regulator of dendrite maintenance and repair, and demonstrate that the structural identity of a dendritic arbor shapes how neurons sense and respond to cytoskeletal perturbation.

Macrophage macropinocytosis contributes to wound healing, antigen presentation, and resolution of inflammation. Macropinocytosis also facilitates nutrient uptake and growth in macrophages, T cells, and cancer cells. Here, CRISPR/Cas9 whole-genome screens in murine bone-marrow derived macrophages (BMDM) identified genes regulating unstimulated, PMA-, and CSF1-stimulated uptake of the fluorescent pinocytosis solute tracer, Lucifer yellow. UVRAG and other members of VPS34 complex II (VPS34-II), which catalyze PI(3)P formation from phosphatidylinositol, were identified as positive regulators of macropinocytosis. Targeted gene disruption of Uvrag and Pik3r3 revealed that VPS34-II is required for efficient macropinocytosis with Uvrag-disrupted BMDM having fewer but larger macropinosomes. In contrast, depletion of ATG14, a unique component of VPS34 complex I, increased solute uptake and the number of macropinosomes. Live-cell imagining of macrophages expressing 2xFYVE-fluorescent protein fusions showed PI(3)P present on the plasma membrane, nascent macropinosomes, and endosomes. The presence of PI(3)P on the plasma membrane prior to macropinocytic cup closure, indicates a novel role on the plasma membrane. Quantitative imaging of fixed cells shows a decrease in concentration of PI(3)P in Uvrag-disrupted BMDMs while increase in concentration of PI(3)P in Atg14-disrupted BMDM compared to WT BMDMs. Treatment with the VPS34 inhibitor SAR-405 acutely decreased macropinocytosis but maintained AKT phosphorylation suggesting class I PI3K activity and PI(3,4,5)P3 production are independent of class III PI3K activity. These results suggest that PI(3)P is a key phosphoinositide governing macropinocytosis at the plasma membrane and that it is primarily formed via direct phosphorylation of PI rather than via the sequential dephosphorylation of PIP3.

In response to tissue damage, the epidermal barrier must be restored immediately to prevent excessive blood loss and infections. How cells coordinate actin-driven protrusions and membrane transport during reepithelialization is poorly understood. Here, we identified Rab35 as an important driver of epidermal wound closure in Drosophila. Endogenous Rab35 positive vesicles are directed to the wound edge and in protruding lamellipodia. Suppression of Rab35 function results in striking lamellipodia defects and an accumulation of PIP2-positive vesicles. Depletion of Fascin, a known Rab35 effector phenocopies rab35 knockdown. Upstream of Rab35, we identified CG18659 as potential Rab35-GEF protein, which physically interacts and colocalizes with Rab35. Suppression of Rab35 GEF CG18659 or overexpression of Rab35 GAP Whacked results in strong wound healing defects. We propose model in which Rab35 drives epidermal wound closure by promoting Fascin-stabilized actin-driven protrusions and vesicle transport required for cell membrane expansion and local PIP2 delivery at the wound site.

Clathrin-mediated endocytosis requires the coordinated assembly of a highly dynamic protein network that couples membrane remodeling to actin-driven force generation. The essential budding yeast protein, Pan1, scaffolds the endocytic protein network by linking adaptor proteins, and other coat components, and actin assembly regulators. How Pan1 coordinates all these different proteins in space and time is not well understood. We show that Pan1 undergoes biomolecular condensation. Elevated Pan1 expression induced the formation of condensates that exhibited partial rapid molecular exchange, temperature-dependent reversibility, and sensitivity to disruption of weak hydrophobic interactions. The Pan1 assemblies are compositionally selective, preferentially enriching late-stage endocytic factors while excluding early adaptor proteins. Truncation analysis demonstrated that condensation depends on cooperative contributions from intrinsically disordered regions, the EH2 domain, and the oligomerization module, whereas a C-terminal region negatively regulates condensation. Deletion of Pan1 regions promoting its self-assembly perturbed Pan1 assembly and function at endocytic sites. Disruption of interactions between Pan1 and endocytic adaptors did not alter the dynamics of endocytic events but instead reduced the number of endocytic events marked by Pan1 and caused the formation of ectopic Pan1 condensates. This indicates that adaptor-mediated interactions spatially constrain Pan1 localization by seeding Pan1 assembly at endocytic sites. Our findings support a model in which adaptor proteins seed Pan1 assembly at the endocytic sites while multivalent interactions drive Pan1 self-assembly to build the higher-order molecular network of the late endocytic coat.

The axon initial segment (AIS) and nodes of Ranvier (NoR) are essential for action potential initiation and propagation. They share many features of their molecular architecture, and their assembly mechanisms converge on the membrane-associated periodic skeleton (MPS). Here, we identify Paralemmin-2 (Palm2) as a component of both the AIS and NoR. Palm2 depletion shortens the AIS and reduces neuronal excitability. Endogenous Palm2 in the AIS is non-periodic, but upon overexpression it associates with the MPS, localizing to actin rings and reducing {beta}IV-spectrin abundance and periodicity. In NoR of the central and peripheral nervous system, Palm2 localizes to different subdomains - nodal or paranodal, respectively. Palm2, and its homolog Palm1, bind the deubiquitinase USP7, implicating paralemmins in proteostasis at the MPS. The complementary localizations of Palm2 and Palm1 at the AIS/NoR or axon shafts, respectively, parallel the distributions of {beta}-spectrin and ankyrin isoforms between these axonal compartments. We propose that Palm2 modulates the submembrane cytoskeleton and its membrane attachment, and thus contributes to the assembly, functioning and remodeling of the AIS and NoR.

Accurate chromosome segregation requires bipolar kinetochore-microtubule attachments. Error correction mechanisms promote cycles of kinetochore-microtubule release and reattachment until bipolar attachments are established. Although conserved error correction pathways operate in mitosis and meiosis, whether they have distinct requirements during meiosis remains unclear. Here, we investigate the role of Stu2/XMAP215/chTOG in kinetochore-microtubule error correction and compare its function with that of Ipl1/Aurora B during budding yeast mitosis and meiosis. Reducing kinetochore-bound Stu2 causes substantially more chromosome missegregation during meiosis than mitosis, revealing a heightened requirement for Stu2-mediated error correction. Whereas Ipl1-depleted cells fail to release initial improper kinetochore-microtubule attachments, kinetochore-defective stu2 mutants undergo repeated error correction attempts yet missegregate chromosomes. Our findings reveal distinct but complementary functions for Ipl1 and Stu2. Ipl1 releases initial improper attachments, while kinetochore-bound Stu2 promotes the formation and stabilization of productive attachments and enables error correction attempts to establish biorientation. We propose that features of meiotic chromosome segregation increase the requirement for multiple error correction pathways.

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.

Meiosis requires coordination between chromosome and nuclear envelope (NE) dynamics. Cytoskeletal forces transmitted through the NE via linker of nucleoskeleton and cytoskeleton (LINC) complexes drive chromosome movement but can also compromise nuclear integrity if not properly balanced. Here, we identify the conserved inner nuclear membrane protein NEMP-1 as a key regulator that contributes to buffering of excessive force at the meiotic NE in Caenorhabditis elegans. NEMP-1 localizes to the NE throughout meiotic prophase and becomes enriched at the NE in nuclei with a weakened nuclear lamina. Simultaneous depletion of NEMP-1 and the lamin protein LMN-1 results in premature nuclear collapse and defects in chromosome synapsis and homolog pairing. These phenotypes depend on excessive dynein-mediated forces acting on a compromised NE. Furthermore, analysis of homolog pairing in the absence of synapsis revealed that NEMP-1 and LMN-1 contribute to the stabilization of synapsis-independent homolog interactions at the NE. Together, our findings reveal that excessive force at the NE can destabilize homolog interactions, and support a model in which NEMP-1 and LMN-1 cooperatively buffer cytoskeletal forces to maintain nuclear integrity while stabilizing chromosome interactions during meiosis.

Mitotic spindle orientation is tightly controlled in epithelia to preserve polarized tissue architecture. Here, we uncover a previously unrecognized role of the adherens junction protein {beta}-catenin in regulating planar mitotic spindle positioning required for symmetric epithelial cell division. Using CRISPR/Cas9-mediated genome editing in MDCK cells, we found that {beta}-catenin--but not its close homolog {gamma}-catenin (plakoglobin)--is required for proper spindle orientation. Loss of {beta}-catenin disrupts astral microtubule anchorage and impairs the localization of LGN-NuMA spindle orientation machinery. Mechanistically, {beta}-catenin mediates spindle regulation via its N-terminal domain, independently of {beta}-catenin binding. We showed that the cortical recruitment of Afadin during mitosis depends on {beta}-catenin, but surprisingly, overexpression of either Afadin or ZO-1 rescue the spindle orientation in {beta}-catenin deficient cells by restoring cortical LGN. Afadin depletion or dominant-negative Afadin abolished ZO-1-mediated compensation, thereby establishing Afadin as the central cortical integrator, potentiated by {beta}-catenin, and operating through mass-action to recruit cortical LGN. Together, our findings define {beta}-catenin as an upstream regulator of the Afadin-based cortical-spindle linkage that coordinate the crosstalk between adherens junctions and mitotic geometry to ensure epithelial homeostasis.

Microtubules (MTs) serve as intracellular tracks that enable molecular motors to transport cargos to specific cellular destinations. It has been proposed that the signals directing motor-driven transport are encoded on MTs through different isotypes, lattice conformations, and post- translational modifications (PTMs) of tubulin, or MT-associated proteins (MAPs) that decorate the MT surface. However, molecular predictions of these models have not been rigorously tested in vitro. Using isotypically pure recombinant tubulin and biochemical reconstitution, we examined how tubulin PTMs and MT lattice spacing influence MAP binding and kinesin-1 motility. We found that kinesin-1 is largely insensitive to tubulin PTMs but is strongly regulated by MT lattice spacing. Likewise, the MAPs tau, MAP7, MAP4, DCX, and MAP9 exhibit little sensitivity to tubulin PTMs, whereas the MT-binding affinities of tau, DCX, and MAP7 depend on lattice spacing. In the presence of activating (MAP7) and inhibitory (tau) MAPs, lattice spacing determines MAP occupancy and thereby controls kinesin-1 motility. These findings support a two-layer transport code in which MT lattice spacing directs MAP binding, and MAPs determine which motors can move along individual MT tracks.

The sarcoplasmic reticulum (SR) is an elaborate membrane network and the primary intracellular Ca2+ store for muscle contraction. SR abundance, morphology, and spatial organization must be remodeled during muscle atrophy and hypertrophy, yet the mechanisms underlying this remodeling remain poorly understood. Autophagy has been implicated in SR homeostasis and may contribute to this process. We previously showed that a subset of Drosophila larval muscles remodels into adult dorsal internal oblique muscles through an autophagy-dependent process accompanied by extensive SR reorganization. Here, we show that autophagy is essential for maintaining SR abundance and architecture during muscle remodeling. Blocking autophagosome formation caused the accumulation of large aggregates of SR membranes, whereas blocking autophagosome-lysosome fusion led to the accumulation of autophagosomes with SR transmembrane proteins localized to their membranes. Moreover, disrupting LC3-interacting region (LIR)-dependent cargo recognition did not impair SR remodeling, arguing against a major contribution from selective autophagic degradation of the SR. Instead, our findings suggest that SR membrane components are incorporated into autophagosomal membranes during muscle remodeling. We propose that this membrane flow couples the removal of excess SR membrane to autophagosome biogenesis during atrophy, thereby facilitating large-scale SR reorganization during muscle remodeling.

Autophagy is initiated at endoplasmic reticulum (ER)-associated omegasomes, yet how the ER contributes to mitochondrial cargo degradation during early autophagosome formation remains unclear. Gp78 is an ER-resident RING E3 ubiquitin ligase involved in ER-associated degradation (ERAD) that also promotes Parkin-independent mitophagy. However, the spatial relationship between Gp78 ubiquitin ligase activity, omegasome formation, and mitochondrial degradation has yet to be defined. Combining targeted mitochondria labeling and super-resolution STED microscopy, we show that the Gp78 ubiquitin ligase is localized to and regulates DFCP1-positive omegasome formation, where it drives mitochondrial protein breakdown. Using fluorescent mitophagy reporters and acute ivermectin-induced mitophagy, generation of mitophagy intermediates lacking mitochondrial outer membrane (TOMM20) was found to be dependent on the Gp78 RING finger as well as the BAG6-UBL4A-USP13 ubiquitin-ligase module, and independent of PINK1 and Parkin. Object-based analysis of 3D STED images further showed that both DFCP1- and LC3-positive structures associate primarily with intact mitochondrial fragments which show progressive degradation of mitochondrial content. Gp78 localizes to and promotes, via BAG6, the formation of DFCP1-positive omegasomes and mitochondrial protein degradation in these early autophagic structures. ER-derived omegasome biogenesis is therefore coupled to Gp78-dependent ubiquitin ligase activity and local degradation of mitochondrial cargo during mitophagy.

Spatiotemporal allocation and dynamic redistribution of centrosomal proteins are critical for coordinating the structural and molecular events that ensure successful cell division. While depletion of the multifunctional protein Optineurin (OPTN) impairs cell-cycle progression, our previous report of the presence of OPTN at key cell-division assemblies raised the question of how its trafficking is coordinated during cell division. In this study, using cell-cycle synchronisation coupled with flow cytometric analysis and confocal microscopy, we report that OPTN progressively dissociates from centrosomes during mitosis, redistributes to cytokinetic structures, accumulates at the spindle midzone, and then concentrates at the midbody during cytokinesis. Moreover, following this transition, OPTN progressively re-emerges at centrosomes during G1 of the subsequent cell cycle. Further, microtubule perturbation and regrowth assays provide evidence that OPTN recruitment and retention at the pericentriolar matrix (PCM) can occur independently of microtubules, whereas its exit is microtubule-dependent. Collectively, this study depicts, for the first time, a detailed movement trail of OPTN, highlighting its centrosome-linked redistribution and microtubule dependency. This context-dependent localisation and trafficking of OPTN across the centrosome-midbody axis establish a definitive spatiotemporal roadmap that can be strategically utilised to study its functional role in cell division.

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.