Journal of Cell Biology

Accurate chromosome segregation requires kinetochores to form robust, load-bearing attachments to dynamic spindle microtubules, mediated primarily by the Ndc80 complex. Two receptors, Dsn1 and Cnn1 (CENP-T), recruit multiple copies of Ndc80c to the kinetochore, but whether they confer functional differences to Ndc80 behavior is unclear. We previously demonstrated that kinetochore components co-purifying with the yeast Dsn1 protein can maintain persistent load-bearing attachments that track with microtubule tip growth and shortening. Using an optical trapping-based assay, we show that Cnn1 purifications also sustain dynamic microtubule attachments under load. Mutation of a conserved region within the disordered N-terminal tail of Cnn1 weakened attachment strength in vitro and caused a growth defect when Dsn1 function was impaired. The Cnn1 mutation reduced Stu2 kinetochore levels without altering other kinetochore proteins. Restoring Stu2, either by direct addition in vitro or by tethering it to Ndc80c in vivo, rescued both attachment strength and cellular viability. These findings reveal a biophysical role for Cnn1 in enabling Stu2-dependent stabilization of kinetochore-microtubule attachments.

The Arp2/3 complex has long been considered to only assemble branched actin structures in the cell (lamellipodia, endocytic patches, comet tails, and many more). We show for the first time by single-molecule tracking (SMT) that the Arp2/3 complex and SPIN90, which activates Arp2/3 complex to nucleate unbranched filaments, bind to and move in the basal cortex with stress fibers and focal adhesions (FA) that, unlike known sites of Arp2/3 enrichment, employ linear actin bundles. SPIN90 knockout in U2OS cells significantly increases the rate of collective cell migration while decreasing cellular traction (myosin-II and actin speeds) and adhesion (FA size and maturation markers). SPIN90's SH3 domain, similar to its adapter protein Nck1, shows enrichment in FAs, suggesting a possible location for SPIN90-Arp2/3 complex activity. Together, our findings indicate that SPIN90-Arp2/3 nucleated filaments also function in stress fibers where they help define the mechanics of traction and adhesion to regulate cell motility.

Microtubules are polarised polymers that assemble into highly specialised networks in a cell-specific manner. This is controlled in part by microtubule organising centres (MTOCs), which concentrate factors necessary for microtubule nucleation and the organisation of microtubule minus ends. Neurons rely on oppositely polarised microtubule networks, with axons containing mostly plus-end-out microtubules, and dendrites contain many minus-end-out microtubules. How minus-end-out microtubule polarity is established in dendrites remains an important question. Here, we identify a new type of MTOC within the dendrites of Drosophila class I dendritic arborisation neurons, a common model for the neuronal cytoskeleton. We show that membrane swellings distributed intermittently along dendrite shafts, which we term "dendritic varicosities", contain the principal component of the microtubule nucleating complex and repeatedly generate microtubules whose plus ends grow back towards the soma. Varicosities located specifically in distal regions also contain MTOC proteins implicated in minus end anchoring, and this correlates with the accumulation of minus ends specifically in distal varicosities. Depletion of these MTOC proteins leads to major defects in minus end organisation, with microtubule buckles and loops deforming the neuronal membrane. Thus, dendritic varicosities are an important new type of neuronal MTOC that contribute to the generation and organisation of the minus-end-out microtubule network within dendrites.

The microtubule plus-end tracking proteins (+TIPs) CLIP-170/Bik1 and EB/Bim1 form a condensate, the +TIP body, at the plus-end of most microtubules in vivo. Remarkably, however, these +TIP bodies typically impart different dynamics and interaction profiles to distinct microtubules, according to their cellular function. The molecular mechanisms underlying the functional versatility of the +TIP body are unknown. Here, we show that the +TIP Kar9 utilizes repeats of a lysine-aspartate-lysine (KDK)-centered short linear motif (SLiM) to interact with Bik1 on a restricted subset of cytoplasmic microtubules during yeast mitosis. Furthermore, these multivalent Kar9-Bik1 interactions tune the material behavior of the +TIP body to specify proper microtubule function. Indicating that KDK serves as generic Bik1-interaction motif, similar motifs are also present in Kip2, where they mediate Bik1-dependent recruitment of Kip2 to the +TIP body. Together, our study provides insights into how low-affinity Bik1 interactors diversify microtubule function by locally specializing the content and behavior of +TIP bodies.

Centrosomes organize microtubules, coordinate ciliogenesis, and support accurate cell division. At the mature mother centriole, distal and subdistal appendages confer specialized functions in ciliary docking, microtubule organization, and intracellular trafficking. Nuclear distribution element 1 (NDE1) is a centrosome-associated regulator of dynein-dependent processes and ciliogenesis, but its nanoscale organization and contribution to centrosome architecture remain incompletely understood. Here, using expansion microscopy and STED super-resolution imaging, we show that endogenous NDE1 forms a ring-like structure at the subdistal appendages in mouse embryonic fibroblasts and human RPE-1 cells. NDE1 occupies an intermediate radial position between the more centriole-proximal CEP128 layer and the more peripheral ninein layer. Depletion of ODF2 or CEP128 reduces centrosomal NDE1, whereas CEP170 depletion has little effect, placing NDE1 within an ODF2- and CEP128-dependent branch of the subdistal appendage organization network. NDE1 depletion compromises centrosome integrity, reduces the centrosomal enrichment of core centriolar proteins, increases the separation between paired centrioles, and generates ectopic foci containing multiple centriolar markers. Loss of NDE1 also disrupts pericentriolar material organization and impairs the establishment of focused, centrosome-associated microtubule arrays. Furthermore, NDE1 depletion increases LC3B- and p62-positive structures and reduces autophagic flux. Together, our findings establish NDE1 as a subdistal appendage-associated factor that supports centrosome architecture and microtubule-organizing activity. More broadly, they support an emerging view of subdistal appendages as a molecularly layered platform in which distinct but cooperating components connect mother centriole maturation to microtubule organization, ciliary regulation, and intracellular trafficking.

Centrosome amplification is frequently associated with chromosomal instability and tumor progression, but how cells coordinate centriole assembly with the control of centrosome numbers and quality remains poorly understood. TIAM1 is a RAC1 guanine nucleotide exchange factor previously implicated in centrosome-associated signaling and {beta}TrCP-dependent control of PLK4 abundance. Here, we examined how Tiam1 regulates autophagy-lysosome homeostasis in mouse embryonic fibroblasts induced to overexpress PLK4. In contrast to a previous model in which Tiam1 loss promotes productive centriole overduplication, we found, by super-resolution imaging and expansion microscopy, an abnormal distribution of PLK4 on the centrioles centriole-associated structures following TIAM1 depletion, suggesting that TIAM1 may support the organization or maturation of centrioles. TIAM1 depletion also resulted in increased LC3B-positive puncta and enlarged LAMP1-positive compartments, but this was not accompanied by increased LC3B-II accumulation after bafilomycin A1 treatment. These findings suggest that TIAM1 may act at the interface between centriole assembly and endolysosomal/autolysosomal organization, linking TIAM1 to lysosome-associated centrosome quality-control pathways.

Proper cell physiology requires the co-assembly of multiple actin cytoskeletal networks that are tailored for specific functions. To maintain and promote the different functions of these networks, cells decorate them with distinct types of actin binding proteins (ABPs). While various models have been proposed to explain this selective sorting of ABPs, the role of actin disassembly factors is less well understood. Here, we used inducible CRISPR interference and quantitative live-cell imaging to test how disassembly factors control the ABP composition of different networks. We found that knockdown of cofilin (Cof1), a potent and highly conserved disassembly factor, disrupts the size, organization, and ABP composition of actin networks. Specifically, defects in Cof1-mediated disassembly disrupt intracellular transport due to the assembly of overgrown and disordered branched actin networks that are inappropriately decorated by tropomyosin (Tpm1). Contrary to prevailing models of ABP sorting, these networks are co-decorated by Tpm1 and fimbrin (Sac6), and their assembly is independent of formin activity. Instead, our findings support a model wherein failure to maintain the proper architecture of branched actin networks drives mis-localization of network-specific ABPs. Together, this work demonstrates that actin disassembly factors play a critical role in maintaining cytoskeletal structure and function to regulate ABP sorting across distinct networks.

Cell wound repair requires rapid and coordinated remodeling of the actin cytoskeleton to restore cortex integrity. Here, we show that a Rab35-Mical-SelR pathway regulates actomyosin ring dynamics through reversible actin redox. We find that Rab35 is recruited to wounds and is essential for proper actin ring assembly and disassembly. Rab35 regulates the recruitment of Mical, an actin-oxidizing enzyme, and SelR, a reductase that reverses oxidation, to the cell wound. Mical and SelR knockdowns disrupt actin ring formation and wound closure, whereas double knockdown partially rescues these defects, indicating a balanced redox cycle is required. Super-resolution microscopy reveals that Mical and SelR differentially regulate F-actin architecture and orientation. Mutation of actin at Methionine 44 does not fully recapitulate Mical knockdown phenotypes, suggesting the presence of additional targets and enzymes. Taken together, our results indicate that reversible actin modifications dynamically regulate F-actin architecture and orientation for actin ring assembly and disassembly.

In eukaryotes, our understanding of clathrin-independent endocytosis (CIE) lags far behind that of clathrin-mediated endocytosis (CME). CIE plays key roles in internalizing receptors, viruses, bacterial toxins, and pathogens; thus, deeper mechanistic insights are critical for understanding cellular strategies for plasma membrane regulation. Yeast CIE requires a signal relay between the stress sensor Mid2, the guanine nucleotide exchange factor (GEF) Rom1, the Rho1 GTPase, and the formin Bni1. While GEFs promote GTPase activity, GTPase-activating proteins (GAPs) conversely stimulate nucleotide hydrolysis and GTPase inactivation. Here, we provide new insight into CIE, adding the RhoGAP Sac7 as a regulator. SAC7 deletion in CME-deficient cells improved cargo internalization, and Sac7 localizes primarily to the mother cortex. Cells lacking SAC7 accumulate active Rho1 and retain Bni1 at the plasma membrane, where Bni1 retention may subsequently enhance actin assembly needed for CIE. Our results thus demonstrate that Sac7 negatively regulates CIE by restricting cortical Rho1 activity.

Autophagy is a catabolic process that degrades damaged organelles and aggregation-prone proteins and plays key roles during development and in maintaining cellular homeostasis. It can be induced by stress including starvation, oxidative stress, or accumulation of misfolded proteins. Autophagy declines with age and there is great interest in manipulating autophagy to improve neurodegenerative diseases, as its stimulation shows promise to improve diseases including Huntington, Alzheimer, and Parkinson. Endosomal microautophagy (e-MI) is a type of autophagy in which cytosolic proteins are delivered to late endosomes and degraded upon incorporation into intraluminal vesicles of multivesicular bodies. Here, we report that the actin nucleation-promoting factors (NPFs) known to activate the Arp2/3 complex to promote branched actin assembly can alter the dynamics of e-MI. We found that upon stress exposure, overexpression of the NPFs WASp, Wash, or SCAR results in an expedited induction of e-MI. Strikingly, Wash is uniquely required for physiological e-MI induction implying that NPFs are not functionally redundant for e-MI. We show that the WASH complex regulates e-MI on late endosomes acting via Arp2/3 and thus likely branched actin. Surprisingly, the regulation of e-MI by Wash is independent of retromer that is known to recruit Wash to early endosomes for its role in recycling of membrane proteins and rather reflects a novel degradative aspect of Wash function. Taken together, we identified a novel function of NPFs as upstream regulators of e-MI that could be used to activate e-MI ectopically to improve aggregate clearance during neurodegeneration.

Quantitative image analysis of subcellular organization requires sufficient spatial resolution to resolve individual organelles and sample size to capture heterogeneity both within cells and between cells. Existing imaging approaches often force a tradeoff between spatial resolution and throughput, limiting the ability to measure organelle-level phenotypes across cell populations. Here, we establish high-throughputs expansion microscopy (HiExM) as a scalable pipeline for single-organelle analysis. As a benchmark, we focus on mapping late endosomes and lysosomes (LELs), a heterogeneous organelle class whose small size, dense intracellular distribution, and functional diversity make it difficult to quantify accurately using conventional light microscopy. HiExM increases effective spatial resolution while preserving compatibility with large-scale image acquisition, enabling robust segmentation and quantitative profiling of individual LELs across large cell populations. Using this pipeline, we identified differences in intracellular trafficking behavior among anti-transferrin receptor antibodies that could not be captured by conventional colocalization analysis alone. We further integrate spatial and morphological features with learned image-based representations that can define relationships between LEL morphology and subcellular position as well as how these relationships respond to perturbations. Together, our work establishes HiExM as a generalizable platform for scalable single-organelle profiling, enabling an analytical framework for quantifying discrete organelles across cells and conditions.

Nuclear pore complexes (NPCs) control molecular exchange across the nuclear envelope, but how they tailor their selective permeability to meet the needs of specific cell types and/or environments remains poorly understood. We demonstrate that the strength of the NPC diffusion barrier differs across cell types, is particularly stringent in cultured neurons, and correlates with the O-linked N-acetylglucosamine (GlcNAc) modification of nucleoporins. Using conditional tools that specifically control nucleoporin GlcNAcylation, we show that GlcNAc modulates NPC permeability. Interestingly, nucleoporin GlcNAcylation is mechanosensitive, increasing in cells plated on stiff substrates, a condition where nuclear pores dilate. Indeed, we demonstrate that increasing or decreasing GlcNAcylation dilates and constricts NPCs, respectively. Further, O-linked N-acetylglucosamine transferase is recruited to modify NPCs upon their acute constriction during osmotic shock. Thus, cells employ GlcNAc to modulate steady-state NPC permeability in response to mechanical inputs and to counteract critical changes to their osmotic environment.

Rab GTPase cascades drive endosomal membrane maturation by sequentially activating and inactivating Rab proteins. These transitions in Rab signaling require the coordinated actions of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). The yeast VINE complex is an endosomal VPS9-family GEF that stimulates a GAP to inactivate the Rab5 homolog Vps21, suggesting a role for VINE in coordinating Rab transitions. Here we report that VINE acts through its catalytic GEF domain to promote signaling by the Rab5-related GTPase Ypt10 and establish a pool of Ypt10 at late endosomes. Ypt10 activation occurs downstream of Vps21 activity, placing Ypt10 within a late endosomal Rab cascade. Genome-wide protein proximity screens revealed a VINE-dependent interaction between Ypt10 and the GEF Mon1-Ccz1. Our data suggest that VINE and Ypt10 regulate late endosomal recruitment of Mon1-Ccz1 to enhance the activation of its substrate, the Rab7 homolog Ypt7. Together, these findings define a Vps21-VINE-Ypt10 regulatory module that adds a layer of control within the late endosomal Vps21-to-Ypt7 cascade and establish VINE as a dual Rab regulator. Through opposing activities on Vps21 and Ypt10, VINE may couple Rab5 inactivation to Mon1-Ccz1 recruitment to provide more precise control of degradative protein traffic to the vacuole. Significance statementFour Rab5-family GTPases direct protein sorting and membrane maturation in the yeast endolysosomal system, yet their individual functions, and the role of the little-studied Rab Ypt10, are unclear. Using genome-wide proximity screens, we find that the GEF complex VINE establishes a pool of Ypt10 at late endosomes downstream of Vps21, where Ypt10 recruits Mon1-Ccz1, the activator of the Rab7 homolog Ypt7. Because VINE also drives GAP-mediated suppression of Vps21, our results suggest it acts as a dual Rab regulator, coupling Vps21 inactivation to Ypt10 activation to fine-tune the endosomal Rab cascade.

Kinesins are microtubule-dependent motors, yet the functions of the kinesin-4 family member KIF27 remain poorly understood. Here, we demonstrate a dynamic and cell-cycle-dependent localization of KIF27, consistent with its functional roles in mitotic progression. Upon mitotic entry, KIF27 relocates to condensed chromosomes. During anaphase, a fraction of KIF27 accumulates at the spindle midzone, and in telophase and late stages of cytokinesis it localizes at the midbody, colocalizing with key cytokinetic regulators at both structures. Recruitment of KIF27 to the midbody depends on KIF23 and CEP55. KIF27 depletion results in profound cell division defects, altered midbody and microtubule morphology, delayed cytokinesis and cytokinesis failure. Beyond cell division, KIF27 depletion directly compromises nuclear morphology, and pan-cancer transcriptomic analyses correlate low KIF27 expression with aneuploidy and poor patient survival in several cancer types. Together, our results identify KIF27 as a novel regulator of mitotic fidelity and genome stability.

Membrane contact sites (MCS) between the endoplasmic reticulum (ER) and the plasma membrane (PM) enable direct intermembrane exchange of signals and metabolites. The Extended Synaptotagmins (E-Syts) are an evolutionary conserved family of ER-PM tethers essential to maintain PM integrity under stress conditions. To investigate the underlying molecular mechanisms, we employed cellular reconstitution experiments in yeast and plants. We show that E-Syt-mediated stress tolerance relies on ER-PM MCS targeting, which requires the E-Syt N-terminal membrane anchor, a minimal set of two C2 domains and an SMP domain. C2 domains are sufficiently conserved that interspecies domains can sustain both PM localization and stress response. The role of the SMP domain in ER-PM localization is also conserved, but SMP function in stress resistance is species-specific. Furthermore, cryo-electron tomography uncovers a scaffolding role for the SMP domain in maintaining ER-PM distance, and in the formation of ER membrane peaks with extreme curvature that appear necessary for stress tolerance. Collectively, our findings reveal the individual and synergistic roles of all E-Syt modules in maintaining cellular homeostasis under stress.

Angstrom-scale changes in microtubule (MT) lattice spacing regulate the selective recruitment of MT-associated proteins, yet how these structural states operate in cells remains poorly understood. Here, we show that MT lattice expansion, induced by protein-based expanders or microtubule-stabilizing agents such as Taxol and epothilone D, drives the relocalization of compact lattice-binding proteins, including tau, doublecortin (DCX), and the C1 domain-containing signaling protein GEF-H1, into highly curved MT-associated domains, whereas the compaction-inducing agent laulimalide suppresses this response. In contrast, the tumor suppressor RASSF1A preferentially associates with expanded lattice states, revealing differential lattice sensitivity among closely related C1 domain-containing proteins. These short, curved assemblies are enriched at MT intersections and discrete MT segments, revealing spatially heterogeneous lattice states within individual microtubules. At substoichiometric levels, compact lattice-binding proteins behave as both MT compactors and curvature sensors. Changes in osmotic pressure selectively promote dissociation of compact lattice-binding proteins, whereas expanded lattice-binding proteins remain largely unaffected. Using curved filament formation as an in-cellulo readout of compact lattice regions, we identify widespread lattice-state sensitivity across diverse MT-associated and signaling proteins. Finally, we show that these principles extend to neurons, where somatic, but not axonal, tau exhibits sensitivity to lattice expansion despite the expanded lattice architecture of distal axonal microtubules, suggesting additional neuron-specific regulation of lattice accessibility. Together, our findings identify the MT lattice as a dynamic mechanochemical platform whose nanoscale structural states spatially organize protein recruitment and signaling in cells and neurons.

Beyond the mechanical capacity of canonical actomyosin-driven amoeboid motility in permissive extracellular environments, the nucleus becomes the principal barrier to T cell migration in confining tissues. We establish the dynein-microtubule (MT) force-transmission axis as an essential mechanism for nuclear translocation during confined T cell migration and transmigration. We argue that dynein acts both as a motor and as an F-actin-anchored force-transmission element (fulcrum), sliding MTs and the MT-coupled nucleus along the cell cortex to drive nucleokinesis and productive cell displacement. Dynein is the primary driver of nucleokinesis: its inhibition arrests nucleus movement independently of myosin II activity, while F-actin dynamics remain spatiotemporally decoupled from nuclear oscillations. During transmigration, dynein and actomyosin act cooperatively and non-redundantly, and only combined inhibition abolishes nuclear passage. Computational modeling demonstrates that dynein-mediated pulling, together with volume exclusion imposed by the nucleus, is sufficient to generate self-organized nuclear oscillations. Dynein inhibition in zebrafish Langerhans cells impairs protrusion dynamics in situ, identifying the dynein-MT axis as an evolutionarily conserved mechanobiological program. Collectively, these findings identify the dynein-MT mechanical unit as a potential target for engineering T cells with enhanced solid-tumor infiltration.

The polarization of motile cilia requires that the centrioles, from which cilia are formed, display rotational asymmetry. This property is manifested in the presence of asymmetrically distributed appendages and relies on evolutionary conserved mechanisms. These mechanisms are also at play in cells that form primary cilia despite the lack of ciliary motility and asymmetric centriole appendages in this context. Here, we find that a complex consisting of CCDC61, KIAA1328 (K1328), and Centlein (CNTLN) contributes to the establishment of centriole rotational asymmetry. In cells with a primary cilium, this complex is required for assembling a linker that repositions the daughter centriole close to and orthogonal to the proximal end of the mother centriole/basal body. The CCDC61/K1328/CNTLN complex also triggers the asymmetric recruitment of pericentriolar matrix components around newly assembled centrioles, which likely facilitates the later attachment of the basal body-daughter centriole linker. Overall, our results establish that rotational asymmetry relies on the coordinated recruitment of asymmetric landmarks along centrioles and is necessary for positioning the centrioles in a configuration that is widely conserved in ciliated cells.

Entry of intracellular pathogenic bacteria is widely considered an actin-driven process, potentially overlooking contributions of the microtubule cytoskeleton. Here, we identify a host microtubule state as a determinant of early infection efficiency by Chlamydia pneumoniae. Human cells enriched in acetylated microtubules are preferentially infected, whereas detyrosinated microtubules show no such association. Pharmacological stabilization of microtubules via Taxol enhances infection, while selective elevation of acetylation with Tubacin does not, indicating that microtubule stability rather than acetylation alone is critical. Thus, a pre-existing interphase microtubule architecture supports C. pneumoniae entry. Consistently, mitotic cells, characterized by a reorganized microtubule architecture, remain permissive but show severely reduced infection efficiency. In addition, infection induces a dose-dependent increase in microtubule acetylation that requires bacterial viability and is not observed during uptake of Yersinia pseudotuberculosis effector protein Invasin-coated beads, indicating that entry/internalization alone is insufficient to trigger this response. To probe how early chlamydial secreted effectors might engage the microtubule cytoskeleton, we focused on the conserved TarP family member CPn0572, an actin and microtubule regulator, which increases microtubule acetylation when ectopically expressed in human cells. Controlled expression of microtubule-localized CPn0572 in the yeast Schizosaccharomyces pombe leads to altered microtubule dynamic and mechanical behaviour, promoting force-bearing microtubules. Together, these findings show that distinct interphase microtubules define a permissive cellular state for bacterial entry and suggest that early chlamydial effector activities might promote a specific microtubule persistence phenotype.

p53-binding protein 1 (53BP1) is a key mediator of the DNA damage response and genome stability. While its interphase function is well-characterized, its mitotic role remains less understood. Here we show that aberrant activation of 53BP1 through the loss of its negative regulator, TIRR, leads to mitotic abnormalities including altered spindle geometry, kinetochore-microtubule (k-MT) attachment errors and whole chromosome missegregation. We demonstrate that loss of TIRR results in excess interaction between 53BP1 and the key mitotic kinase Polo-like kinase 1 (PLK1), altering PLK1s activation, spatial distribution, and its interaction with known PLK1 substrates at multiple mitotic stages. Moreover, due to PLK1s established role in CENP-A loading, hyperactivation of 53BP1 compromises CENP-A loading, triggers gradual loss of CENP-A from centromeres and generates severe kinetochore assembly defects. These findings uncover a non-canonical mitotic function of 53BP1 as a key regulator of PLK1 activity and chromosome segregation fidelity.