Journal of Cell Science

Cilia are assembled by intraflagellar transport (IFT), which relies on two protein complexes: IFT-A and IFT-B. It is generally assumed that IFT-B and IFT-A are critical for anterograde and retrograde transport, respectively. However, full deletion of IFT-A genes in several organisms suggests a possible contribution to anterograde transport. In many species, cilia collapse when IFT is altered, hindering functional studies. Here, we investigated the role of IFT-A in the protist Trypanosoma brucei, where IFT is not required for cilium maintenance. Following the inducible knockdown of IFT88 (an IFT-B member) or IFT140 (an IFT-A member), we monitored the fate of several IFT proteins in preassembled cilia using live imaging and evaluated the consequences on train formation by volumetric electron microscopy. Surprisingly, both IFT88 and IFT140 turned out to be essential for anterograde train assembly. Their depletion initially led to the formation of shorter trains and subsequently to an inhibition of train injection. We propose a model to reconcile the diverging phenotypes reported in the literature.

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.

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.

Cellular senescence is characterized by stable cell-cycle arrest, cytoskeletal remodeling, and altered secretion of senescence-associated secretory phenotype (SASP) factors, including SERPINE1/plasminogen activator inhibitor-1 (PAI-1). Although extracellular matrix (ECM) stiffening has been linked to fibroblast mechanotransduction and SERPINE1-associated remodeling, the molecular pathway connecting substrate stiffness to SERPINE1 regulation in senescent fibroblasts remains incompletely understood. Here, we investigated how defined substrate stiffness affects fibroblast morphology, mechanical phenotype, and SERPINE1 expression, and examined whether the clathrin adaptor AP2A1 participates in this response in replicative senescent human fibroblasts. Using tunable polyacrylamide hydrogels, we found that increasing substrate stiffness enhanced fibroblast spreading, stress fiber thickening, focal adhesion maturation, cellular stiffness, and senescence-associated marker expression. Stiff substrates also increased SERPINE1 expression and its colocalization with actin fibers, with stronger responses observed in senescent than in young fibroblasts. Functional perturbation experiments further suggested that SERPINE1 contributes to stress fiber organization in senescent cells. In addition, AP2A1 colocalized with SERPINE1, and modulation of AP2A1 under knockdown and overexpression conditions altered SERPINE1 signal intensity. Conversely, perturbation of SERPINE1 also affected AP2A1, supporting a potential bidirectional relationship between these two components. Together, these findings identify SERPINE1 as a stiffness-responsive factor associated with senescence-linked cytoskeletal remodeling and support a functional relationship between AP2A1 and SERPINE1 in senescent fibroblasts. These results suggest that the AP2A1-SERPINE1 axis may contribute to the link between extracellular mechanical cues and senescence-associated fibroblast remodeling.

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.

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.

Summary: Single-molecule localization microscopy techniques, such as (direct) stochastic optical reconstruction microscopy ((d)STORM) and photo-activated localization microscopy (PALM) enable the visualization of subcellular molecular organization beyond the diffraction limit of conventional light microscopy. Not only is data acquisition rather slow, but the downstream analysis of localization datasets often remains computationally challenging and time-consuming. Consequently, the complexity and duration of data processing often limit experiments to the acquisition and analysis of only small numbers of cells or regions of interest, thereby restricting the statistical power and biological reliability of SMLM studies. To address this limitation, we developed an open-source Python-based package for automated, high-throughput post-processing and quantitative analysis of SMLM localization data, enabling efficient and straightforward handling of extensive datasets with minimal manual intervention. Availability and implementation: dSTORMQuant (source code and documentation) are freely available on GitHub at https://github.com/BCMM-Bielefeld-University/dSTORMQuant under GPL v3 license.

The primary cilium has been implicated in multiple developmental processes, such as cell migration and asymmetric cell division of stem- and progenitor cells. While most in vitro model systems examine ciliogenesis induced by serum starvation, it is not fully understood how de- and re-ciliation are regulated in proliferating stem- and progenitor cells. Here we employ the hierarchically organized C2C12 skeletal muscle cell line to examine how K-Ras4B participates in de- and re-ciliation processes of ciliated stem- and progenitor cells. We show that MAPK-pathway activation supports ciliogenesis through phosphorylation of centrosomal protein CEP55, which can then no longer stabilize the master regulator of de-ciliation Aurora kinase A. K-Ras4B localizes to the primary cilium aided by the ciliary trafficking chaperone PDE6D, which promotes ciliation. In line with this, depletion of components of the PDE6D machinery, RPGR and RPGRIP1L, decreases ciliation. Activation of the ciliary AMPK-PKG2-pathway increases S181-phosphorylation of K-Ras4B, which negatively regulates its binding to PDE6D, its ciliary abundance and promotes differentiation. Our work integrates a major mediator of mitogenic signaling into the regulation of ciliogenesis of proliferating muscle stem- and progenitor cells.

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.

Microtubules are cytoskeletal polymers that play essential roles in eukaryotic cells, including structural support, cell division, and intracellular transport. During intracellular transport, kinesin motor proteins move cargo along microtubule tracks via their processive stepping. Recent studies have shown that the kinesin-1 KIF5C can damage the microtubule lattice while stepping. Microtubule damage can be repaired through incorporation of new tubulin subunits, however, excessive lattice damage results in microtubule breakage and disassembly. To identify cellular factors involved in microtubule repair, we performed an siRNA screen targeting microtubule-associated proteins (MAPs) known to regulate microtubule dynamics and stability. Based on the results, we investigated whether the end binding protein EB1 and cytoplasmic linker-associated protein 2 (CLASP2) contribute to repair of microtubule damage. To test this, we used a microtubule destruction assay in which damage was induced in microtubules gliding over surfaces coated with wild-type or mutant KIF5C proteins. Our findings suggest that CLASP2 directly facilitates microtubule repair, whereas EB1 does not. We further examined CLASP function using a microtubule repair assay and found that CLASP2 promotes repair by enhancing tubulin incorporation and reducing microtubule breakage. Together, these findings demonstrate that CLASP proteins play an important role in repairing and protecting against lattice damage caused by kinesin-1 motor activity. Our results further suggest that MAPs can directly regulate microtubule lattice integrity under mechanical stress generated by motor protein-driven intracellular transport.

Mechanical forces and signaling pathways are increasingly recognized as interdependent regulators of epithelial morphogenesis, yet their combined role in cell fate acquisition remains poorly understood. Here, we investigate the interplay between adherens junction mechanics and Notch receptor signaling during the asymmetric division of sensory organ precursors in the Drosophila pupal notum epithelium. Using quantitative live imaging and laser ablation, we identify the newly formed interface between SOP daughter cells as a mechanically specialized junction, characterized by persistently low membrane tension, distinct adhesive organization, and a unique cortical actomyosin architecture. We propose that low membrane tension may facilitate efficient Notch activation, as ligand-mediated endocytosis promotes Notch signaling by generating traction forces of a few piconewtons, oriented perpendicular to the plasma membrane. Perturbations of Notch pathway activity systematically alter junctional recoil following laser ablation, with reduced Notch signaling correlating with increased tension. Conversely, constitutive Notch activation in a Notch loss-of-function context is sufficient to restore a low-tension state. These findings suggest that Notch signaling actively shapes the mechanical properties of its signaling interface, indicating reciprocal interactions between mechanics and signaling. Together, our results support a model in which Notch activity and junctional mechanics are coupled during asymmetric cell division, highlighting how local mechanical states may contribute to the robustness of cell fate specification in epithelia.

The nuclear envelope (NE) surrounds the genetic material and is continuous with the endoplasmic reticulum (ER). In yeast and other organisms undergoing closed mitosis, nuclear envelope expansion (NME) is strictly required to accommodate spindle elongation and ensure proper chromosome segregation within a single nuclear compartment. Failure to expand the NE during mitosis leads to chromosome missegregation. Here, we show that deletion of the unstructured N-terminal domain of Rgf3, a Rho1-specific guanine nucleotide exchange factor (GEF), causes early mitotic defects that produce the characteristic "cut" phenotype of untimely cell division. The rgf3{Delta}N2 mutant displays spindle buckling, a hallmark of anaphase nuclei unable to properly expand the NE. From yeast to mammals, phosphatidic acid (PA)--a key precursor in phospholipid biosynthesis--is metabolized via two competing pathways, the cytidine diphosphate-diacylglycerol (CDP-DAG) and the Kennedy pathways, both contributing to lipid membrane homeostasis. We provide evidence that impaired Rho1 activation in rgf3{Delta}N2 selectively disrupts phospholipid synthesis through the CDP-choline branch of the Kennedy pathway. Thus, Rho1 promotes mitotic progression by modulating phospholipid biosynthesis to enable efficient NME during anaphase. HighlightsThe N-terminus of Rgf3 is required for proper nuclear envelope expansion (NME) during anaphase. The structurally flexible N-terminal domain of Rgf3 is essential for localized Rho1 activation. Active Rho1 drives mitotic membrane growth by modulating phospholipid synthesis through the Kennedy pathway.

CO2 opens a subset of connexin hemichannels by binding to a site in the cytoplasmic domain of the channel. From outside the cell, CO2 must cross at least one membrane to reach this site. We have used Neuro-2A cells, which exhibit very low expression of CO2 permeable aquaporins (AQPs) and do not express any of the connexins (Cxs) known to be CO2 sensitive, to evaluate the minimal complement of molecular components required to recapitulate whole cell CO2 sensitivity mediated by connexins (assayed by either whole cell patch clamp recordings or real time recordings of ATP release via a co-expressed genetically encoded ATP sensor). Neuro-2A cells that expressed either Cx26, Cx32 or Cx43 on their own did not exhibit CO2-dependent connexin hemichannel gating. Expression of AQP1 or AQP5 either with or without carbonic anhydrase 2 (CA2) did not reveal any endogenous CO2 sensitivity of Neuro-2A cells. Only by expressing one of Cx26, Cx32 or Cx43 with either AQP1 or AQP5, plus CA2 were we able to reconstitute whole cell CO2 sensitivity. We found that expression of Cx26 with either AQP1 or AQP5 resulted in high levels of cell death. This was prevented by co-expression of CA2. Simulations of the influx and diffusion of CO2 show that CA2 prevents accumulation of intracellular CO2 and excessive activation of Cx26, thus protecting the cells from death. Surveying the transcriptome of cells that express CO2 sensitive connexins shows that many also express CO2 permeable aquaporins and CA2. We suggest that connexins, aquaporins and carbonic anhydrases represent the minimal trifecta of components required for cellular CO2 sensing.

The endoplasmic reticulum (ER) and its muscle-specialized form, the sarcoplasmic reticulum (SR), are crucial organelles in muscle cells, involved notably in protein synthesis, calcium regulation and muscle contraction. A well-known process involved in ER remodeling and homeostasis is ER-phagy, also called reticulophagy, a selective form of autophagic process in which ER-phagy receptors mediate the delivery of ER portions to lysosomes for degradation. SH3KBP1 is an adaptor protein involved in membrane trafficking. Recently, it was shown to control ER morphology and SR formation in striated skeletal muscle. In this study, we demonstrate that SH3KBP1 can bind to LC3B and CKAP4 proteins, bridging ER to autophagosome membranes, and is degraded by autophagy, in developing muscle fibers. Moreover, SH3KBP1 down-regulation impacts basal autophagy efficiency and ER-phagy stimulation; it also impairs the turnover of numerous ER-resident proteins. Our work highlights a new role for SH3KBP1 as a soluble ER-phagy receptor in striated skeletal muscle.

High-resolution 3D imaging is an important strategy for visualizing and analysing complex skeletal tissue architecture and the bone marrow microenvironment. However, multicolor immunolabeling and imaging of intact skeletal tissues are technologically challenging. The current immunolabeling and clearing methods for intact skeletal elements are very limited, time-consuming and generate low-resolution data or depend on the use of reporter mice. Here, we describe a protocol for efficient clearing and immunolabeling of intact calcified tissues that enables superfast, single-cell resolution, and quantitative 3D light-sheet imaging of intact skeletal elements and teeth. A key aspect of our protocol is the addition of a collagenase digestion step after fixation and decalcification. This step enhances antibody penetration, resulting in deep, comprehensive staining throughout immunostained bones and other calcified tissues. The protocol includes soft tissue removal, fixation, decalcification, bone dehydration, and bleaching, followed by antigen retrieval and permeabilization before the collagenase digestion step. This procedure is performed to prepare the samples for the tissue clearing process that improves bone tissue transparency prior to light-sheet imaging. The entire protocol, from bone collection to image analysis and quantification, takes about 4 days to complete, thus offering significant improvements over previous methods. This protocol is broadly applicable to the visualization of bone microstructure, bone marrow analysis, vascular and neural network mapping, and the study of signaling molecules in bone development and growth. The protocol requires experience with standard tissue processing and immunostaining techniques, and prior experience in tissue clearing and light-sheet imaging is beneficial but not essential. Key pointsO_LIA protocol for efficient clearing and immunolabeling of intact calcified tissues that enables superfast, high-resolution, and quantitative 3D imaging of various intact bones and teeth. C_LIO_LIThe entire protocol takes only 4 days to complete the comprehensive staining and perfect transparency throughout the intact bones, offering significant improvements over previous methods. C_LI Key referencesBiswas, L. et al. Cell 186, 382-397.e24 (2023): https://doi.org/10.1016/j.cell.2022.12.031

While bioelectricity is increasingly recognized as an important regulator of cell function and morphogenesis, the field has almost exclusively focused on plasma membrane states. Voltage across the inner nuclear membrane (INM) has been proposed as a potential regulator of nuclear function, but how it responds to extracellular ionic perturbations and whether it relates to chromatin organization remain unclear. Here, we targeted the ratiometric genetically encoded voltage indicator ASAP3-R3 to SUN2-associated nuclear membranes in intact NRK cells and combined INM voltage measurements with Gray-Level Co-Occurrence Matrix (GLCM)-based chromatin texture analysis. Reporter localization was confirmed by fluorescence imaging and electron microscopy, and functional validation in isolated nuclei showed that sodium-potassium pump inhibition produced INM depolarization consistent with Goldman-Hodgkin-Katz (GHK)-based prediction. We then used our validated construct to determine the response of Vnuc and chromatin texture to changing ionic conditions via two exposure methods, gradual (ramped) exposure or direct application. In intact cells, ramping different sets of ionic solutions of decreasing sodium/increasing potassium, decreasing sodium, increasing potassium, or decreasing chloride induced INM hyperpolarization and coordinated changes in chromatin texture, including increased contrast and entropy, reduced homogeneity, and reduced nuclear area. These effects were strongly path-dependent, with nuclear responses shaped by the history and order of ionic exposure: sodium and potassium responses emerged most clearly during ramping exposure, whereas reducing chloride by direct exposure showed a more pronounced response profile. Direct changes in sodium exposure produced limited electrical and chromatin-texture effects, while direct potassium exposure altered chromatin texture and nuclear area without significantly changing VNuc. Importantly, shifting baseline chromatin state in either direction, through Trichostatin-A (TSA)-induced chromatin relaxation or sodium azide/2-deoxy-D-glucose-induced compaction, blunted ion-associated Vnuc and chromatin responses across sodium, potassium, and chloride conditions. Together, these findings identify the nucleus as a dynamic, ion-responsive electro-structural system in which INM voltage and chromatin organization are functionally coupled, and in which both ionic trajectory and pre-existing chromatin state shape the magnitude of the nuclear response.

Microproteins translated from short open reading frames are increasingly understood to play important roles in cell biology and development. Here, we describe a microprotein in Drosophila that is expressed in ovarian follicle cells which surround the developing oocyte. The Dafcin microprotein is predicted to form an amphipathic alpha-helix, a structure known to interact with lipid bilayers. The structure of Dafcin most resembles the influenza HA fusion peptide, which induces negative curvature of endosomal membranes. Dafcin tagged with GFP localizes to the Golgi and is ultimately secreted from the follicle cells. Remarkably, this occurs without the microprotein having a secretory signal sequence. The protein is taken up into the oocyte by endocytosis, localizing to the inner face of storage lysosomes called yolk granules. Mutant analysis shows that Dafcin is required to limit the size of yolk granules. This may occur by inducing negative membrane curvature like HA peptide. In support, liposomes formed in vitro with both Dafcin and HA peptides are smaller in size.

Mechanotransduction via the actin cytoskeleton is linked to fundamental cellular processes such as morphogenesis, cell division, and motility, requiring the control of tensile forces mediated by the motor protein non-muscle myosin 2 (NM2). Formins such as mDia1 have been shown to elongate actin structures that are under mechanical tension; conversely, mDia1s elongation rates are modulated by the applied force. Despite their relevance at the membrane/cortex interface, reported values for tension in formin-elongated actin filaments stem from theoretical estimates and simulations, but have not been amenable experimentally so far. Thus, we developed a Forster resonance energy transfer (FRET)-based, tension-sensitive probe (mDia1TS) and quantified the measured tension in live U2OS cells using fluorescence lifetime imaging microscopy (FLIM). Through whole-cell ROI analysis we show a short and long lifetime component, reporting an intensity-weighted, averaged lifetime corresponding to [~]3.5 pN. Upon mitogen stimulation of cells using EGF, we show that the tension homeostasis changed significantly, with a measurable increase in tension in the cells periphery and relaxation in its center. Furthermore, the reported average tension relaxed by 2 pN after adding the NM2 inhibitor para-nitroblebbistatin. We utilized siRNA knockdowns of individual NM2 paralogs (NM2-A, NM2-B, or NM2-C) to measure their individual contribution, revealing NM2-A as the main paralog to produce tensile force in this system. Taken together, we demonstrate that mDia1TS is able to directly determine that active mDia1 in cells is under tension, and that subcellular quantification with pN precision is possible. SignificanceDespite the fundamental importance of formins in regulating actin-based processes, reported values for tension in formin-mediated actin structures stem from simulations and theoretical estimates. In this study we developed a FRET-based, tension-sensitive reporter probe for formin mDia1, which we termed mDia1TS. Given the expanding clinical spectrum of DIAPH1/mDia1 mutations, our tool mDia1TS provides a quantitative tool for elucidation of changes in cytoskeletal assemblies.

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.

Eukaryotic chromosomes occupy ordered configurations within the nucleus, an organization that must be re-established in daughter cells as the nuclear envelope reforms at the end of mitosis. The conserved enzyme VPS4 and ESCRT-III proteins mediate nuclear envelope reformation, yet their role in post-mitotic centromere positioning remains unclear. Here, we develop a chemical genetics approach to analyze the role of VPS4 in human cells. VPS4 inhibition prevents the clearance of CHMP7 from centromeres, which remain constrained in ring-like configurations established during mitosis. Without VPS4 activity, CHMP7, but not other ESCRT-III proteins, forms nuclear foci, nuclear envelope protein distribution is altered and inner nuclear membrane invaginations appear. Following these defects, DNA damage is observed in the vicinity of centromeres. Depletion of CHMP7, but not CHMP4B, suppresses this damage. We propose that VPS4-mediated turnover of CHMP7 releases centromeres from transient nuclear envelope contacts, ensuring their proper positioning after mitosis and maintaining genome integrity.