The ESCRT machinery counteracts Nesprin-2G-mediated mechanical forces during nuclear envelope repair.
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ABSTRACT:
SUBMITTER: Wallis SS
PROVIDER: S-EPMC8657813 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Active positioning of the nucleus is integral to division, migration and differentiation of mammalian cells. Fibroblasts polarizing for migration orient their centrosomes by actin-dependent nuclear movement. This nuclear movement depends on nesprin-2 giant (N2G), a large, actin-binding outer nuclear membrane component of transmembrane actin-associated (TAN) lines that couple nuclei to moving actin cables. Here, we identify the diaphanous formin FHOD1 as an interaction partner of N2G. Silencing FHOD1 expression or expression of fragments containing binding sites for N2G or FHOD1 disrupted nuclear movement and centrosome orientation in polarizing fibroblasts. Unexpectedly, silencing of FHOD1 expression did not affect the formation or rearward flow of dorsal actin cables required for nuclear positioning. Rather, N2G-FHOD1 interaction provided a second connection to actin cables essential for TAN line formation and thus nuclear movement. These results reveal a unique function for a formin in coupling an organelle to actin filaments for translocation, and suggest that TAN lines require multi-point attachments to actin cables to resist the large forces necessary to move the nucleus.
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Project description:Recent work done exclusively in tissue culture cells revealed that the nuclear envelope (NE) ruptures and repairs in interphase. The duration of NE ruptures depends on lamins; however, the underlying mechanisms and relevance to in vivo events are not known. Here, we use the Caenorhabditis elegans zygote to analyze lamin's role in NE rupture and repair in vivo. Transient NE ruptures and subsequent NE collapse are induced by weaknesses in the nuclear lamina caused by expression of an engineered hypomorphic C. elegans lamin allele. Dynein-generated forces that position nuclei enhance the severity of transient NE ruptures and cause NE collapse. Reduction of dynein forces allows the weakened lamin network to restrict nucleo-cytoplasmic mixing and support stable NE recovery. Surprisingly, the high incidence of transient NE ruptures does not contribute to embryonic lethality, which is instead correlated with stochastic chromosome scattering resulting from premature NE collapse, suggesting that C. elegans tolerates transient losses of NE compartmentalization during early embryogenesis. In sum, we demonstrate that lamin counteracts dynein forces to promote stable NE repair and prevent catastrophic NE collapse, and thus provide the first mechanistic analysis of NE rupture and repair in an organismal context.
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Project description:The nucleus of a cell has long been considered to be subject to mechanical force. Despite the observation that mechanical forces affect nuclear geometry and movement, how forces are applied onto the nucleus is not well understood. The nuclear LINC (linker of nucleoskeleton and cytoskeleton) complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton onto the nucleus. Previously used techniques for studying nuclear forces have been unable to resolve forces across individual proteins, making it difficult to clearly establish if the LINC complex experiences mechanical load. To directly measure forces across the LINC complex, we generated a fluorescence resonance energy transfer-based tension biosensor for nesprin-2G, a key structural protein in the LINC complex, which physically links this complex to the actin cytoskeleton. Using this sensor we show that nesprin-2G is subject to mechanical tension in adherent fibroblasts, with highest levels of force on the apical and equatorial planes of the nucleus. We also show that the forces across nesprin-2G are dependent on actomyosin contractility and cell elongation. Additionally, nesprin-2G tension is reduced in fibroblasts from Hutchinson-Gilford progeria syndrome patients. This report provides the first, to our knowledge, direct evidence that nesprin-2G, and by extension the LINC complex, is subject to mechanical force. We also present evidence that nesprin-2G localization to the nuclear membrane is altered under high-force conditions. Because forces across the LINC complex are altered by a variety of different conditions, mechanical forces across the LINC complex, as well as the nucleus in general, may represent an important mechanism for mediating mechanotransduction.
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Project description:During telophase, the nuclear envelope (NE) reforms around daughter nuclei to ensure proper segregation of nuclear and cytoplasmic contents. NE reformation requires the coating of chromatin by membrane derived from the endoplasmic reticulum, and a subsequent annular fusion step to ensure that the formed envelope is sealed. How annular fusion is accomplished is unknown, but it is thought to involve the p97 AAA-ATPase complex and bears a topological equivalence to the membrane fusion event that occurs during the abscission phase of cytokinesis. Here we show that the endosomal sorting complex required for transport-III (ESCRT-III) machinery localizes to sites of annular fusion in the forming NE in human cells, and is necessary for proper post-mitotic nucleo-cytoplasmic compartmentalization. The ESCRT-III component charged multivesicular body protein 2A (CHMP2A) is directed to the forming NE through binding to CHMP4B, and provides an activity essential for NE reformation. Localization also requires the p97 complex member ubiquitin fusion and degradation 1 (UFD1). Our results describe a novel role for the ESCRT machinery in cell division and demonstrate a conservation of the machineries involved in topologically equivalent mitotic membrane remodelling events.
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Project description:Endolysosomes can be damaged by diverse materials. Terminally damaged compartments are degraded by lysophagy, but pathways that repair salvageable organelles are poorly understood. Here we found that the endosomal sorting complex required for transport (ESCRT) machinery, known to mediate budding and fission on endolysosomes, also plays an essential role in their repair. ESCRTs were rapidly recruited to acutely injured endolysosomes through a pathway requiring calcium and ESCRT-activating factors that was independent of lysophagy. We used live-cell imaging to demonstrate that ESCRTs responded to small perforations in endolysosomal membranes and enabled compartments to recover from limited damage. Silica crystals that disrupted endolysosomes also triggered ESCRT recruitment. ESCRTs thus provide a defense against endolysosomal damage likely to be relevant in physiological and pathological contexts.
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Project description:Eukaryotic genomes are organized within the nucleus through interactions with inner nuclear membrane (INM) proteins. How chromatin tethering to the INM is controlled in interphase and how this process contributes to subsequent mitotic nuclear envelope (NE) remodeling remains unclear. We have probed these fundamental questions using the fission yeast Schizosaccharomyces japonicus, which breaks and reforms the NE during mitosis. We show that attachments between heterochromatin and the transmembrane Lem2-Nur1 complex at the INM are remodeled in interphase by the ESCRT-III/Vps4 machinery. Failure of ESCRT-III/Vps4 to release Lem2-Nur1 from heterochromatin leads to persistent association of chromosomes with the INM throughout mitosis. At mitotic exit, such trapping of Lem2-Nur1 on heterochromatin prevents it from re-establishing nucleocytoplasmic compartmentalization. Our work identifies the Lem2-Nur1 complex as a substrate for the nuclear ESCRT machinery and explains how the dynamic tethering of chromosomes to the INM is linked to the establishment of nuclear compartmentalization.
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Project description:Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disorder, caused by mutation in the gene encoding lamin A/C, which produces a truncated protein called progerin. In cells from HGPS patients, progerin accumulates at the nuclear membrane (NM), where it causes NM deformations. In this study, we investigated whether progerin-induced NM deformation involved ESCRT-III, a protein complex that remodels nuclear and cytoplasmic membranes. The ESCRT-III protein CHMP4B was recruited to sites of aberrant NM proliferation in human cells ectopically expressing progerin and in patient-derived HGPS fibroblasts. Derepression of NM deformation in these cells was observed following depletion of CHMP4B or an ESCRT-III adaptor, ALIX. Treatment with rapamycin (which induce autophagic clearance of progerin and reverse progerin-induced cellular phenotypes) down-regulated progerin-induced NM deformation, whereas treatment with bafilomycin A1 (an inhibitor of autophagy and lysosome-based degradation) or CHMP4B depletion antagonized the effects of rapamycin. These results indicate that the ALIX-mediated ESCRT-III pathway plays a suppressive role in progerin-induced NM deformation and suggest that autophagy down-regulates progerin-induced NM deformation in a manner dependent on ESCRT-III machinery.
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Project description:During cell division, remodelling of the nuclear envelope enables chromosome segregation by the mitotic spindle1. The reformation of sealed nuclei requires ESCRTs (endosomal sorting complexes required for transport) and LEM2, a transmembrane ESCRT adaptor2-4. Here we show how the ability of LEM2 to condense on microtubules governs the activation of ESCRTs and coordinated spindle disassembly. The LEM motif of LEM2 binds BAF, conferring on LEM2 an affinity for chromatin5,6, while an adjacent low-complexity domain (LCD) promotes LEM2 phase separation. A proline-arginine-rich sequence within the LCD binds to microtubules and targets condensation of LEM2 to spindle microtubules that traverse the nascent nuclear envelope. Furthermore, the winged-helix domain of LEM2 activates the ESCRT-II/ESCRT-III hybrid protein CHMP7 to form co-oligomeric rings. Disruption of these events in human cells prevented the recruitment of downstream ESCRTs, compromised spindle disassembly, and led to defects in nuclear integrity and DNA damage. We propose that during nuclear reassembly LEM2 condenses into a liquid-like phase and coassembles with CHMP7 to form a macromolecular O-ring seal at the confluence between membranes, chromatin and the spindle. The properties of LEM2 described here, and the homologous architectures of related inner nuclear membrane proteins7,8, suggest that phase separation may contribute to other critical envelope functions, including interphase repair8-13 and chromatin organization14-17.
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Project description:The late-acting endosomal sorting complex required for transport (ESCRT) machinery has been implicated in facilitating the resealing of the nuclear envelope (NE) after mitosis, enabling compartmentalization of the genome away from the cytoplasm. Here, we leverage the stereotypic first division of the C. elegans embryo to identify additional functions of the ESCRT machinery in maintaining the structure of the inner nuclear membrane. Specifically, impaired ESCRT function results in a defect in the pruning of inner nuclear membrane invaginations, which arise normally during NE reformation and expansion. Additionally, in combination with a hypomorphic mutation that interferes with assembly of the underlying nuclear lamina, inhibition of ESCRT function significantly perturbs NE architecture and increases chromosome segregation defects, resulting in penetrant embryonic lethality. Our findings highlight links between ESCRT-mediated inner nuclear membrane remodeling, maintenance of nuclear envelope morphology, and the preservation of the genome during early development.
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Project description:Nesprins and emerin are structural nuclear envelope proteins that tether nuclei to the cytoskeleton. In this work, we identified the cytoskeleton-associated ?-N/E-catenins as novel nesprin-2-binding partners. The association involves the C termini of nesprin-2 giant and ?-N/E-catenins. ?-E/T/N-catenins are known primarily for their roles in cadherin-mediated cell-cell adhesion. Here, we show that, in addition, ?-catenin forms complexes with nesprin-2 that include ?-catenin and emerin. We demonstrate that the depletion of nesprin-2 reduces both the amount of active ?-catenin inside the nucleus and T-cell factor/lymphoid-enhancing factor-dependent transcription. Taken together, these findings suggest novel nesprin-2 functions in cellular signaling. Moreover, we propose that, in contrast to emerin, nesprin-2 is a positive regulator of the Wnt signaling pathway.
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