CC2D1B Coordinates ESCRT-III Activity during the Mitotic Reformation of the Nuclear Envelope.
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ABSTRACT:
SUBMITTER: Ventimiglia LN
PROVIDER: S-EPMC6286407 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:In addition to its role in membrane abscission during cytokinesis, viral budding, endosomal sorting, and plasma membrane repair [1], the endosomal sorting complex required for transport-III (ESCRT-III) machinery has recently been shown to seal holes in the reforming nuclear envelope (NE) during mitotic exit [2, 3]. ESCRT-III also acts during interphase to repair the NE upon migration-induced rupture [4, 5], highlighting its key role as an orchestrator of membrane integrity at this organelle. While NE localization of ESCRT-III is dependent upon the ESCRT-III component CHMP7 [3], it is unclear how this complex is able to engage nuclear membranes. Here we show that the N terminus of CHMP7 acts as a novel membrane-binding module. This membrane-binding ability allows CHMP7 to bind to the ER, an organelle continuous with the NE, and it provides a platform to direct NE recruitment of ESCRT-III during mitotic exit. CHMP7's N terminus comprises tandem Winged-Helix domains [6], and, by using homology modeling and structure-function analysis, we identify point mutations that disrupt membrane binding and prevent both ER localization of CHMP7 and its subsequent enrichment at the reforming NE. These mutations also prevent assembly of downstream ESCRT-III components at the reforming NE and proper establishment of post-mitotic nucleo-cytoplasmic compartmentalization. These data identify a novel membrane-binding activity within an ESCRT-III subunit that is essential for post-mitotic nuclear regeneration.
| S-EPMC5069351 | biostudies-literature
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.
| S-EPMC4471131 | biostudies-literature
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.
| S-EPMC7321842 | biostudies-literature
Project description:Repo-Man targets protein phosphatase 1 ? (PP1?) to chromatin at anaphase onset and regulates chromosome structure during mitotic exit. Here, we show that a Repo-Man:PP1 complex forms in anaphase following dephosphorylation of Repo-Man. Upon activation, the complex localizes to chromosomes and causes the dephosphorylation of histone H3 (Thr3, Ser10, and Ser28). In anaphase, Repo-Man has both catalytic and structural functions that are mediated by two separate domains. A C-terminal domain localizes Repo-Man to bulk chromatin in early anaphase. There, it targets PP1 for the dephosphorylation of histone H3 and possibly other chromosomal substrates. An N-terminal domain localizes Repo-Man to the chromosome periphery later in anaphase. There, it is responsible for the recruitment of nuclear components such as Importin ? and Nup153 in a PP1-independent manner. These observations identify Repo-Man as a key factor that coordinates chromatin remodeling and early events of nuclear envelope reformation during mitotic exit.
| S-EPMC3480639 | biostudies-literature
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.
| S-EPMC7139201 | biostudies-literature
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.
| S-EPMC7606583 | biostudies-literature
Project description:Through membrane sealing and disassembly of spindle microtubules, the Endosomal Sorting Complex Required for Transport-III (ESCRT-III) machinery has emerged as a key player in the regeneration of a sealed nuclear envelope (NE) during mitotic exit, and in the repair of this organelle during interphase rupture. ESCRT-III assembly at the NE occurs transiently during mitotic (M) exit and is initiated when CHMP7, an ER-localised ESCRT-II/ESCRT-III hybrid protein, interacts with the Inner Nuclear Membrane (INM) protein LEM2. Whilst classical nucleocytoplasmic transport mechanisms have been proposed to separate LEM2 and CHMP7 during interphase, it is unclear how CHMP7 assembly is suppressed in mitosis when NE and ER identities are mixed. Here, we use live cell imaging and protein biochemistry to examine the biology of these proteins during M-exit. Firstly, we show that CHMP7 plays an important role in the dissolution of LEM2 clusters that form at the NE during M-exit. Secondly, we show that CDK1 phosphorylates CHMP7 upon M-entry at Ser3 and Ser441 and that this phosphorylation reduces CHMP7's interaction with LEM2, limiting its assembly during M-phase. We show that spatiotemporal differences in the dephosphorylation of CHMP7 license its assembly at the NE during telophase, but restrict its assembly on the ER at this time. Without CDK1 phosphorylation, CHMP7 undergoes inappropriate assembly in the peripheral ER during M-exit, capturing LEM2 and downstream ESCRT-III components. Lastly, we establish that a microtubule network is dispensable for ESCRT-III assembly at the reforming nuclear envelope. These data identify a key cell-cycle control programme allowing ESCRT-III-dependent nuclear regeneration.
| S-EPMC8324300 | biostudies-literature
Project description:Mitotic cells must form a single nucleus during telophase or exclude part of their genome as damage-prone micronuclei. While research has detailed how micronuclei arise from cells entering anaphase with lagging chromosomes, cellular mechanisms allowing late-segregating chromosomes to rejoin daughter nuclei remain underexplored. Here, we find that late-segregating acentric chromosome fragments that rejoin daughter nuclei are associated with nuclear membrane but devoid of lamin and nuclear pore complexes in Drosophila melanogaster. We show that acentrics pass through membrane-, lamin-, and nuclear pore-based channels in the nuclear envelope that extend and retract as acentrics enter nuclei. Membrane encompassing the acentrics fuses with the nuclear membrane, facilitating integration of the acentrics into newly formed nuclei. Fusion, mediated by the membrane fusion protein Comt/NSF and ESCRT-III components Shrub/CHMP4B and CHMP2B, facilitates reintegration of acentrics into nuclei. These results suggest a previously unsuspected role for membrane fusion, similar to nuclear repair, in the formation of a single nucleus during mitotic exit and the maintenance of genomic integrity.
| S-EPMC7054997 | biostudies-literature
Project description:Quantitative crosslinking analysis to probe in vitro conformational dynamics governing ESCRT-mediated nuclear envelope formation. CHMP7 was crosslinked with D12-BS3 while CHMP7 in the presence of LEM2 winged helix domain was crosslinked with H12-BS3. Samples were mixed after crosslinking and analyzed according to standard procedures. The raw files included here correspond to four SEC fractions from a single experiment that contain crosslinked peptides.
2020-01-24 | MSV000084837 | MassIVE
Project description:The final stage of mitosis is cytokinesis, which results in 2 independent daughter cells. Cytokinesis has 2 phases: membrane ingression followed by membrane abscission. IQGAP1 is a scaffold protein that interacts with proteins implicated in mitosis, including F-actin, myosin and CaM. IQGAP1 in yeast recruits actin and myosin II filaments to the contractile ring for membrane ingression. In contrast, we show that mammalian IQGAP1 is not required for ingression, but coordinates nuclear pore complex (NPC) reassembly and completion of abscission. Depletion of IQGAP1 disrupts Nup98 and mAb414 nuclear envelope localization and delays abscission timing. IQGAP1 phosphorylation increases 15-fold upon mitotic entry at S86, S330 and T1434, with the latter site being targeted by CDK2/Cyclin A and CDK1/Cyclin A/B in vitro. Expressing the phospho-deficient mutant IQGAP1-S330A impairs NPC reassembly in cells undergoing abscission. Thus, mammalian IQGAP1 functions later in mitosis than its yeast counterpart to regulate nuclear pore assembly in a S330 phosphorylation-dependent manner during the abscission phase of cytokinesis.
| S-EPMC4613854 | biostudies-literature