Membrane Binding by CHMP7 Coordinates ESCRT-III-Dependent Nuclear Envelope Reformation

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.

Hypopharyngeal squamous cell carcinoma (HPSCC) is a common malignant tumor of the head and neck, characterized by a high degree of malignancy and significantly poor outcome. Charged multivesicular body protein 4C (CHMP4C) is an important subunit of the Endosomal Sorting Complex Required for Transport-III (ESCRT-III), which is mainly involved in cytokinetic abscission. Moreover, CHMP4C plays an important role in cancer development. However, the mechanism of CHMP4C's role in hypopharyngeal squamous cell carcinoma is unclear. Therefore, the aim of this study was to investigate the biological role and potential mechanism of CHMP4C in hypopharyngeal squamous cell carcinoma. First, we evaluated the expression of CHMP4C in hypopharyngeal squamous cell carcinoma tissues and normal tissues using HPA database. Knockdown of CHMP4C in cell lines was achieved using lentivirus, and the effects of CHMP4C on cell proliferation were detected by CCK-8 and cell colony formation assay; cell migration and invasion abilities were assessed by Transwell assay; cell cycle distribution and apoptosis levels were detected by flow cytometry. Finally, the expression of the epithelial-mesenchymal transition (EMT)-related proteins and the Wnt/β-catenin signaling pathway-related proteins was investigated using Western blot. The results revealed that CHMP4C expression was upregulated in hypopharyngeal squamous cell carcinoma tissues compared with normal tissues. Knockdown of CHMP4C inhibited Fadu cell proliferation, migration, and invasion. Knockdown of CHMP4C could promote apoptosis by blocking the S-phase of the cell cycle. In addition, we found that knockdown of CHMP4C resulted in upregulation of E-cadherin protein expression and downregulation of N-cadherin and Vimentin protein expression; meanwhile, knockdown of CHMP4C resulted in the downregulation of β-catenin and C-myc protein expression, suggesting that CHMP4C is involved in the Wnt/β-catenin signaling pathway. CHMP4C may promote the progression of hypopharyngeal squamous cell carcinoma through the Wnt/β-catenin signaling pathway. CHMP4C is a very promising target for tumor therapy.

Lamin B receptor (LBR), a chromatin and lamin B-binding protein in the inner nuclear membrane, has been proposed to target the membrane precursor vesicles to chromatin mediated by importin β during the nuclear envelope (NE) assembly. However, the mechanisms for the binding of LBR with importin β and the membrane targeting by LBR in NE assembly remain largely unknown. In this report, we show that the amino acids (aa) 69-90 of LBR sequences are required to bind with importin β at aa 45-462, and the binding is essential for the NE membrane precursor vesicle targeting to the chromatin during the NE assembly at the end of mitosis. We also show that this binding is cell cycle-regulated and dependent on the phosphorylation of LBR Ser-71 by p34(cdc2) kinase. RNAi knockdown of LBR causes the NE assembly failure and abnormal chromatin decondensation of the daughter cell nuclei, leading to the daughter cell death at early G(1) phase by apoptosis. Perturbation of the interaction of LBR with importin β by deleting the LBR N-terminal spanning region or aa 69-73 also induces the NE assembly failure, the abnormal chromatin decondensation, and the daughter cell death. The first transmembrane domain of LBR promotes the NE production and expansion, because overexpressing this domain is sufficient to induce membrane overproduction of the NE. Thus, these results demonstrate that LBR targets the membrane precursor vesicles to chromatin by interacting with importin β in a LBR phosphorylation-dependent manner during the NE assembly at the end of mitosis and that the first transmembrane domain of LBR promotes the LBR-bearing membrane production and the NE expansion in interphase.

In higher eukaryotic cells, the nuclear envelope (NE) is composed of double nuclear membranes studded with nuclear pore complexes (NPCs) and undergoes dynamic disassembly and reassembly during the cell cycle. However, how the NE and NPC reassemble remains largely unclear. Here, using HeLa, HEK293, and Drosophila cells, along with immunofluorescence microscopy and transmission EM methods, we found that postmitotic annulate lamellae (AL) assembly contributes to NE and NPC assembly. We observed that the AL are parallel membrane-pair stacks and possess regularly spaced AL pore complexes (ALPCs) that are morphologically similar to the NPCs. We found that the AL assemble in the cytoplasm during mitotic exit simultaneously with NE re-formation in daughter cells. Then, the assembled AL either bound the decondensing chromatin to directly transform into the NE or bound and fused with the outer nuclear membrane to join the assembling NE. The AL did not colocalize with sheet and tubular endoplasmic reticulum (ER) marker proteins on the ER or the lamin B receptor-localized membrane in the cytoplasm, suggesting that postmitotic AL assembly occurs independently of the chromatin and ER. Collectively, our results indicate that postmitotic AL assembly is a common cellular event and an intermediate step in NE and NPC assembly and in NE expansion in higher eukaryotic cells.

In mitotic exit, proteins that were highly phosphorylated are sequentially targeted by the phosphatase PP2A-B55, but what underlies substrate selection is unclear. In this issue, Cundell et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201606033) identify the determinants of PP2A-B55’s dephosphorylation program, thereby influencing spindle disassembly, nuclear envelope reformation, and cytokinesis.

Nuclear structure and chromatin changes are very useful biomarkers in cancer diagnosis. Despite this, their biological significance and relevance to cancer progression are still not well understood. The identification of new proteins that link the nuclear envelope to chromatin organization and the understanding of the molecular mechanisms underlying these connections have begun to provide some important clues. This review discusses the role of the nuclear protein Repo-Man (CDCA2) in the maintenance of genome stability. Repo-Man (CDCA2) is a targeting subunit for the protein phosphatase 1 involved in the dephosphorylation of histone H3 during mitotic exit. In this role, it is important for the chromatin organization in post-mitotic nuclei. Repo-Man (CDCA2) is also essential for proper nuclear envelope reformation and the regulation of DNA damage responses. The relevance of this complex for cancer biology is also corroborated by emerging evidence that provides a correlation between Repo-Man (CDCA2) expression levels and cancer progression; several studies now suggest that Repo-Man (CDCA2) represents a very strong prognostic marker for poor patient survival.

Nuclear pore complexes (NPCs) are channels within the nuclear envelope that mediate nucleocytoplasmic transport. NPCs form within the closed nuclear envelope during interphase or assemble concomitantly with nuclear envelope reformation in late stages of mitosis. Both interphase and mitotic NPC biogenesis require coordination of protein complex assembly and membrane deformation. During early stages of mitotic NPC assembly, a seed for new NPCs is established on chromatin, yet the factors connecting the NPC seed to the membrane of the forming nuclear envelope are unknown. Here, we report that the reticulon homology domain protein REEP4 not only localizes to high-curvature membrane of the cytoplasmic endoplasmic reticulum but is also recruited to the inner nuclear membrane by the NPC biogenesis factor ELYS. This ELYS-recruited pool of REEP4 promotes NPC assembly and appears to be particularly important for NPC formation during mitosis. These findings suggest a role for REEP4 in coordinating nuclear envelope reformation with mitotic NPC biogenesis.

Aqueous channels are at the core of the translocase of the outer membrane (TOM) and the translocase of the inner membrane for the transport of preproteins (TIM23), the translocases mediating the transport of proteins across the outer and inner mitochondrial membranes. Yet, the existence of a channel associated to the translocase of the inner membrane for the insertion of multitopic protein (TIM22) complex has been arguable, as its function relates to the insertion of multispanning proteins into the inner membrane. For the first time, we report conditions for detecting a channel activity associated to the TIM22 translocase in organelle, i.e. intact mitoplasts. An internal signal peptide in the intermembrane space of mitochondria is a requisite to inducing this channel, which is otherwise silent. The channel showed slightly cationic and high conductance activity of 1000 pS with a predominant half-open substate. Despite their different composition, the channels of the three mitochondrial translocases were thus remarkably similar, in agreement with their common task as pores transiently trapping proteins en route to their final destination. The opening of the TIM22 channel was a step-up process depending on the signal peptide concentration. Interestingly, low membrane potentials kept the channel fully open, providing a threshold level of the peptide is present. Our results portray TIM22 as a dynamic channel solely active in the presence of its cargo proteins. In its fully open conformation, favored by the combined action of internal signal peptide and low membrane potential, the channel could embrace the in-transit protein. As insertion progressed and initial interaction with the signal peptide faded, the channel would close, sustaining its role as a shunt that places trapped proteins into the membrane.

Sea urchin eggs are used to investigate the involvement of spindle microtubules in the mechanisms that control the timing of cell cycle events. Eggs are treated for 4 min with Colcemid at prophase of the first mitosis. No microtubules are assembled for at least 3 h, and the eggs do not divide. These eggs show repeated cycles of nuclear envelope breakdown (NEB) and nuclear envelope reformation (NER). Mitosis (NEB to NER) is twice as long in Colcemid-treated eggs as in the untreated controls. Interphase (NER to NEB) is the same in both. Thus, each cycle is prolonged entirely in mitosis. The chromosomes of treated eggs condense and eventually split into separate chromatids which do not move apart. This "canaphase" splitting is substantially delayed relative to anaphase onset in the control eggs. Treated eggs are irradiated after NEB with 366-nm light to inactivate the Colcemid. This allows the eggs to assemble normal spindles and divide. Up to 14 min after NEB, delays in the start of microtubule assembly give equal delays in anaphase onset, cleavage, and the events of the following cell cycle. Regardless of the delay, anaphase follows irradiation by the normal prometaphase duration. The quantity of spindle microtubules also influences the timing of mitotic events. Short Colcemid treatments administered in prophase of second division cause eggs to assemble small spindles. One blastomere is irradiated after NEB to provide a control cell with a normal-sized spindle. Cells with diminished spindles always initiate anaphase later than their controls. Telophase events are correspondingly delayed. This work demonstrates that spindle microtubules are involved in the mechanisms that control the time when the cell will initiate anaphase, finish mitosis, and start the next cell cycle.

The endosomal sorting complex required for transport (ESCRT) is a conserved protein machine mediating membrane remodeling and scission. In the context of viral infection, different components of the ESCRT-III complex, which serve as the core machinery to catalyze membrane fission, are involved in diverse viruses' entry, replication, and/or budding. However, the interplay between ESCRT-III and viral factors in the virus life cycle, especially for that of large enveloped DNA viruses, is largely unknown. Recently, the ESCRT-III components Vps2B, Vps20, Vps24, Snf7, Vps46, and Vps60 were determined for entry and/or egress of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Here, we identified the final three ESCRT-III components Chm7, Ist1, and Vps2A of Spodoptera frugiperda. Overexpression of the dominant-negative forms of these proteins or RNAi downregulation of their transcripts significantly reduced infectious budded viruses (BVs) production of AcMNPV. Quantitative PCR together with confocal and transmission electron microscopy analysis revealed that these proteins were required for internalization and trafficking of BV during entry and egress of nucleocapsids. In infected Sf9 cells, nine ESCRT-III components were distributed on the nuclear envelope and plasma membrane, and except for Chm7, the other components were also localized to the intranuclear ring zone. Y2H and BiFC analysis revealed that 42 out of 64 BV-related proteins including 35 BV structural proteins and 7 non-BV structural proteins interacted with single or multiple ESCRT-III components. By further mapping the interactome of 64 BV-related proteins, we established the interaction networks of ESCRT-III and the viral protein complexes involved in BV entry and egress.IMPORTANCEFrom archaea to eukaryotes, the endosomal sorting complex required for transport (ESCRT)-III complex is hijacked by many enveloped and nonenveloped DNA or RNA viruses for efficient replication. However, the mechanism of ESCRT-III recruitment, especially for that of large enveloped DNA viruses, remains elusive. Recently, we found the ESCRT-III components Vps2B, Vps20, Vps24, Snf7, Vps46, and Vps60 are necessary for the entry and/or egress of budded viruses (BVs) of Autographa californica multiple nucleopolyhedrovirus. Here, we demonstrated that the other three ESCRT-III components Chm7, Ist1, and Vps2A play similar roles in BV infection. By determining the subcellular localization of ESCRT-III components in infected cells and mapping the interaction of nine ESCRT-III components and 64 BV-related proteins, we built the interaction networks of ESCRT-III and the viral protein complexes involved in BV entry and egress. These studies provide a fundamental basis for understanding the mechanism of the ESCRT-mediated membrane remodeling for replication of baculoviruses.

The nuclear envelope in the plant cell cycle: structure, function and regulation.

During cleavage of cystogenous plasmodia, nuclei of Woronina pythii underwent one meiosis I-like division. The division was centric, closed with polar fenestrations, and characterized by the formation of synaptonemal complexes. Pachytene nuclei exhibited nuclear projections which were heretofore undocumented structural modifications of the nuclear membrane. Hypothetical functions of these structures are proposed. During diakinesis, fragmentation of the nuclear envelope occurred at the poles through the apparent growth of microtubules. Intranuclear membranous vesicles appeared to bud from the inner membrane of the original nuclear envelope during metaphase and anaphase, and coalesced on the surfaces of the separating chromatin masses, thereby forming new daughter nuclear envelopes within the intact original nuclear envelope. Similar descriptions of the spindle apparatus and events relating to nuclear envelope breakdown and reformation have been reported for the mitotic divisions of W. pythii. Nuclei of some mature cysts contained intranuclear cisternae which were continuous with the inner membrane of the nuclear envelope, and these cisternae frequently partitioned the nuclei into two or three separate compartments. The formation of intranuclear cisternae was presumed to result in the eventual fission of nuclei, which may represent functionally the second meiotic division.

The nuclear envelope (NE) is a double lipid bilayer that separates nucleus and cytoplasm. In metazoa, NE breakdown (NEBD) occurs during prophase and NE reformation around segregated chromatids occurs at anaphase-telophase. We identified Caenorhabditis elegans Lipin homologue (called Lpin-1) as an essential factor with roles in endoplasmic reticulum (ER) organization and NEBD. RNAi-mediated downregulation of Lpin-1 had no effect on timely entry into mitosis or on the early steps of NEBD, but Lpin-1 was required for disassembly of the nuclear lamina during late NEBD. This Lpin-1 requirement appears to be separable from the effect of Lpin-1 on the peripheral ER.

In all higher animal and plant forms so far studied, the nucleus is surrounded by an envelope consisting of two membranes separated by a variable distance of the order of 20 to 40 mμ. The continuity of the membranes is interrupted by circular differentiations which have been called “pores” (1) and “annuli” (2, 3). Four lines of evidence have been presented in support of the concept that the nuclear membranes are part of, and at times continuous with the vesicular component of the cytoplasm (endoplasmic reticulum) : a) the cytoplasmic surface of the envelope is occasionally covered with dense particles similar to those that cover the flattened vesicles of the endoplasmic reticulum in actively synthesizing cells, as in the pancreas (1); b) actual continuity between the outer membrane and the endoplasmic reticulum has been observed in which the cisternal cavity of the reticulum is continuous with the space between inner and outer nuclear membranes (1); c) strong resemblance has been reported between the nuclear envelope and elements of the stacks of basophilic “annulate lamellae” particularly prominent in the cytoplasm of egg and sperm-forming cells of certain invertebrates (4) ; d) outpocketings of the nuclear envelope in Drosophila salivary gland cells (5) and in spermatocytes and spermatids of crayfish (6) have been observed which could conceivably give rise to membranous elements of the cytoplasm.

During mitotic exit in metazoans, the properly segregated chromosome mass is typically enclosed by the newly formed nuclear envelope to form a single nucleus in each daughter cell. On the other hand, mis-segregated chromosomes that lag behind can also undergo nuclear envelope assembly, leading to the formation of atypical nuclear structures such as micronuclei. Micronuclei are commonly observed in cancer and are known to cause extensive genome alterations such as chromothripsis. Recent studies highlight that micronuclei frequently exhibit fragile nuclear envelopes and undergo defective nuclear envelope assembly, which are linked to numerous adverse consequences, including abnormal DNA replication, DNA damage, transcriptional defects, and proinflammatory immune responses. In this chapter, I describe methods to examine nuclear envelope assembly on micronuclei during chromosome mis-segregation.