Junctions between the endoplasmic reticulum (ER) and the outer membrane of the nuclear envelope (NE) physically connect both organelles. These ER-NE junctions are essential for supplying the NE with lipids and proteins synthesized in the ER. However, little is known about the structure of these ER-NE junctions. Here, we systematically study the ultrastructure of ER-NE junctions in cryo-fixed mammalian cells staged in anaphase, telophase, and interphase by correlating live cell imaging with three-dimensional electron microscopy. Our results show that ER-NE junctions in interphase cells have a pronounced hourglass shape with a constricted neck of 7-20 nm width. This morphology is significantly distinct from that of junctions within the ER network, and their morphology emerges as early as telophase. The highly constricted ER-NE junctions are seen in several mammalian cell types, but not in budding yeast. We speculate that the unique and highly constricted ER-NE junctions are regulated via novel mechanisms that contribute to ER-to-NE lipid and protein traffic in higher eukaryotes.

Midbodies function during telophase to regulate the abscission step of cytokinesis. Until recently, it was thought that abscission-regulating proteins, such as ESCRT-III complex subunits, accumulate at the MB by directly or indirectly binding to the MB resident protein, CEP55. However, recent studies have shown that depletion of CEP55 does not fully block ESCRT-III targeting the MB. Here, we show that MBs contain mRNAs and that these MB-associated mRNAs can be locally translated, resulting in the accumulation of abscission-regulating proteins. We demonstrate that localized MB-associated translation of CHMP4B is required for its targeting to the abscission site and that 3\' UTR-dependent CHMP4B mRNA targeting to the MB is required for successful completion of cytokinesis. Finally, we identify regulatory cis-elements within RNAs that are necessary and sufficient for mRNA trafficking to the MB. We propose a novel method of regulating cytokinesis and abscission by MB-associated targeting and localized translation of selective mRNAs.

Cell mechanics is a factor that determines cell growth, migration, proliferation, or differentiation, as well as trafficking inside the cytoplasm and organization of organelles. Knowledge about cell mechanics is critical to gaining insight into these biological processes. Here, we used atomic force microscopy to examine the elasticity, an important parameter of cell mechanics, of non-adherent Jurkat leukemic T-cells in both interphase and mitotic phases. We found that the elasticity of an individual cell does not significantly change at interphase. When a cell starts to divide, its elasticity increases in the transition from metaphase to telophase during normal division while the cell is stiffened right after it enters mitosis during abnormal division. At the end of the division, the cell elasticity gradually returned to the value of the mother cell. These changes may originate from the changes in cell surface tension during modulating actomyosin at the cleavage furrow, redistributing cell organelles, and constricting the contractile ring to sever mother cell to form daughters. The difference in elasticity patterns suggests that there is a discrepancy in the redistribution of the cell organelles during normal and abnormal division.

Cell divisions are accurately positioned to generate cells of the correct size and shape. In plant cells, the new cell wall is built in the middle of the cell by vesicles trafficked along an antiparallel microtubule and a microfilament array called the phragmoplast. The phragmoplast expands toward a specific location at the cell cortex called the division site, but how it accurately reaches the division site is unclear. We observed microtubule arrays that accumulate at the cell cortex during the telophase transition in maize (Zea mays) leaf epidermal cells. Before the phragmoplast reaches the cell cortex, these cortical-telophase microtubules transiently interact with the division site. Increased microtubule plus end capture and pausing occur when microtubules contact the division site-localized protein TANGLED1 or other closely associated proteins. Microtubule capture and pausing align the cortical microtubules perpendicular to the division site during telophase. Once the phragmoplast reaches the cell cortex, cortical-telophase microtubules are incorporated into the phragmoplast primarily by parallel bundling. The addition of microtubules into the phragmoplast promotes fine-tuning of the positioning at the division site. Our hypothesis is that division site-localized proteins such as TANGLED1 organize cortical microtubules during telophase to mediate phragmoplast positioning at the final division plane.

Several studies have suggested a role for the LEM-domain protein emerin and the DNA binding factor BAF in nuclear envelope reformation after mitosis, but the exact molecular mechanisms are not understood. Using HeLa cells deficient for emerin or both emerin and lamin A, we show that emerin deficiency induces abnormal aggregation of lamin A at the nuclear periphery in telophase. As a result, nuclear membrane expansion is impaired and BAF accumulates at the core region, the middle part of telophase nuclei. Aggregates do not form when lamin A carries the mutation R435C in the immunoglobulin fold known to prevent interaction of lamin A with BAF suggesting that aggregation is caused by a stabilized association of lamin A with BAF bound to chromosomal DNA. Reintroduction of emerin in the cells prevents formation of lamin A clusters and BAF accumulation at the core region. Therefore emerin is required for the expansion of the nuclear membrane at the core region to enclose the nucleus and for the rapid reformation of the nuclear lamina based on lamin A/C in telophase. Finally, we show that LEM-domain and lumenal domain are required for the targeting of emerin to exert its function at the core region.

The cell cycle is a series of events leading to cell replication. When plated at low cell densities in serum-containing medium, cultured cells start to proliferate, moving through the four phases of the cell cycle: G1, S, G2, and M. Mitosis is the most dynamic period of the cell cycle, involving a major reorganization of virtually all cell components. Mitosis is further divided into prophase, prometaphase, metaphase, anaphase, and telophase, which can be easily distinguished from one another by protein markers and/or comparing their chromosome morphology under fluorescence microscope. The progression of the cell cycle through these mitotic subphases is tightly regulated by complicated molecular mechanisms. Synchronization of cells to the mitotic subphases is important for understanding these molecular mechanisms. Here, we describe a protocol to synchronize Hela cells to prometaphase, metaphase, and anaphase/telophase. In this protocol, Hela cells are first synchronized to the early S phase by a double thymidine block. Following the release of the block, the cells are treated with nocodazole, MG132, and blebbistatin to arrest them at prometaphase, metaphase, and anaphase/telophase, respectively. Successful synchronization is assessed using Western blot and fluorescence microscopy.

DNA transfection is an important technology in life sciences, wherein nuclear entry of DNA is necessary to express exogenous DNA. Non-viral vectors and their transfection reagents are useful as safe transfection tools. However, they have no effect on the transfection of non-proliferating cells, the reason for which is not well understood. This study elucidates the mechanism through which transfected DNA enters the nucleus for gene expression. To monitor the behavior of transfected DNA, we introduce plasmid bearing lacO repeats and RFP-coding sequences into cells expressing GFP-LacI and observe plasmid behavior and RFP expression in living cells. RFP expression appears only after mitosis. Electron microscopy reveals that plasmids are wrapped with nuclear envelope (NE)‒like membranes or associated with chromosomes at telophase. The depletion of BAF, which is involved in NE reformation, delays plasmid RFP expression. These results suggest that transfected DNA is incorporated into the nucleus during NE reformation at telophase.

Dephosphorylation of lamin A, which triggers nuclear lamina reconstitution, is crucial for the completion of mitosis. However, the specific phosphatase and regulatory mechanism that allow timely lamin A dephosphorylation remain unclear. Here, we report that RepoMan (also known as CDCA2), a regulatory subunit of protein phosphatase 1γ (PP1γ) is transiently modified with SUMO-2 at K762 during late telophase. SUMOylation of RepoMan markedly enhanced its binding affinity with lamin A. Moreover, SUMOylated RepoMan contributes to lamin A recruitment to telophase chromosomes and dephosphorylation of the mitotic lamin A phosphorylation. Expression of a SUMO-2 mutant that has a defective interaction with the SUMO-interacting motif (SIM) resulted in failure of the lamin A and RepoMan association, along with abrogation of lamin A dephosphorylation and subsequent nuclear lamina formation. These findings strongly suggest that RepoMan recruits lamin A through SUMO-SIM interaction. Thus, transient SUMOylation of RepoMan plays an important role in the spatiotemporal regulation of lamin A dephosphorylation and the subsequent nuclear lamina formation at the end of mitosis.

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.

Several lines of evidence suggest the existence in the eukaryotic cells of a tight, yet largely unexplored, connection between DNA replication and sister chromatid cohesion. Tethering of newly duplicated chromatids is mediated by cohesin, an evolutionarily conserved hetero-tetrameric protein complex that has a ring-like structure and is believed to encircle DNA. Cohesin is loaded onto chromatin in telophase/G1 and converted into a cohesive state during the subsequent S phase, a process known as cohesion establishment. Many studies have revealed that down-regulation of a number of DNA replication factors gives rise to chromosomal cohesion defects, suggesting that they play critical roles in cohesion establishment. Conversely, loss of cohesin subunits (and/or regulators) has been found to alter DNA replication fork dynamics. A critical step of the cohesion establishment process consists in cohesin acetylation, a modification accomplished by dedicated acetyltransferases that operate at the replication forks. Defects in cohesion establishment give rise to chromosome mis-segregation and aneuploidy, phenotypes frequently observed in pre-cancerous and cancerous cells. Herein, we will review our present knowledge of the molecular mechanisms underlying the functional link between DNA replication and cohesion establishment, a phenomenon that is unique to the eukaryotic organisms.