Dissociation of membrane-chromatin contacts is required for proper chromosome segregation in mitosis.

The nuclear envelope (NE) aids in organizing the interphase genome by tethering chromatin to the nuclear periphery. During mitotic entry, NE-chromatin contacts are broken. Here, we report on the consequences of impaired NE removal from chromatin for cell division of human cells. Using a membrane-chromatin tether that cannot be dissociated when cells enter mitosis, we show that a failure in breaking membrane-chromatin interactions impairs mitotic chromatin organization, chromosome segregation and cytokinesis, and induces an aberrant NE morphology in postmitotic cells. In contrast, chromosome segregation and cell division proceed successfully when membrane attachment to chromatin is induced during metaphase, after chromosomes have been singularized and aligned at the metaphase plate. These results indicate that the separation of membranes and chromatin is critical during prometaphase to allow for proper chromosome compaction and segregation. We propose that one cause of these defects is the multivalency of membrane-chromatin interactions.

Cellular Reorganization during Mitotic Entry.

The preparation of eukaryotic cells for division requires an extensive cellular reorganization, affecting cytoskeletal elements, chromatin, and organelles. These drastic changes in cellular architecture ensure the proper segregation of chromosomes and inheritance of organelles. The morphological alterations occurring during mitotic entry are tightly coordinated with the cell cycle, mainly through the action of mitotic kinases. Conversely, the fidelity of these processes impacts mitotic progression and is important for organismal homeostasis and cell fate. Here, we provide an overview of major architectural changes observed during early mitosis and review recent progress in understanding their regulatory mechanisms, focusing on processes accompanying mitotic cell rounding and restructuring of organelles in mammalian cells.

An in vitro system to study nuclear envelope breakdown.

During mitosis in vertebrate cells, the nuclear compartment is completely disintegrated in the process of nuclear envelope breakdown (NEBD). NEBD comprises the disassembly of nuclear pore complexes, disintegration of the nuclear lamina, and the retraction of nuclear membranes into the endoplasmic reticulum. Deciphering of the mechanisms that underlie these dynamic changes requires the identification of the involved molecular components and appropriate experimental tools to define their mode of action. Here, we describe an in vitro, imaging-based experimental system, which recapitulates NEBD. In our assay, we induce NEBD on nuclei of semi-permeabilized HeLa cells expressing fluorescently tagged nuclear envelope (NE) marker proteins by addition of mitotic cell extract that is supplemented with fluorescently labeled dextran. Time-lapse confocal microscopy is used to monitor the fate of the selected NE marker protein, and loss of the NE permeability barrier is deduced by influx of the fluorescent dextran into the nucleus. This in vitro system provides a powerful tool to follow NEBD and to characterize factors required for the reorganization of the NE during mitosis.

SUN proteins facilitate the removal of membranes from chromatin during nuclear envelope breakdown.

SUN proteins reside in the inner nuclear membrane and form complexes with KASH proteins of the outer nuclear membrane that connect the nuclear envelope (NE) to the cytoskeleton. These complexes have well-established functions in nuclear anchorage and migration in interphase, but little is known about their involvement in mitotic processes. Our analysis demonstrates that simultaneous depletion of human SUN1 and SUN2 delayed removal of membranes from chromatin during NE breakdown (NEBD) and impaired the formation of prophase NE invaginations (PNEIs), similar to microtubule depolymerization or down-regulation of the dynein cofactors NudE/EL. In addition, overexpression of dominant-negative SUN and KASH constructs reduced the occurrence of PNEI, indicating a requirement for functional SUN-KASH complexes in NE remodeling. Codepletion of SUN1/2 slowed cell proliferation and resulted in an accumulation of morphologically defective and disoriented mitotic spindles. Quantification of mitotic timing revealed a delay between NEBD and chromatin separation, indicating a role of SUN proteins in bipolar spindle assembly and mitotic progression.

Rec8-containing cohesin maintains bivalents without turnover during the growing phase of mouse oocytes.

During female meiosis, bivalent chromosomes are thought to be held together from birth until ovulation by sister chromatid cohesion mediated by cohesin complexes whose ring structure depends on kleisin subunits, either Rec8 or Scc1. Because cohesion is established at DNA replication in the embryo, its maintenance for such a long time may require cohesin turnover. To address whether Rec8- or Scc1-containing cohesin holds bivalents together and whether it turns over, we created mice whose kleisin subunits can be cleaved by TEV protease. We show by microinjection experiments and confocal live-cell imaging that Rec8 cleavage triggers chiasmata resolution during meiosis I and sister centromere disjunction during meiosis II, while Scc1 cleavage triggers sister chromatid disjunction in the first embryonic mitosis, demonstrating a dramatic transition from Rec8- to Scc1-containing cohesin at fertilization. Crucially, activation of an ectopic Rec8 transgene during the growing phase of Rec8(TEV)(/TEV) oocytes does not prevent TEV-mediated bivalent destruction, implying little or no cohesin turnover for ≥2 wk during oocyte growth. We suggest that the inability of oocytes to regenerate cohesion may contribute to age-related meiosis I errors.