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Summary During interphase, the nuclear envelope (NE) serves as a selective barrier between cytosol and nucleoplasm. When vertebrate cells enter mitosis, the NE is dismantled in the process of nuclear envelope breakdown (NEBD). Disassembly of nuclear pore complexes (NPCs) is a key aspect of NEBD, required for NE permeabilization and formation of a cytoplasmic mitotic spindle. Here, we show that both CDK1 and polo-like kinase 1 (PLK1) support mitotic NPC disintegration by hyperphosphorylation of Nup98, the gatekeeper nucleoporin, and Nup53, a central nucleoporin linking the inner NPC scaffold to the pore membrane. Multisite phosphorylation of Nup53 critically contributes to its liberation from its partner nucleoporins, including the pore membrane protein NDC1. Initial steps of NPC disassembly in semi-permeabilized cells can be reconstituted by a cocktail of mitotic kinases including cyclinB-CDK1, NIMA, and PLK1, suggesting that the unzipping of nucleoporin interactions by protein phosphorylation is an important principle underlying mitotic NE permeabilization., Graphical Abstract, Highlights • PLK1 localizes to NPCs during prophase and promotes mitotic NPC disassembly • CDK1 and PLK1 mediate hyperphosphorylation of the central linker nucleoporin Nup53 • Multisite phosphorylation of Nup53 promotes the disintegration of the central NPC • Mitotic kinases induce NPC disassembly in a reconstituted system, The disassembly of nuclear pore complexes (NPCs) is a key process for nuclear envelope breakdown at the onset of open mitosis in metazoan cells. Linder et al. show that multisite phosphorylation of the key interconnecting nucleoporin Nup53 by CDK1 and PLK1 is required for the timely disassembly of the central NPC framework.

The Journal of Cell Biology, 198 (5), ISSN:0021-9525, ISSN:1540-8140

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.

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.

Chromokinesins are chromosome-bound proteins during mitosis that play multiple important roles in chromosome segregation. The chromokinesin Kid has been shown to be involved in chromosome congression during mitosis and meiosis. Here we have generated a monoclonal antibody specific for the human chromokinesin hKid by immunizing BALB/c mice with a recombinant protein fragment corresponding to the C-terminal 250-amino acid residues of hKid. All five immunized mice responded excellently and gave high, nearly monospecific, antibody titers. One hybridoma, 8C12, was generated from spleen cells of a selected mouse, which recognized hKid on immunoblots and in immunofluorescence experiments. As hKid is an important regulator of chromosome segregation, this monoclonal antibody will be a useful tool for further analysis of this chromokinesin.

Human Spindly is required for kinetochore localization of cytoplasmic dynein, which is essential for poleward movement of chromosomes and for kinetochore protein streaming. In addition, Spindly controls the activity and kinetochore abundance of the RZZ complex, which contributes to microtubule attachment and mitotic checkpoint activity., Spindly recruits a fraction of cytoplasmic dynein to kinetochores for poleward movement of chromosomes and control of mitotic checkpoint signaling. Here we show that human Spindly is a cell cycle–regulated mitotic phosphoprotein that interacts with the Rod/ZW10/Zwilch (RZZ) complex. The kinetochore levels of Spindly are regulated by microtubule attachment and biorientation induced tension. Deletion mutants lacking the N-terminal half of the protein (NΔ253), or the conserved Spindly box (ΔSB), strongly localized to kinetochores and failed to respond to attachment or tension. In addition, these mutants prevented the removal of the RZZ complex and that of MAD2 from bioriented chromosomes and caused cells to arrest at metaphase, showing that RZZ-Spindly has to be removed from kinetochores to terminate mitotic checkpoint signaling. Depletion of Spindly by RNAi, however, caused cells to arrest in prometaphase because of a delay in microtubule attachment. Surprisingly, this defect was alleviated by codepletion of ZW10. Thus, Spindly is not only required for kinetochore localization of dynein but is a functional component of a mechanism that couples dynein-dependent poleward movement of chromosomes to their efficient attachment to microtubules.

The anaphase-promoting complex/cyclosome (APC/C) is essential for progression through mitosis. At anaphase onset, the APC/C requires the activator protein CDC20 to target securin and cyclin B1 for proteasome-dependent degradation, but then depends on the CDC20-related protein FZR1 (also known as CDH1) to remain active until the onset of the next S phase. To investigate the role of FZR1 in mammalian cells, we used RNAi in human cell lines and conditional gene targeting in mouse embryonic fibroblasts. In neither case was FZR1 required for exit from mitosis, but in cells lacking FZR1, the G1 phase was shortened and the S phase was prolonged. In several normal and transformed human cell lines, loss of FZR1 function induced DNA-damage responses and impaired proliferation independently of the p53 status. Constitutive knockdown of p53 in U2OS cells with inducible FZR1 siRNA also failed to restore their proliferative capacity. Thus, the proliferation defects are a direct consequence of the genetic damage inflicted by loss of FZR1 function and are largely independent of p53. In summary, mammalian FZR1 is not required for the completion of mitosis, but is an important regulator of G1 phase and is required for efficient DNA replication in human and mouse somatic cells.

During mitosis in vertebrate cells, the nuclear envelope undergoes extensive structural reorganization, starting with the retraction of nuclear membranes into the ER at mitotic onset and ending with the re-enclosure of chromatin by ER-derived membranes during mitotic exit. Here, we review our current understanding of postmitotic nuclear assembly.