Mitotic Disassembly of Nuclear Pore Complexes Involves CDK1- and PLK1-Mediated Phosphorylation of Key Interconnecting Nucleoporins.

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.

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.

Enclosing chromatin: reassembly of the nucleus after open mitosis.

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.

Human chromokinesins promote chromosome congression and spindle microtubule dynamics during mitosis.

Chromokinesins are microtubule plus end-directed motor proteins that bind to chromosome arms. In Xenopus egg cell-free extracts, Xkid and Xklp1 are essential for bipolar spindle formation but the functions of the human homologues, hKID (KIF22) and KIF4A, are poorly understood. By using RNAi-mediated protein knockdown in human cells, we find that only co-depletion delayed progression through mitosis in a Mad2-dependent manner.

Spindly/CCDC99 is required for efficient chromosome congression and mitotic checkpoint regulation.

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.

Loss of the mammalian APC/C activator FZR1 shortens G1 and lengthens S phase but has little effect on exit from mitosis.

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.

Dose-dependent effects of stable cyclin B1 on progression through mitosis in human cells.

The disassembly of the mitotic spindle and exit from mitosis require the inactivation of Cdk1. Here, we show that expression of nondegradable cyclinB1 causes dose-dependent mitotic arrest phenotypes. By monitoring chromosomes in living cells, we determined that pronounced overexpression of stable cyclinB1 entailed metaphase arrest without detectable sister chromatid separation, while moderate overexpression arrested cells in a pseudometaphase state, in which separated sister chromatids were kept at the cellular equator by a bipolar 'metaphase-like' spindle.

Generation and characterization of an hKid-specific monoclonal antibody.

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.