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.
Human chromokinesins hKID and KIF4A contribute to proper attachment of chromosomes by controlling the positioning of the chromosome arms and microtubule dynamics, respectively.
SummaryDuring 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.
The disassembly of the mitotic spindle and exit from mitosis require the inactivation of Cdk1. Here, we show that expression of nondegradable cyclinB1 causes dosedependent 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. Chromosomes that left the pseudometaphase plate became pulled back and individual kinetochores were found to be merotelically attached to both spindle poles in stable cyclinB1 arrested cells. Inactivation of the chromokinesin hKid, by RNAi or antibody microinjection, prevented the formation of stable bipolar spindles and the 'metaphase-like' alignment of chromosomes in cells expressing stable cyclinB1. These experiments show that cyclinB1 is able to maintain a bipolar spindle even after sister chromatids had become separated and suggest an important role of hKid in this process. Cells expressing low levels of nondegradable cyclinB1 progressed further in mitosis and arrested in telophase.
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.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.