Cdc37 is essential for chromosome segregation and cytokinesis in higher eukaryotes

Cdc37 has been shown to be required for the activity and stability of protein kinases that regulate different stages of cell cycle progression. However, little is known so far regarding interactions of Cdc37 with kinases that play a role in cell division. Here we show that the loss of function of Cdc37 in Drosophila leads to defects in mitosis and male meiosis, and that these phenotypes closely resemble those brought about by the inactivation of Aurora B. We provide evidence that Aurora B interacts with and requires the Cdc37/Hsp90 complex for its stability. We conclude that the Cdc37/Hsp90 complex modulates the function of Aurora B and that most of the phenotypes brought about by the loss of Cdc37 function can be explained by the inactivation of this kinase. These observations substantiate the role of Cdc37 as an upstream regulatory element of key cell cycle kinases.     

Role of chromosomal passenger complex in chromosome segregation and cytokinesis

Chromosomal passenger proteins associate with chromosomes early in mitosis and transfer to the spindle at ana/telophase. Recent results show that aurora B/AIM-1 (aurora and Ipl1-like midbody-associated protein kinase), which is responsible for mitotic histone H3 phosphorylation, INCENP (Inner Centromere protein) and Survivin/BIR are in a macromolecular complex as novel chromosomal passenger proteins. Aurora B/AIM-1 can bind to Survivin and the C-terminal region of INCENP, respectively, and colocalizes with both proteins to the centromeres, midzone and midbody. Disruption of either aurora B/AIM-1 or INCENP function leads to sever defects in chromosome segregation and cytokinesis. Moreover, the formation of the central spindle through anaphase to cytokinesis is also disrupted severely. These data suggest that chromosomal passenger complex is required for proper chromosome segregation by phosphorylating histone H3, and cytokinesis by ensuring the correct assembly of the midzone and midbody microtubule. Chromosomal passenger protein complex may couple chromosome segregation with cytokinesis.     

Inhibition of cyclin-dependent kinase 1 induces cytokinesis without chromosome segregation in an ECT2 and MgcRacGAP-dependent manner

Cleavage furrow formation marks the onset of cell division during early anaphase. The small GTPase RhoA and its regulators ECT2 and MgcRacGAP have been implicated in furrow ingression in mammalian cells, but the signaling upstream of these molecules remains unclear. We now show that the inhibition of cyclin-dependent kinase (Cdk)1 is sufficient to initiate cytokinesis. When mitotically synchronized cells were treated with the Cdk-specific inhibitor BMI-1026, the initiation of cytokinesis was induced precociously before chromosomal separation. Cytokinesis was also induced by the Cdk1-specific inhibitor purvalanol A but not by Cdk2/Cdk5- or Cdk4-specific inhibitors. Consistent with initiation of precocious cytokinesis by Cdk1 inhibition, introduction of anti-Cdk1 monoclonal antibody resulted in cells with aberrant nuclei. Depolymerization of mitotic spindles by nocodazole inhibited BMI-1026-induced precocious cytokinesis. However, in the presence of a low concentration of nocodazole, BMI-1026 induced excessive membrane blebbing, which appeared to be caused by formation of ectopic cleavage furrows. Depletion of ECT2 or MgcRacGAP by RNA interference abolished both of the phenotypes (precocious furrowing after nocodazole release and excessive blebbing in the presence of nocodazole). RNA interference of RhoA or expression of dominant-negative RhoA efficiently reduced both phenotypes. RhoA was localized at the cleavage furrow or at the necks of blebs. We propose that Cdk1 inactivation is sufficient to activate a signaling pathway leading to cytokinesis, which emanates from mitotic spindles and is regulated by ECT2, MgcRacGAP, and RhoA. Chemical induction of cytokinesis will be a valuable tool to study the initiation mechanism of cytokinesis.     

DBF8, an essential gene required for efficient chromosome segregation in Saccharomyces cerevisiae.

To investigate chromosome segregation in Saccharomyces cerevisiae, we examined a collection of temperature-sensitive mutants that arrest as large-budded cells at restrictive temperatures (L. H. Johnston and A. P. Thomas, Mol. Gen. Genet. 186:439-444, 1982). We characterized dbf8, a mutation that causes cells to arrest with a 2c DNA content and a short spindle. DBF8 maps to chromosome IX near the centromere, and it encodes a 36-kDa protein that is essential for viability at all temperatures. Mutational analysis reveals that three dbf8 alleles are nonsense mutations affecting the carboxy-terminal third of the encoded protein. Since all of these mutations confer temperature sensitivity, it appears that the carboxyl-terminal third of the protein is essential only at a restrictive temperature. In support of this conclusion, an insertion of URA3 at the same position also confers a temperature-sensitive phenotype. Although they show no evidence of DNA damage, dbf8 mutants exhibit increased rates of chromosome loss and nondisjunction even at a permissive temperature. Taken together, our data suggest that Dbf8p plays an essential role in chromosome segregation.     

Trypanosoma brucei Polo-like kinase is essential for basal body duplication, kDNA segregation and cytokinesis

Polo-like kinases (PLKs) are conserved eukaryotic cell cycle regulators, which play multiple roles, particularly during mitosis. The function of Trypanosoma brucei PLK was investigated in procyclic and bloodstream-form parasites. In procyclic trypanosomes, RNA interference (RNAi) of PLK, or overexpression of TY1-epitope-tagged PLK (PLKty), but not overexpression of a kinase-dead variant, resulted in the accumulation of cells that had divided their nucleus but not their kinetoplast (2N1K cells). Analysis of basal bodies and flagella in these cells suggested the defect in kinetoplast division arose because of an inhibition of basal body duplication, which occurred when PLK expression levels were altered. Additionally, a defect in kDNA replication was observed in the 2N1K cells. However, the 2N1K cells obtained by each approach were not equivalent. Following PLK depletion, the single kinetoplast was predominantly located between the two divided nuclei, while in cells overexpressing PLKty, the kinetoplast was mainly found at the posterior end of the cell, suggesting a role for PLK kinase activity in basal body and kinetoplast migration. PLK RNAi in bloodstream trypanosomes also delayed kinetoplast division, and was further observed to inhibit furrow ingression during cytokinesis. Notably, no additional roles were detected for trypanosome PLK in mitosis, setting this protein kinase apart from its counterparts in other eukaryotes.     

The Aurora/Ipl1p kinase family: regulators of chromosome segregation and cytokinesis.

Members of the Aurora/Ipl1p family of mitotically regulated serine/threonine kinases are emerging as key regulators of chromosome segregation and cytokinesis. Proper chromosome segregation and cytokinesis ensure that each daughter cell receives the full complement of genetic material. Defects in these processes can lead to aneuploidy and the propagation of genetic abnormalities. This review discusses the Aurora/Ipl1p kinases in terms of their protein structure and proposed function in mitotic cells and also the potential role of aurora2 in human cancer.     

Eukaryotic initiation factor 4E-3 is essential for meiotic chromosome segregation, cytokinesis and male fertility in Drosophila

Gene expression is translationally regulated during many cellular and developmental processes. Translation can be modulated by affecting the recruitment of mRNAs to the ribosome, which involves recognition of the 5' cap structure by the cap-binding protein eIF4E. Drosophila has several genes encoding eIF4E-related proteins, but the biological role of most of them remains unknown. Here, we report that Drosophila eIF4E-3 is required specifically during spermatogenesis. Males lacking eIF4E-3 are sterile, showing defects in meiotic chromosome segregation, cytokinesis, nuclear shaping and individualization. We show that eIF4E-3 physically interacts with both eIF4G and eIF4G-2, the latter being a factor crucial for spermatocyte meiosis. In eIF4E-3 mutant testes, many proteins are present at different levels than in wild type, suggesting widespread effects on translation. Our results imply that eIF4E-3 forms specific eIF4F complexes that are essential for spermatogenesis.     

The regulated assembly of a PKCepsilon complex controls the completion of cytokinesis

The cell cycle is exquisitely controlled by multiple sequential regulatory inputs to ensure fidelity. Here we demonstrate that the final step in division, the physical separation of daughter cells, is controlled by a member of the PKC gene superfamily. Specifically, we have identified three phosphorylation sites within PKCepsilon that control its association with 14-3-3. These phosphorylations are executed by p38 MAP kinase (Ser 350), GSK3 (Ser 346) and PKC itself (Ser 368). Integration of these signals is essential during mitosis because mutations that prevent phosphorylation of PKCepsilon and/or PKCepsilon binding to 14-3-3 also cause defects in the completion of cytokinesis. Using chemical genetic and dominant-negative approaches it is shown that selective inhibition of PKCepsilon halts cells at the final stages of separation. This arrest is associated with persistent RhoA activation at the midbody and a delay in actomyosin ring dissociation. This study therefore identifies a new regulatory mechanism that controls exit from cytokinesis, which has implications for carcinogenesis.     

sepB: an Aspergillus nidulans gene involved in chromosome segregation and the initiation of cytokinesis.

In Aspergillus nidulans conidia, cytokinesis (septation) is delayed until three rounds of nuclear division have been completed. This has permitted the identification of essential genes that are involved in the coordination of cytokinesis with nuclear division. Conditional mutations in the sepB gene block septation but allow germinating spores to complete the first three rounds of nuclear division at restrictive temperature. sepB3 mutants demonstrate transient delays in M-phase, accumulate aneuploid nuclei and show defects in chromosome segregation. Molecular analysis of the sepB gene reveals that it is essential and possesses limited similarity to the CTF4 gene of Saccharomyces cerevisiae. Using temperature-shift analysis we show that sepB is required after the first nuclear division but before the onset of cytokinesis. A failure to execute the sepB function results in a block to nuclear division and leads to cell death at a time when wild-type cells would be undergoing cytokinesis. Finally, we demonstrate that sepB is also required for the uninucleate cell divisions of developing conidiophores. Our results suggest that sepB3 mutants accumulate specific nuclear defects that do not arrest mitosis, but block the initiation of septum formation. Thus, proper chromosome segregation and a functional sepB gene are required to initiate cytokinesis.     

Phosphatase inhibitor-2 balances protein phosphatase 1 and aurora B kinase for chromosome segregation and cytokinesis in human retinal epithelial cells

Mitosis in Saccharomyces cerevisiae depends on IPL1 kinase, which genetically interacts with GLC8. The metazoan homologue of GLC8 is inhibitor-2 (I-2), but its function is not understood. We found endogenous and ectopic I-2 localized to the spindle, midzone, and midbody of mitotic human epithelial ARPE-19 cells. Knockdown of I-2 by RNA interference produced multinucleated cells, with supernumerary centrosomes, multipolar spindles and lagging chromosomes during anaphase. These defects did not involve changes in levels of protein phosphatase-1 (PP1), and the multinuclear phenotype was rescued by overexpression of I-2. Appearance of multiple nuclei and supernumerary centrosomes required progression through the cell cycle and I-2 knockdown cells failed cytokinesis, as observed by time-lapse microscopy. Inhibition of Aurora B by hesperadin produced multinucleated cells and reduced H3S10 phosphorylation. I-2 knockdown enhanced this latter effect. Partial knockdown of PP1Cα prevented multiple nuclei caused by either knockdown of I-2 or treatment with hesperadin. Expression of enhanced green fluorescent protein-I-2 or hemagglutinin-I-2 made cells resistant to hesperadin. We propose that I-2 acts to enhance Aurora B by inhibiting specific PP1 holoenzymes that dephosphorylate Aurora B substrates necessary for chromosome segregation and cytokinesis. Conserved together throughout eukaryotic evolution, I-2, PP1 and Aurora B function interdependently during mitosis.     

Conditional mutations in gamma-tubulin reveal its involvement in chromosome segregation and cytokinesis

gamma-Tubulin is a conserved essential protein required for assembly and function of the mitotic spindle in humans and yeast. For example, human gamma-tubulin can replace the gamma-tubulin gene in Schizosaccharomyces pombe. To understand the structural/functional domains of gamma-tubulin, we performed a systematic alanine-scanning mutagenesis of human gamma-tubulin (TUBG1) and studied phenotypes of each mutant allele in S. pombe. Our screen, both in the presence and absence of the endogenous S. pombe gamma-tubulin, resulted in 11 lethal mutations and 12 cold-sensitive mutations. Based on structural mapping onto a homology model of human gamma-tubulin generated by free energy minimization, all deleterious mutations are found in residues predicted to be located on the surface, some in positions to interact with alpha- and/or beta-tubulins in the microtubule lattice. As expected, one class of tubg1 mutations has either an abnormal assembly or loss of the mitotic spindle. Surprisingly, a subset of mutants with abnormal spindles does not arrest in M phase but proceeds through anaphase followed by abnormal cytokinesis. These studies reveal that in addition to its previously appreciated role in spindle microtubule nucleation, gamma-tubulin is involved in the coordination of postmetaphase events, anaphase, and cytokinesis.     

CENP-A is phosphorylated by Aurora B kinase and plays an unexpected role in completion of cytokinesis.

Aurora B is a mitotic protein kinase that phosphorylates histone H3, behaves as a chromosomal passenger protein, and functions in cytokinesis. We investigated a role for Aurora B with respect to human centromere protein A (CENP-A), a centromeric histone H3 homologue. Aurora B concentrates at centromeres in early G2, associates with histone H3 and centromeres at the times when histone H3 and CENP-A are phosphorylated, and phosphorylates histone H3 and CENP-A in vitro at a similar target serine residue. Dominant negative phosphorylation site mutants of CENP-A result in a delay at the terminal stage of cytokinesis (cell separation). The only molecular defects detected in analysis of 22 chromosomal, spindle, and regulatory proteins were disruptions in localization of inner centromere protein (INCENP), Aurora B, and a putative partner phosphatase, PP1gamma1. Our data support a model where CENP-A phosphorylation is involved in regulating Aurora B, INCENP, and PP1gamma1 targeting within the cell. These experiments identify an unexpected role for the kinetochore in regulation of cytokinesis.     

New topoisomerase essential for chromosome segregation in E. coli

The nucleotide sequence of the parC gene essential for chromosome partition in E. coli was determined. The deduced amino acid sequence was homologous to that of the A subunit of gyrase. We found another new gene coding for about 70 kd protein. The gene was sequenced, and the deduced amino acid sequence revealed that the gene product was homologous to the gyrase B subunit. Mutants of this gene were isolated and showed the typical Par phenotype at nonpermissive temperature; thus the gene was named parE. Enhanced relaxation activity of supercoiled plasmid molecules was detected in the combined crude cell lysates prepared from the ParC and ParE overproducers. A topA mutation defective in topoisomerase I could be compensated by increasing both the parC and the parE gene dosage. It is suggested that the parC and parE genes code for the subunits of a new topoisomerase, named topo IV.     

Two-pore channel 1 interacts with citron kinase,regulating completion of cytokinesis

Two-pore channels (TPC1, 2, and 3) are recently identified endolysosmal ion channels, but remain poorly characterized. In this study, we show for the first time a role for TPC1 in cytokinesis, the final step in cell division. HEK 293 T-REx cells inducibly overexpressing TPC1 demonstrated a lack of proliferation accompanied by multinucleation and an increase in G2/M cycling cells. Increased TPC1 was associated with a concomitant accumulation of active RhoGTP and a decrease in phosphorylated myosin light chain (MLC). Finally, we demonstrated a novel interaction between TPC1 and citron kinase (CIT). These results identify TPC1 as a central component of cytokinetic control, specifically during abscission, and introduce a means by which the endolysosomal system may play an active role in this process.     

Local change in phospholipid composition at the cleavage furrow is essential for completion of cytokinesis

Cell division ends up with the membrane separation of two daughter cells, presumably by a membrane fusion that requires dynamic changes of the distribution and the composition of membrane lipids. We have previously shown that a membrane lipid phosphatidylethanolamine (PE) is exposed on the cell surface of the cleavage furrow during late cytokinesis and that this PE movement is involved in regulation of the contractile ring disassembly. Here we show that immobilization of cell surface PE by a PE-binding peptide blocks the RhoA inactivation in the late stage of cytokinesis. Phosphatidylinositol 4-phosphate 5-kinase (PIP5K), but not other RhoA effectors, is co-localized with RhoA in the peptide-treated cells. Indeed, PIP5K and its product phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) are localized to the cleavage furrow of normally dividing cells. Both overexpression of a kinase-deficient PIP5K mutant and microinjection of anti-PI(4,5)P(2) antibodies compromise cytokinesis by preventing local accumulation of PI(4,5)P(2) in the cleavage furrow. These findings demonstrate that the localized production of PI(4,5)P(2) is required for the proper completion of cytokinesis and that the possible formation of a unique lipid domain in the cleavage furrow membrane may play a crucial role in coordinating the contractile rearrangement with the membrane remodeling during late cytokinesis.     

Two-pore channel 1 interacts with citron kinase,regulating completion of cytokinesis

Two-pore channels (TPC1, 2, and 3) are recently identified endolysosmal ion channels, but remain poorly characterized. In this study, we show for the first time a role for TPC1 in cytokinesis, the final step in cell division. HEK 293 T-REx cells inducibly overexpressing TPC1 demonstrated a lack of proliferation accompanied by multinucleation and an increase in G2/M cycling cells. Increased TPC1 was associated with a concomitant accumulation of active RhoGTP and a decrease in phosphorylated myosin light chain (MLC). Finally, we demonstrated a novel interaction between TPC1 and citron kinase (CIT). These results identify TPC1 as a central component of cytokinetic control, specifically during abscission, and introduce a means by which the endolysosomal system may play an active role in this process.     

TMAP/CKAP2 is essential for proper chromosome segregation

Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2), is a novel mitotic spindle-associated protein which is frequently up-regulated in various malignances. However, its cellular functions remain unknown. Previous reports suggested that the cellular functions of TMAP/CKAP2 pertain to regulation of the dynamics and assembly of the mitotic spindle. To investigate its role in mitosis, we studied the effects of siRNA-mediated depletion of TMAP/CKAP2 in cultured mammalian cells. Unexpectedly, TMAP/CKAP2 knockdown did not result in significant alterations of the spindle apparatus. However, TMAP/CKAP2-depleted cells often exhibited abnormal nuclear morphologies, which were accompanied by abnormal organization of the nuclear lamina, and chromatin bridge formation between two daughter cell nuclei. Time lapse video microscopy revealed that the changes in nuclear morphology and chromatin bridge formations observed in TMAP/CKAP2-depleted cells are the result of defects in chromosome segregation. Consistent with this, the spindle checkpoint activity was significantly reduced in TMAP/CKAP2-depleted cells. Moreover, chromosome missegregation induced by depletion of TMAP/CKAP2 ultimately resulted in reduced cell viability and increased chromosomal instability. Our present findings demonstrate that TMAP/CKAP2 is essential for proper chromosome segregation and for maintaining genomic stability.     

Phosphorylation of the mitotic regulator protein Hec1 by Nek2 kinase is essential for faithful chromosome segregation

Hec1 (highly expressed in cancer) plays essential roles in chromosome segregation by interacting through its coiled-coil domains with several proteins that modulate the G(2)/M phase. Hec1 localizes to kinetochores, and its inactivation either by genetic deletion or antibody neutralization leads to severe and lethal chromosomal segregation errors, indicating that Hec1 plays a critical role in chromosome segregation. The mechanisms by which Hec1 is regulated, however, are not known. Here we show that human Hec1 is a serine phosphoprotein and that it binds specifically to the mitotic regulatory kinase Nek2 during G(2)/M. Nek2 phosphorylates Hec1 on serine residue 165, both in vitro and in vivo. Yeast cells are viable without scNek2/Kin3, a close structural homolog of Nek2 that binds to both human and yeast Hec1. When the same yeasts carry an scNek2/Kin3 (D55G) or Nek2 (E38G) mutation to mimic a similar temperature-sensitive nima mutation in Aspergillus, their growth is arrested at the nonpermissive temperature, because the scNek2/Kin3 (D55G) mutant binds to Hec1 but fails to phosphorylate it. Whereas wild-type human Hec1 rescues lethality resulting from deletion of Hec1 in Saccharomyces cerevesiae, a human Hec1 mutant or yeast Hec1 mutant changing Ser(165) to Ala or yeast Hec1 mutant changing Ser(201) to Ala does not. Mutations changing the same Ser residues to Glu, to mimic the negative charge created by phosphorylation, partially rescue lethality but result in a high incidence of errors in chromosomal segregation. These results suggest that cell cycle-regulated serine phosphorylation of Hec1 by Nek2 is essential for faithful chromosome segregation.     

The Schizosaccharomyces pombe aurora-related kinase Ark1 interacts with the inner centromere protein Pic1 and mediates chromosome segregation and cytokinesis

The chromosomal passenger proteins aurora-B, survivin, and inner centromere protein (INCENP) have been implicated in coordinating chromosome segregation with cell division. This work describes the interplay between aurora, survivin, and INCENP orthologs in the fission yeast Schizosaccharomyces pombe and defines their roles in regulating chromosome segregation and cytokinesis. We describe the cloning and characterization of the aurora-related kinase gene ark1(+), demonstrating that it is an essential gene required for sister chromatid segregation. Cells lacking Ark1p exhibit the cut phenotype, DNA fragmentation, and other defects in chromosome segregation. Overexpression of a kinase-defective version of Ark1, Ark1-K147R, inhibits cytokinesis, with cells exhibiting an elongated, multiseptate phenotype. Ark1p interacts physically and/or genetically with the survivin and INCENP orthologs Bir1p and Pic1p. We identified Pic1p in a two-hybrid screen for Ark1-K147R interacting partners and went on to map domains in both proteins that mediate their binding. Pic1p residues 925-972 are necessary and sufficient for Ark1p binding, which occurs through the kinase domain. As with Ark1-K147R, overexpression of Ark1p-binding fragments of Pic1p leads to multiseptate phenotypes. We also provide evidence that the dominant-negative effect of Ark1-K147R requires Pic1p binding, indicating that the formation of Ark1p-Pic1p complexes is required for the execution of cytokinesis.     

An Arabidopsis mitogen-activated protein kinase kinase kinase gene family encodes essential positive regulators of cytokinesis

The signal transduction pathways that control cytokinesis in plants are largely uncharacterized. Here, we provide genetic evidence that mitogen-activated protein kinase kinase kinases (MAPKKKs) play a role in the control of plant cell division. Using a reverse-genetic approach, we isolated plants carrying knockout alleles of the Arabidopsis MAPKKK genes ANP1, ANP2, and ANP3. The resulting single-mutant plants displayed no obvious abnormal phenotypes; two of the three double-mutant combinations displayed defects in cell division and growth; and the triple-mutant combination was not transmitted through either male or female gametes. The molecular and structural phenotypes displayed by the double mutants support a model in which the ANP family of MAPKKKs positively regulates cell division and growth and may negatively regulate stress responses.