Initiation Of Cytokinesis Research Articles

Molecular Cloning of the Gene for p85 That Regulates the Initiation of Cytokinesis in Tetrahymena

Tetrahymena p85 is localized to the presumptive division plane before division furrow formation; its molecular weight in SDS–polyacrylamide gel electrophoresis differs in wild-type and temperature-sensitive cell-division-arrest mutant cdaA1 cells. At the restrictive temperature, p85 localization and division furrow formation are not observed in cdaA1 cells. In this study, we purified p85 and cloned a wild-type p85 cDNA. The deduced amino acid sequence of p85 was composed mainly of two kinds of repeat sequences. One of these contained regions homologous to a calmodulin-binding site and a part of actin, and the other contained a region homologous to a part of a cdc2 kinase homologue. Moreover, we cloned a cDNA encoding the cdaA1 p85. There was no difference in the predicted amino acid sequences of wild-type and cdaA1 p85, suggesting that the difference in molecular weight between p85 in wild-type and mutant cells is caused by a disorder of posttranslational-modification mechanisms of p85 in the cdaA1 cell.

A putative exchange factor for Rho1 GTPase is required for initiation of cytokinesis in Drosophila.

Cytokinesis ensures the successful completion of the cell cycle and distribution of chromosomes, organelles, and cytoplasm between daughter cells. It is accomplished by formation and constriction of an actomyosin contractile ring that drives the progression of a cleavage furrow. Microinjection experiments and in vitro transfection assays have suggested a requirement for small GTPases of the Rho family in cytokinesis. Yet, the identity of proteins regulating Rho signaling pathways during cytokinesis remains unknown. Here we show that in Drosophila, Pebble (Pbl), a putative exchange factor for Rho GTPases (RhoGEF), is required for the formation of the contractile ring and initiation of cytokinesis. The dynamics of Pbl expression and its distribution during mitosis, as well as structure-function analysis, indicate that it is a key regulatory component of the pathway. pbl interacts genetically with Rho1, but not with Rac1 or Cdc42, and Pbl and Rho1 proteins interact in vivo in yeast. Similar to mutations in pbl, loss of Rho1 or expression of a dominant-negative Rho1 blocks cytokinesis. Our results identify Pbl as a RhoGEF specifically required for cytokinesis and linked through Rho1 activity to the reorganization of the actin cytoskeleton at the cleavage furrow.

The myosin ATPase inhibitor 2,3-butanedione-2-monoxime (BDM) inhibits tip growth and cytokinesis in the fission yeast, Schizosaccharomyces pombe.

The growth of fission yeast cultures was reversibly inhibited by exposure to the myosin-ATPase inhibitor 2,3-butanedione-2-monoxime (BDM). Wild-type cells treated with 20 mM BDM for approximately two generation times were smaller than untreated controls and had a septation index approximately twice that seen in the absence of the inhibitor. The organization of actin at the cell poles was somewhat disorganized in the presence of BDM; however, cells formed a cytokinetic actin ring. When nitrogen-starved stationary-phase cells were reinoculated into fresh medium in the presence of BDM, the time taken to repolarize the actin cytoskeleton and to resume the characteristic vegetative cell shape before initiation of the first cell division were both substantially delayed. BDM significantly inhibited the increase in cell length of cdc25.22 cells arrested for cell cycle progress by incubation at the restrictive temperature and substantially delayed the initiation of both mitosis and cytokinesis in arrested cdc25.22 cells after release of the temperature block. These results suggest that tip growth and cytokinesis--processes in fission yeast that involve the actin cytoskeleton--also require myosin activity.

The control of septum formation in fission yeast.

If cell division is to produce two daughter cells that are viable, faithful copies of the parent cell, the landmark events of the cell division cycle, S phase, mitosis, and cytokinesis, must be executed in the correct order and with high fidelity. Though considerable advances have been made toward understanding the mechanisms that control the onset of S phase and mitosis, regulation of the events that occur at the end of the cell cycle, such as the reorganization of the cytoskeleton and the initiation of cell separation or cytokinesis, are less well understood. The fission yeast Schizosaccharomyces pombe provides a simple eukaryotic model for the study of cytokinesis. S. pombe cells are rod shaped, grow mainly by elongation at their ends, and divide by binary fission after forming a centrally placed division septum. Study of S. pombe mutants has begun to shed light on how septum formation and cytokinesis are regulated both spatially and temporally, to achieve proper co-ordination with mitosis. Some of the genes defined by these mutants have been functionally conserved through eukaryotic evolution, suggesting that aspects of this control will be common to all eukaryotic cells.

The fission yeast dma1 gene is a component of the spindle assembly checkpoint, required to prevent septum formation and premature exit from mitosis if spindle function is compromised.

Premature initiation of cytokinesis can lead to loss of chromosomes, and 'cutting' of the nucleus. Therefore, the proper spatial and temporal co-ordination of mitosis and cytokinesis is essential for maintaining the integrity of the genome. The fission yeast cdc16 gene is implicated both in the spindle assembly checkpoint and control of septum formation. To identify other proteins involved in these controls, we have isolated multicopy suppressors of the cdc16-116 mutation, and the characterization of one of these, dma1 (defective in mitotic arrest), is presented here. dma1 is not an essential gene, but in a dma1 null background (dma1-D1) the function of the spindle assembly checkpoint is compromised. If assembly of the spindle is prevented, dma1-D1 cells do not arrest, the activity of cdc2 kinase decays and cells form a division septum without completing a normal mitosis. dma1-D1 cells also show an increased rate of chromosome loss during exponential growth. Upon ectopic expression from an inducible promoter, dma1p delays progress through mitosis and inhibits septum formation, giving rise to elongated, multinucleate cells. We propose that dma1 is a component of the spindle assembly checkpoint, required to prevent septum formation and premature exit from mitosis if spindle function is impaired.

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.

The Drosophila kinesin-like protein KLP3A is a midbody component required for central spindle assembly and initiation of cytokinesis.

We describe here a new member of the kinesin superfamily in Drosophila, KLP3A (Kinesin-Like-Protein-at-3A). The KLP3A protein localizes to the equator of the central spindle during late anaphase and telophase of male meiosis. Mutations in the KLP3A gene disrupt the interdigitation of microtubules in spermatocyte central spindles. Despite this defect, anaphase B spindle elongation is not obviously aberrant. However, cytokinesis frequently fails after both meiotic divisions in mutant testes. Together, these findings strongly suggest that the KLP3A presumptive motor protein is a critical component in the establishment or stabilization of the central spindle. Furthermore, these results imply that the central spindle is the source of signals that initiate the cleavage furrow in higher cells.

The timing of protein kinase activation events in the cascade that regulates mitotic progression in Tradescantia stamen hair cells.

Stamen hair cells of the spiderwort plant Tradescantia virginiana exhibit unusually predictable rates of progression through mitosis, particularly from the time of nuclear envelope breakdown (NEBD) through the initiation of cytokinesis. The predictable rate of progression through prometaphase and metaphase has made these cells a useful model system for the determination of the timing of regulatory events that trigger entry into anaphase. A number of studies suggest that the elevation of one or more protein kinase activities is a necessary prerequisite for entry into anaphase. The current experiments employ two strategies to test when these elevations in protein kinase activity actually occur during metaphase. In perfusions, we added the protein kinase inhibitors K-252a, staurosporine, or calphostin C to living stamen hair cells for 10-min intervals at known times during prometaphase or metaphase and monitored the subsequent rate of progression into anaphase. Metaphase transit times were altered as a function of the time of addition of K-252a or staurosporine to the cells; metaphase transit times were extended significantly by treatments initiated in prometaphase through early metaphase and again late in metaphase. Transit times were normal after treatments initiated in mid-metaphase, approximately 15 to 21 min after NEBD. Calphostin C had no significant effect on the metaphase transit times. In parallel, cells were microinjected with known quantities of a general-purpose protein kinase substrate peptide, VRKRTLRRL, at predefined time points during prometaphase and metaphase. At a cytosolic concentration of 100 nM to 1 microM, the peptide doubled or tripled the metaphase transit times when injected into the cytosol of mitotic cells within the first 4 min after NEBD, at any point from 7.5 to 9 min after NEBD, at any point from 14 to 16 min after NEBD, at 21 min after NEBD, or at 24 min after NEBD. At the concentration used and during these brief intervals, the peptide appeared to act as a competitive inhibitor to reveal inflection points when protein kinase activation was occurring or when endogenous substrate levels approached levels of the peptide. The timing of these inflection points coincides with the changes in protein kinase activities during prometaphase and metaphase, as indicated by our perfusions of cells with the broad spectrum kinase inhibitors. Collectively, our results suggest that the cascade that culminates in anaphase is complex and involves several successive protein kinase activation steps punctuated by the activation of one or more protein phosphatases in mid-metaphase.

The cdc7 protein kinase is a dosage dependent regulator of septum formation in fission yeast.

Mutation of the Schizosaccharomyces pombe cdc7 gene prevents formation of the division septum and cytokinesis. We have cloned the cdc7 gene and show that it encodes a protein kinase which is essential for cell division. In the absence of cdc7 function, spore germination, DNA synthesis and mitosis are unaffected, but cells are unable to initiate formation of the division septum. Overexpression of p120cdc7 causes cell cycle arrest; cells complete mitosis and then undergo multiple rounds of septum formation without cell cleavage. This phenotype, which is similar to that resulting from inactivation of cdc16 protein, requires the kinase activity of p120cdc7. Mutations inactivating the early septation gene, cdc11, suppress the formation of multiple septa and allow cells to proliferate normally. If formation of the division septum is prevented by inactivation of either cdc14 or cdc15, p120cdc7 overproduction does not interfere with other events in the mitotic cell cycle. Septation is not induced by overexpression of p120cdc7 in G2 arrested cells, indicating that it does not bypass the normal dependency of septation upon initiation of mitosis. These findings indicate that the p120cdc7 protein kinase plays a key role in initiation of septum formation and cytokinesis in fission yeast and suggest that p120cdc7 interacts with the cdc11 protein in the control of septation.

Identification and characterization of Aspergillus nidulans mutants defective in cytokinesis.

Filamentous fungi undergo cytokinesis by forming crosswalls termed septa. Here, we describe the genetic and physiological controls governing septation in Aspergillus nidulans. Germinating conidia do not form septa until the completion of their third nuclear division. The first septum is invariantly positioned at the basal end of the germ tube. Block-and-release experiments of nuclear division with benomyl or hydroxyurea, and analysis of various nuclear division mutants demonstrated that septum formation is dependent upon the third mitotic division. Block-and-release experiments with cytochalasin A and the localization of actin in germlings by indirect immunofluorescence showed that actin participated in septum formation. In addition to being concentrated at the growing hyphal tips, a band of actin was also apparent at the site of septum formation. Previous genetic analysis in A. nidulans identified four genes involved in septation (sepA-D). We have screened a new collection of temperature sensitive (ts) mutants of A. nidulans for strains that failed to form septa at the restrictive temperature but were able to complete early nuclear divisions. We identified five new genes designated sepE, G, H, I and J, along with one additional allele of a previously identified septation gene. On the basis of temperature shift experiments, nuclear counts and cell morphology, we sorted these cytokines mutants into three phenotypic classes. Interestingly, one class of mutants fails to form septa and fails to progress past the third nuclear division. This class of mutants suggests the existence of a regulatory mechanism in A. nidulans that ensures the continuation of nuclear division following the initiation of cytokinesis.

Coordination of division events in theChlamydomonas cell cycle

At concentrations that did not affect growth, hydroxyurea and 21-deoxyadenosine inhibited DNA synthesis inChlamydomonas. Evidence that initiation of mitosis is dependent upon completion of DNA replication was provided by the arrest of inhibited cells with undivided nuclei containing undispersed nucleoli. Initiation of cytokinesis is not dependent upon progress of nuclear division since, in arrested cells, cleavage microtubules became deployed in a phycoplast and a cleavage furrow developed fully, until obstructed by the undivided nucleus. Chloroplast constriction and division also continued independently of nuclear division. It is concluded that nuclear division, cytoplasmic cleavage and chloroplast division are in separate sequences of dependent events. This is supported by flexibility of their relative timing in successive divisions, since after the first commitment to divide nuclear division is followed by initiation of cleavage and then chloroplast division, whereas following subsequent commitments these events occur in reverse time order. This flexibility of order indicates changing rates of progress through separate sequences of events.

Chapter 23 The Mitotic Apparatus: Methods for Isolation

This chapter describes the methods for isolation of the mitotic apparatus (MA). The mitotic apparatus (spindle–aster–chromosome complex) functions during cell division to bring about an equal distribution of nuclear material to the daughter cells and is responsible for initiation of cytokinesis. The rationale for the DMSO–glycerol method is to isolate functional MA with stable microtubules. As the functional activity (chromosome movement) cannot be used to assess the merits of the MA isolation method, other criteria must be employed. These involve comparisons of optical and physicochemical properties of isolated MA with MA in vivo . Using a compound microscope with bright-field optics, the MA in vivo cannot be seen and isolates are barely visible. The MA in vivo are cold-labile, pressure-labile, and colchicine-sensitive. Depending upon the method of isolation, in vitro MA also displays lability to cold, hydrostatic pressure, and sensitivity to colchicine.

Cytokinetic Aberrations in <i>Impatiens sultani</i> Mutants and Their Significance in Cytoplasmic Control of Pollen Wall Development

A comparative study of the development of normal and aberrant microspores of two mutants of Impatiens sultani shows that initiation of cytokinesis is not restrained by aberrations in chromosomal cycle. Although cytokinetic cleavage begins simultaneously in normal and aberrant meiocytes, delay or blockage of the progression of furrowing occurs in atypical meiocytes resulting in lobed monads and dyads. Random cytoplasmic divisions in meiocytes with scattered chromosome groups of telophase II produce spores of various sizes enclosing 0-8 or more chromosomes. Despite these aberrations in the cleavage phase, all spores-even those with a single chromosome or no chromosome at all-develop pollen walls with identical exine pattern and chemical constitution, lending support to the suggested role of extrachromosomal factors in pollen wall development.

The cell surface in relation to the growth cycle

The cell surface bears an intimate relation to events during the cell cycle. The following postulates are presented as working hypotheses in order to clarify current observations and to stimulate new experimental approaches. o (1) Initiation of karyokinesis is a necessary prerequisite for the initiation of cytokinesis; the reverse is not true. (2) In spherical cells that double in volume prior to mitosis, the ratio of surface area: volume is maintained by the elaboration of microvilli; the extra surface area generated by cytokinesis is provided by an unfolding of microvilli. (3) The state of the cell surface during interphase and in G 0 is different from that during mitosis; the difference is due to the involvement of intracellular elements that otherwise participate in the formation of the mitotic spindle and cleavage furrow. The association of such elements with the cell surface is greater in normal than in cancer cells.

Polar lobe formation and cytokinesis in fertilized eggs of Ilyanassa obsoleta: I. Ultrastructure and effects of cytochalasin B and colchicine

Formation of polar lobe constrictions and cleavage furrows in fertilized eggs of the marine mudsnail, Ilyanassa obsoleta, is associated with localized concentrations of microfilaments in the cortical cytoplasm. These microfilaments disappear after treatment with cytochalasin B, with concomitant regression of polar lobe constrictions and cleavage furrows. Microtubules are present in the noncortical cytoplasm of the polar lobe and disappear after treatment with colchicine. Colchicine application early in lobe development inhibits both polar lobe formation and cytokinesis; however, drug treatment later in lobe development, although still inhibiting cytokinesis, fails to inhibit the continued constriction of the polar lobe neck and subsequent lobe resorption. The data therefore suggest that separate colchicine-sensitive steps are required for the complete constriction of the polar lobe neck and for initiation of cytokinesis. Polar lobe necks constrict at two markedly different rates in contrast to the smooth, single-phase progression of cleavage furrows. As with cytokinesis, polar lobe formation may occur by the contraction of a microfilament ring whose polymerization or activity is regulated by microtubules.

The effects of p-DL-fluorophenylalanine on chromosome movement and cytokinesis of human amnion cells in culture.

An analysis is presented of time lapse motion picture films showing the effects of DL-p-fluorophenylalanine (PFPA) on chromosome movement in cultured human amnion cells. The data indicate that PFPA has the unusual effect of slowing the rate at which chromosomes move in anaphases which occur after the beginning of treatment. Irrespective of treatment, cytokinesis begins when chromsome movement has progressed to a relatively fixed point. Thus, a slowdown in the rate of chromosome movement leads to a delay in initiation of cytokinesis. Cytokinesis, once it begins, is not affected by treatment with the analog.

The behaviour and localization of intracellular relaxing system during cleavage in the sea urchin egg

A glycerinated model of cleaving sea urchin eggs was prepared using a new technique. The localization of the relaxing system in these eggs was determined by means of fluorescein-labeled antibodies. Some kind of cytoplasmic structure which reacts with anti-“relaxing granule”-serum was found to exist and show a directed migration during the cleavage in the sea urchin egg. As a result of directed movement of the “relaxing system” within the cell towards the centers of the mitotic apparatus during metaphase and anaphase, the presumptive furrowing region became temporarily poor in the relaxing system as compared with the polar regions. It was suggested that this relative deficiency of the relaxing system at the presumptive furrowing region will be the cause of the initiation of cytokinesis.