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Cell division is characterized by orchestrated events of chromosome segregation, distribution of cellular organelles, and the eventual partitioning and separation of the two daughter cells. Mitotic kinases, including polo-like kinases (Plk), influence multiple events in mitosis. In yeast two-hybrid screens using mammalian Plk C-terminal domain baits, we have identified Golgi peripheral protein GRASP65 (Golgi reassembly stacking protein of 65 kDa) as a Plk-binding protein. GRASP65 appears to function in the post-mitotic reassembly of Golgi stacks. In this report we demonstrate binding between Plk and GRASP65 and provide in vitro and in vivo evidence that Plk is a GRASP65 kinase. Moreover, we show that Cdc2 can also phosphorylate GRASP65. In addition, we present data which support the observation that the conserved C terminus of Plk is important for its function. Deletion or frameshift mutations in the conserved C-terminal domain of Plk greatly diminish its ability to phosphorylate GRASP65. These and previous findings suggest that phosphorylation of Golgi components by mitotic kinases may regulate mechanisms of Golgi inheritance during cell division.

The nucleus is surrounded by a double membrane, the nuclear envelope, which is in turn lined by a network of proteins known as lamins. In mammalian cells, the nuclear envelope [...]

The spindle checkpoint prevents errors in chromosome segregation by inhibiting anaphase onset until all chromosomes have aligned at the spindle equator through attachment of their sister kinetochores to microtubules from opposite spindle poles. A key checkpoint component is the mitotic arrest-deficient protein 2 (Mad2), which localizes to unattached kinetochores and inhibits activation of the anaphase-promoting complex (APC) through an interaction with Cdc20. Recent studies have suggested a catalytic model for kinetochore function where unattached kinetochores provide sites for assembling and releasing Mad2-Cdc20 complexes, which sequester Cdc20 and prevent it from activating the APC. To test this model, we examined Mad2 dynamics in living PtK1 cells that were either injected with fluorescently labeled Alexa 488-XMad2 or transfected with GFP-hMAD2. Real-time, digital imaging revealed fluorescent Mad2 localized to unattached kinetochores, spindle poles, and spindle fibers depending on the stage of mitosis. FRAP measurements showed that Mad2 is a transient component of unattached kinetochores, as predicted by the catalytic model, with a [t.sub.1/2] of ~24-28 s. Cells entered anaphase ~10 min after Mad2 was no longer detectable on the kinetochores of the last chromosome to congress to the metaphase plate. Several observations indicate that Mad2 binding sites are translocated from kinetochores to spindle poles along microtubules. First, Mad2 that bound to sites on a kinetochore was dynamically stretched in both directions upon microtubule interactions, and Mad2 particles moved from kinetochores toward the poles. Second, spindle fiber and pole fluorescence disappeared upon Mad2 disappearance at the kinetochores. Third, ATP depletion resulted in microtubule-dependent depletion of Mad2 fluorescence at kinetochores and increased fluorescence at spindle poles. Finally, in normal cells, the half-life of Mad2 turnover at poles, 23 s, was similar to kinetochores. Thus, kinetochore-derived sites along spindle fibers and at spindle poles may also catalyze Mad2 inhibitory complex formation. Key words: cell cycle * mitosis * spindle checkpoint * chromosome * microtubule

Subcellular targeting of cAMP-dependent protein kinase (protein kinase A [PKA]) and of type 1 protein phosphatase (PP1) is believed to enhance the specificity of these enzymes. We report that in addition to anchoring PKA, A-kinase anchoring protein AKAP149 recruits PP1 at the nuclear envelope (NE) upon somatic nuclear reformation in vitro, and that PP1 targeting to the NE is a prerequisite for assembly of B-type lamins. AKAP149 is an integral membrane protein of the endoplasmic reticulum/NE network. The PP1-binding domain of AKAP149 was identified as [K.sup.153][GVLF.sup.157]. PP1 binds immobilized AKAP149 in vitro and coprecipitates with AKAP149 from purified NE extracts. Affinity isolation of PPI from solubilized NEs copurifies AKAP149. Upon reassembly of somatic nuclei in interphase extract, PP1 is targeted to the NE. Targeting is inhibited by a peptide containing the PP1-binding domain of AKAP149, abolished in nuclei assembled with membranes immunodepleted of AKAP149, and restored after reincorporation of AKAP149 into nuclear membranes. B-type lamins do not assemble into a lamina when NE targeting of PP1 is abolished, and is rescued upon recruitment of PP1 to the NE. We propose that kinase and phosphatase anchoring at the NE by AKAP149 plays in a role in modulating nuclear reassembly at the end of mitosis. Key words: AKAP * mitosis * nuclear envelope * protein kinase A * protein phosphatase 1

We have investigated the intracellular roles of an Xklp2-related kinesin motor, [KRP.sub.180], in positioning spindle poles during early sea urchin embryonic cell division using quantitative, real-time analysis. Immunolocalization reveals that [KRP.sub.180] concentrates on microtubules in the central spindle, but is absent from centrosomes. Microinjection of inhibitory antibodies and dominant negative constructs suggest that [KRP.sub.180] is not required for the initial separation of spindle poles, but instead functions to transiently position spindle poles specifically during prometaphase. Key words: Xklp2 * kinesin * sea urchin * mitosis * [Gamma]-tubulin

Okamoto, Curtis T., Jeana McKinney, and Young Y. Jeng. Clathrin in mitotic spindles. Am J Physiol Cell Physiol 279: C369-C374, 2000.-- Subconfluent cultures of Madin-Darby canine kidney (MDCK) and CV-1 cells were immunostained with two monoclonal antibodies (MAbs), MAb X-22 and MAb 23, against clathrin heavy chain and with polyclonal antiserum against a conserved region of all mammalian clathrin light chains. In interphase MDCK and CV-1 cells, staining by all three antibodies resulted in the characteristic intracellular punctate vesicular and perinuclear staining pattern. In mitotic cells, all three anti-clathrin antibodies strongly stained the mitotic spindle. Staining of clathrin in the mitotic spindle was colocalized with anti-tubulin staining of microtubular arrays in the spindle. Staining of the mitotic spindle was evident in mitotic cells from prometaphase to telophase and in spindles in mitotic cells released from a thymidine-nocodazole block. In CV-1 cells, staining of clathrin in the mitotic spindle was not affected by brefeldin A. On Western blots, clathrin was detected, but not enriched, in isolated spindles. The immunodetection of clathrin in the mitotic spindle may suggest a novel role for clathrin in mitosis. Alternatively, the recruitment of clathrin to the spindle may suggest a novel regulatory mechanism for localization of clathrin in mitotic cells. Madin-Darby canine kidney cells; CV-1 cells; brefeldin A

Barrier-to-autointegration factor (BAF) is a highly conserved cellular protein that was identified by its activity in protecting retroviral DNA against autointegration. We show that BAF has the property of bridging double-stranded DNA in a highly ordered nucleoprotein complex. Whereas BAF protein alone is a dimer in solution, upon binding DNA, BAF forms a dodecamer with DNA bound at multiple discrete sites in the complex. The interactions between BAF and DNA are entirely nonspecific with respect to DNA sequence. The dual interaction of BAF with DNA and LAP2, a protein associated with the nuclear lamina, suggests a role for LAP2 in chromosome organization. Consistent with this idea, RNA interference experiments with Caenorhabditis elegans reveal a defect in mitosis.

Progression through the mammalian cell cycle is regulated by the sequential activation and inactivation of the cyclin-dependent kinases. In adult cells, cyclin A2-dependent kinases are required for entry into S and M phases, completion of S phase, and centrosome duplication. However, mouse embryos lacking the cyclin A2 gene nonetheless complete preimplantation development, but die soon after implantation. In this report, we investigated whether a contribution of maternal cyclin A2 mRNA and protein to early embryonic cell cycles might explain these conflicting observations. Our data show that a maternal stock of cyclin A2 mRNA is present in the oocyte and persists after fertilization until the second mitotic cell cycle, when it is degraded to undetectable levels coincident with transcriptional activation of the zygotic genome. A portion of maternally derived cyclin A2 protein is stable during the first mitosis and persists in the cytoplasm, but is completely degraded at the second mitosis. The ability of cyclin A2-null mutants to develop normally from the four-cell to the postimplantation stage in the absence of detectable cyclin A2 gene product indicates therefore that cyclin A2 is dispensable for cellular progression during the preimplantation nongrowth period of mouse embryo development. [C] 2000 Academic Press Key Words: cell cycle; mouse oocyte; mitosis; transcription; protein degradation.

Increased nuclear translocation of catalytically active PKC-[Zeta] during mouse colonocyte hyperproliferation. Am J Physiol Gastrointest Liver Physiol 279: G223-G237, 2000.-Protein kinase (PK) C-[Zeta] is implicated in the control of colonic epithelial cell proliferation in vitro. However, less is known about its physiological role in vivo. Using the transmissible murine colonic hyperplasia (TMCH) model, we determined its expression, subcellular localization, and kinase activity during native crypt hyperproliferation. Enhanced mitosis was associated with increased cellular 72-kDa holoenzyme (PKC-[Zeta], 3.2-fold), 48-kDa catalytic subunit (PKM-[Zeta], 3- to 9-fold), and 24-kDa membrane-bound fragment ([M.sub.f]-[Zeta], [is greater than]10-fold) expression. Both PKC-[Zeta] and PKM-[Zeta] exhibited intrinsic kinase activity, and substrate phosphorylation increased 4.5-fold. No change in cellular PKC-[Iota]/PKM-[Iota] expression occurred. The subcellular distribution of immunoreactive PKC-[Zeta] changed significantly: neck cells lost their basal subcellular pole filamentous staining, whereas proliferating cell nuclear antigen-positive cells exhibited elevated cytoplasmic, lateral membrane, and nuclear staining. Subcellular fractionation revealed increased PKC-[Zeta] and PKM-[Zeta] expression and activity within nuclei, which preferentially accumulated PKM-[Zeta]. These results suggest separate cellular and nuclear roles, respectively, for PKC-[Zeta] in quiescent and mitotically active colonocytes. PKM-[Zeta] may specifically act as a modulator of proliferation during TMCH. protein kinase C; cellular mitosis; mouse colon

Budding yeast initiates anaphase by activating the Cdc20-dependent anaphase-promoting complex (APC). The mitotic activity of Cdc28 (Cdk1) is required to activate this form of the APC, and mutants that are impaired in mitotic Cdc28 function have difficulty leaving mitosis. This defect can be explained by a defect in APC phosphorylation, which depends on mitotic Cdc28 activity in vivo and can be catalyzed by purified Cdc28 in vitro. Mutating putative Cdc28 phosphorylation sites in three components of the APC, Cdc16, Cdc23, and Cdc27, makes the APC resistant to phosphorylation both in vivo and in vitro. The nonphosphorylatable APC has normal activity in G1, but its mitotic, Cdc20-dependent activity is compromised. These results show that Cdc28 activates the APC in budding yeast to trigger anaphase. Previous reports have shown that the budding yeast Cdc5 homologue, Plk, can also phosphorylate and activate the APC in vitro. We show that, like cdc28 mutants, cdc5 mutants affect APC phosphorylation in vivo. However, although Cdc5 can phosphorylate Cdc16 and Cdc27 in vitro, this in vitro phosphorylation does not occur on in vivo sites of phosphorylation. Key words: mitosis * budding yeast * Cdc5 * Cks1 * Pds1

The activity of the cyclin-dependent kinase 1 (Cdk1), Cdc28, inhibits the transition from anaphase to G1 in budding yeast. CDC28-T18V, Y19F (CDC28-VF), a mutant that lacks inhibitory phosphorylation sites, delays the exit from mitosis and is hypersensitive to perturbations that arrest cells in mitosis. Surprisingly, this behavior is not due to a lack of inhibitory phosphorylation or increased kinase activity, but reflects reduced activity of the anaphase-promoting complex (APC), a defect shared with other mutants that lower Cdc28/Clb activity in mitosis. CDC28-VF has reduced Cdc20-dependent APC activity in mitosis, but normal Hct1-dependent APC activity in the G1 phase of the cell cycle. The defect in Cdc20-dependent APC activity in CDC28-VF correlates with reduced association of Cdc20 with the APC. The defects of CDC28-VF suggest that Cdc28 activity is required to induce the metaphase to anaphase transition and initiate the transition from anaphase to G1 in budding yeast. Key words: anaphase-promoting complex * Hct1 * Cdc20 * Pds1 * sister chromatid separation

During mitosis, two arrays of microtubules form the bipolar mitotic spindle in order to effect partitioning of the duplicated chromosomes into the daughter cells. The fabrication of the spindle therefore [...]

Abnormal mitosis occurs in maize tapetum, producing binucleate cells that later disintegrate, following a pattern of programmed cell death. FISH allowed us to observe chromosome nondisjunction and micronucleus formation in binucleate cells, using DNA probes specific to B chromosomes (B's), knobbed chromosomes, and the chromosome 6 (NOR) of maize. All chromosome types seem to be involved in micronucleus formation, but the B's form more micronuclei than do knobbed chromosomes and knobbed chromosomes form more than do chromosomes without knobs. Micronuclei were more frequent in 1B plants and in a genotype selected for low B transmission rate. Nondisjunction was observed in all types of FISH-labeled chromosomes. In addition, unlabeled bridges and delayed chromatids were observed in the last telophase before binucleate cell formation, suggesting that nondisjunction might occur in all chromosomes of the maize complement. B nondisjunction is known to occur in the second pollen mitosis and in the endosperm, but it was not previously reported in other tissues. This is also a new report of nondisjunction of chromosomes of the normal set (A's) in tapetal cells. Our results support the conclusion that nondisjunction and micronucleus formation are regular events in the process of the tapetal cell death program, but B's strongly increase A chromosome instability.

hDlg, the human homologue of the Drosophila Discs-large (Dlg) tumor suppressor protein, is known to interact with the tumor suppressor protein APC and the human papillomavirus E6 transforming protein. In a two-hybrid screen, we identified a 322-aa serine/threonine kinase that binds to the PDZ2 domain of hDlg. The mRNA for this PDZ-binding kinase, or PBK, is most abundant in placenta and absent from adult brain tissue. The protein sequence of PBK has all the characteristic protein kinase subdomains and a C-terminal PDZ-binding T/SXV motif. In vitro, PBK binds specifically to PDZ2 of hDlg through its C-terminal T/SXV motif. PBK and hDlg are phosphorylated at mitosis in HeLa cells, and the mitotic phosphorylation of PBK is required for its kinase activity. In vitro, cdc2/cyclin B phosphorylates PBK. This evidence shows how PBK could link hDlg or other PDZ-containing proteins to signal transduction pathways regulating the cell cycle or cellular proliferation.

The cis-Golgi matrix protein GM130 is phosphorylated in mitosis on serine 25. Phosphorylation inhibits binding to p115, a vesicle-tethering protein, and has been implicated as an important step in the mitotic Golgi fragmentation process. We have generated an antibody that specifically recognizes GM130 phosphorylated on serine 25, and used this antibody to study the temporal regulation of phosphorylation in vivo. GM130 is phosphorylated in prophase as the Golgi complex starts to break down, and remains phosphorylated during further breakdown and partitioning of the Golgi fragments in metaphase and anaphase. In telophase, GM130 is dephosphorylated as the Golgi fragments start to reassemble. The timing of phosphorylation and dephosphorylation correlates with the dissociation and reassociation of p115 with Golgi membranes. GM130 phosphorylation and p115 dissociation appear specific to mitosis, since they are not induced by several drugs that trigger nonmitotic Golgi fragmentation. The phosphatase responsible for dephosphorylation of mitotic GM130 was identified as PP2A. The active species was identified as heterotrimeric phosphatase containing the B[Alpha] regulatory subunit, suggesting a role for this isoform in the reassembly of mitotic Golgi membranes at the end of mitosis. Key words: Golgi * mitosis * GM130 * phosphorylation * protein phosphatase 2A

At the onset of mitosis, the Golgi apparatus, which consists of several cisternae, disperses throughout the cell to be partitioned into daughter cells. The molecular mechanisms of this process are now beginning to be understood. To investigate the biochemical requirements and kinetics of mitotic Golgi membrane dynamics in polarized cells, we have reconstituted the disassembly of the Golgi apparatus by introducing Xenopus egg extracts into permeabilized Mardin-Darby canine kidney (MDCK) cells. We used green fluorescence protein (GFP)-tagged galactosyltransferase-expressing MDCK cells to analyze the morphological changes of the Golgi membrane in the semi-intact system. Analyses by fluorescence and electron microscopies showed that the Golgi disassembly can be dissected into two elementary processes morphologically. In the first process, the perinuclear Golgi stacks break into punctate structures, intermediates, which are comprised of mini-stacks of cisternae associating with apical microtubule networks. In the second process, the structures fragment more thoroughly or substantially relocate to the ER. Our analyses further showed that cdc2 kinase and mitogen-activated protein kinase kinase (MAPKK = MEK) are differently involved in these two processes: the first process is mainly regulated by MEK and the second mainly by cdc2. Key words: Golgi complex * mitosis * semi-intact cells * GFP * kinase

Incubation of permeabilized cells with mitotic extracts results in extensive fragmentation of the pericentriolarly organized stacks of cisternae. The fragmented Golgi membranes are subsequently dispersed from the pericentriolar region. We have shown previously that this process requires the cytosolic protein mitogen-activated protein kinase kinase 1 (MEK1). Extracellular signal-regulated kinase (ERK) 1 and ERK2, the known downstream targets of MEK1, are not required for this fragmentation (Acharya et al., 1998). We now provide evidence that MEK1 is specifically phosphorylated during mitosis. The mitotically phosphorylated MEK1, upon partial proteolysis with trypsin, generates a different peptide population compared with interphase MEK1. MEK1 cleaved with the lethal factor of the anthrax toxin can still be activated by its upstream mitotic kinases, and this form is fully active in the Golgi fragmentation process. We believe that the mitotic phosphorylation induces a change in the conformation of MEK1 and that this form of MEK1 recognizes Golgi membranes as a target compartment. Immunoelectron microscopy analysis reveals that treatment of permeabilized normal rat kidney (NRK) cells with mitotic extracts, treated with or without lethal factor, converts stacks of pericentriolar Golgi membranes into smaller fragments composed predominantly of tubuloreticular elements. These fragments are similar in distribution, morphology, and size to the fragments observed in the prometaphase/metaphase stage of the cell cycle in vivo. Key words: mitogen-activated protein kinase kinase 1 * extracellular signal-regulated kinases * mitosis * Golgi fragmentation * phosphorylation

We describe a Drosophila gene, orbit, that encodes a conserved 165-kD microtubule-associated protein (MAP) with GTP binding motifs. Hypomorphic mutations in orbit lead to a maternal effect resulting in branched and bent mitotic spindles in the syncytial embryo. In the larval central nervous system, such mutants have an elevated mitotic index with some mitotic cells showing an increase in ploidy. Amorphic alleles show late lethality and greater frequencies of hyperploid mitotic cells. The presence of cells in the hypomorphic mutant in which the chromosomes can be arranged, either in a circular metaphase or an anaphase-like configuration on monopolar spindles, suggests that polyploidy arises through spindle and chromosome segregation defects rather than defects in cytokinesis. A role for the Orbit protein in regulating microtubule behavior in mitosis is suggested by its association with microtubules throughout the spindle at all mitotic stages, by its copurification with microtubules from embryonic extracts, and by the finding that the Orbit protein directly binds to MAP-free microtubules in a GTP-dependent manner. Key words: mitosis * microtubule-associated protein * Drosophila melanogaster * mitotic spindle * centrosome

RAD51, the eukaryote homolog of the Escherichia coli recA, recombinase, participates in homologous recombination during mitosis, meiosis, and in the repair of double-stranded DNA breaks. The Tetrahymena thermophila RAD51 gene was recently cloned, and the in vitro activities and induction of Rad51p following DNA damage were shown to be similar to that of RAD51 from other species. This study describes the pattern of Tetrahymena RAD51 expression during both the cell cycle and conjugation. Tetrahymena RAD51 mRNA abundance is elevated during macronuclear S phase during vegetative cell growth and with both meiotic prophase and new macronuclear development during conjugation. Gene disruption of the macronuclear RAD51 locus leads to severe abnormalities during both vegetative growth and conjugation. rad51 nulls divide slowly and incur rapid deterioration of their micronuclear chromosomes. Conjugation of two rad5I nulls leads to an arrest early during prezygotic development (meiosis I). We discuss the potential usefulness of the ciliates' characteristic nuclear duality for further analyses of the potentially unique roles of Tetrahymena RAD51.

The superior cervical ganglion (SCG) is a well-characterized model of neural development, in which several regulatory signals have been identified. Vasoactive intestinal peptide (VIP) has been found to regulate diverse ontogenetic processes in sympatheties, though functional requirements for high peptide concentrations suggest that other ligands are involved. We now describe expression and functions of pituitary adenylate cyclase-activating polypeptide (PACAP) during SCG ontogeny, suggesting that the peptide plays critical roles in neurogenesis. PACAP and PACAP receptor ([PAC.sub.1]) mRNA's were detected at embryonic days 14.5 (E14.5) through E17.5 in vivo and virtually all precursors exhibited ligand and receptor, indicating that the system is expressed as neuroblasts proliferate. Exposure of cultured precursors to PACAP peptides, containing 27 or 38 residues, increased mitogenic activity 4-fold. Significantly, PACAP was 1000-fold more potent than VIP and a highly potent and selective antagonist entirely blocked effects of micromolar VIP, consistent with both peptides acting via [PAC.sub.1] receptors. Moreover, PACAP potently enhanced precursor survival more than 2-fold, suggesting that previously defined VIP effects were mediated via [PAC.sub.1] receptors and that PACAP is the more significant developmental signal. In addition to neurogenesis, PACAP promoted neuronal differentiation, increasing neurite outgrowth 4-fold and enhancing expression of neurotrophin receptors trkC and trkA. Since PACAP potently activated cAMP and PI pathways and increased intracellular [Ca.sup.2+], the peptide may interact with other developmental signals. PACAP stimulation of precursor mitosis, survival, and trk receptor expression suggests that the signaling system plays a critical autocrine role during sympathetic neurogenesis. [C] 2000 Academic Press Key Words: neurogenesis; proliferation; mitosis; neurotrophins; trkA; trkC; VIP; trophic factors; survival; [Ca.sup.2+] flux; cAMP; phosphatidyl inositol.

The peptidyl-prolyl isomerase Pin1 has been implicated in regulating cell cycle progression. Pin 1 was found to be required for the DNA replication checkpoint in Xenopus laevis. Egg extracts depleted of Pin1 inappropriately transited from the [G.sub.2] to the M phase of the cell cycle in the presence of the DNA replication inhibitor aphidicolin. This defect in replication checkpoint function was reversed after the addition of recombinant wild-type Pin1, but not an isomerase-inactive mutant, to the depleted extract. Premature mitotic entry in the absence of Pin1 was accompanied by hyperphosphorylation of Cdc25, activation of Cdc2/cyclin B, and generation of epitopes recognized by the mitotic phosphoprotein antibody, MPM-2. Therefore, Pin1 appears to be required for the checkpoint delaying the onset of mitosis in response to incomplete replication., The peptidyl-prolyl isomerase (PPIase) Pin1 affects cell cycle transitions. Originally identified in yeast-two hybrid screens as a protein that binds to and suppresses the toxicity of the fungal mitotic kinase [...]

The orientation of the mitotic spindle along a polarity axis is critical in asymmetric cell divisions. In the budding yeast, Saccharomyces cerevisiae, loss of the S-phase B-type cyclin Clb5p under conditions of limited cyclin-dependent kinase activity (cdc28-4 clb5[Delta] cells) causes a spindle positioning defect that results in an undivided nucleus entering the bud. Based on time-lapse digital imaging microscopy of microtubules labeled with green fluorescent protein fusions to either tubulin or dynein, we observed that the asymmetric behavior of the spindle pole bodies during spindle assembly was lost in the cdc28-4 clb5[Delta] cells. As soon as a spindle formed, both poles were equally likely to interact with the bud cell cortex. Persistent dynamic interactions with the bud ultimately led to spindle translocation across the bud neck. Thus, the mutant failed to assign one spindle pole body the task of organizing astral microtubules towards the mother cell. Our data suggest that Clb5p-associated kinase is required to confer mother-bound behavior to one pole in order to establish correct spindle polarity. In contrast, B-type cyclins, Clb3p and Clb4p, though partially redundant with Clb5p for an early role in spindle morphogenesis, preferentially promote spindle assembly. Key words: spindle polarity * astral microtubules * cell cycle * cyclin * Saccharomyces cerevisiae

Members of the polo subfamily of protein kinases play pivotal roles in cell-cycle control and proliferation. In addition to a high degree of sequence similarity in the kinase domain, polo kinases contain a strikingly conserved motif termed 'polo-box' in the noncatalytic C-terminal domain. We have previously shown that the mammalian polo-like kinase Plk is a functional homolog of Saccharomyces cerevisiae Cdc5. Here, we show that, in a polo-box- and kinase activity-dependent manner, ectopic expression of Plk in budding yeast can induce a class of cells with abnormally elongated buds. In addition to localization at spindle poles and cytokinetic neck filaments, Plk induces and localizes to ectopic septin ring structures within the elongated buds. In contrast, mutations in the polo-box abolish both localization to, and induction of, septal structures. Consistent with the polo-box-dependent subcellular localization, the C-terminal domain of Plk, but not its polo-box mutant, is sufficient for subcellular localization. Our data suggest that Plk may contribute a signal to initiate or promote cytokinetic event(s) and that an intact polo-box is required for regulation of these cellular processes. septin | mitosis | cytokinesis

Persistent activation of p42 mitogen-activated protein kinase (p42 MAPK) during mitosis induces a 'cytostatic factor' arrest, the arrest responsible for preventing the parthenogenetic activation of unfertilized eggs. The protein kinase p90 Rsk is a substrate of p42 MAPK; thus, the role of p90 Rsk in p42 MAPK-induced mitotic arrest was examined. Xenopus laevis egg extracts immunodepleted of Rsk lost their capacity to undergo mitotic arrest in response to activation of the Mos-MEK-1-p42 MAPK cascade of protein kinases. Replenishing Rsk-depleted extracts with catalytically competent Rsk protein restored the ability of the extracts to undergo mitotic arrest. Rsk appears to be essential for cytostatic factor arrest., Masui identified two hypothetical M-phase regulators in his classic studies of Rana pipiens oocyte maturation. The first, maturationpromoting factor (MPF), was an activity present in mature oocytes that was able [...]

Smart molecules that perturb specific protein functions are valuable tools for dissecting complex processes in mammalian cells. A combination of two phenotype-based screens, one based on a specific posttranslational modification, the other visualizing microtubules and chromatin, was used to identify compounds that affect mitosis. One compound, here named monastrol, arrested mammalian cells in mitosis with monopolar spindles, In vitro, monastrol specifically inhibited the motility of the mitotic kinesin Eg5, a motor protein required for spindle bipolarity. All previously known small molecules that specifically affect the mitotic machinery target tubulin. Monastrol will therefore be a particularly useful tool for studying mitotic mechanisms., Cell-permeable small molecules can rapidly perturb the function of their targets and are therefore powerful tools to dissect dynamic cellular processes. However, such modulators are not available for most of [...]

One of the defining events during cell division (mitosis) is the separation of sister chromatids that are attached to each other and to the mitotic spindle, a process called cohesion [...]

Polypterus development is similar to sturgeons, i.e. amphibian-like. Small (1.4 mm) telolecithal eggs develop rapidly and undergo unequal holoblastic cleavage. In the late blastula, an animal hemispheric cap of small (10-25 [micro]m) yolk-free cells 34 layers thick at the pole and 5-6 layers thick at the equatorial marginal zone encloses a small blastocoel and surmounts a sphere of large (75-200 [micro]m) yolk-rich vegetal cells. Adjacent surface cells are tightly joined to form an epithelium. Mitosis is common among animal hemisphere cells. Gastrulation begins with epiboly of the animal hemisphere and formation by bottle cells of a shallow blastoporal groove in the equatorial zone of junction. The groove becomes a blastopore that opens into an archenteron. Overgrowth of the vegetal cells coincides with involution of surface animal pole cells through the blastopore to form the roof of the archenteron. A cleft of Brachet forms between pre-and post-involution cell layers. The floor of the archenteron is formed by ingression of large, yolk-rich vegetal pole cells. Supported by NSF.

Abstract: In the root meristem cells of the rice line AMR, which causes loss of heterozygosity in its hybrids, both normal and assortment mitoses were observed. During normal mitosis, chromosomes [...]

No one would expect an orchestra to perform Beethoven's ninth symphony without a conductor. It is therefore no surprise that when cells perform their most dramatic tour de force--division by [...]

The thermal splitting reaction of bis-Cu(II) complex of octaphyrin (1.1.1.1.1.1.1.1) in two Cu(II) porphyrins is reported. The conclusion states that this Cu(II) gives rise to the facile splitting reaction triggered by increased distortion in the bis-metal complex, which provides a rare example of molecular mitosis for expanded porphyrins.

This paper concerns the mechanism of mitosis. I briefly review the history of mitosis research, emphasizing the role of microscopic observation. The polymerization and depolymerization of microtubules play central roles in both the assembly of the spindle, and in the way it moves chromosomes. I describe recent advances in understanding the dynamic behavior of individual microtubules, and of microtubules attached to kinetochores. The kinetochore is able to hold on to microtubule ends while allowing them to grow and shrink, reactions that can be studied both in living cells and in the test tube. Experiments in which microtubules in living cells are marked and then followed allow us to relate these individual dynamic processes to the overall mechanism of chromosome movement. I end the review by speculating about biochemical mechanisms for producing force on chromosomes in anaphase, and for balancing chromosomes in the center of the metaphase spindle.

I review recent advances in our knowledge of the eucaryotic cell cycle; the set of processes by which cells grow and divide. Genetic approaches to the cell cycle of somatic cells identified a pathway of events where the initiation of each event was dependent on the successful completion of the preceding event, as well as a single key gene, cdc2, that is required both at the beginning and at the end of the cell cycle. The alternative approach of studying the cell cycle biochemically in early embryos provided evidence for a cytoplasmic oscillator which alternated between mitosis-inducing and interphase-inducing states and identified the mitosis-inducing component as maturation promoting factor (MPF). These two very different views of the cell cycle initially seemed irreconcilable. However, a link between the somatic and embryonic cell cycles was provided by the recent discovery that the cdc2 protein is one of the components of MPF. In the embryonic cell cycle the activiation of MPF and induction of mitosis is triggered by the accumulation of a protein named cyclin which becomes a component of MPF. Somehow, MPF induces the proteolytic degradation of cyclin, which inturn allows MPF to be inactivated and allows the cell cycle to pass from mitosis into interphase. The more complex cell cycle of somatic cells is probably derived from the embryonic cyclin-based oscillator by imposing a system of checks and balances on the accumulation and destruction of cyclin. I also present some thoughts on the relationships between science and society, and comment on the way in which scientists describe their work to the lay world.

The Structure of Sister Minichromosome DNA Before Anaphase in Saccharomyces cerevisiae SISTER CHROMATIDS ARE HELD TOGETHER from the time of their replication to the beginning of anaphase. This interaction is [...]

Early Signals in the Mitogenic Response GROWTH FACTORS ARE IMPLICATED IN A WIDE VARIETY OF physiological and pathological processes. These include embryogenesis, growth and development, selective cell survival, hemopoiesis, tissue [...]

Calcium Rises Abruptly and Briefly Throughout the Cell at the Onset of Anaphase THE ONSET OF ANAPHASE IS AN abrupt transition characterized by the separation of chromatids at the metaphase [...]