PtdIns(4,5)P2 functions at the cleavage furrow during cytokinesis

Phosphoinositides play important roles in regulating the cytoskeleton and vesicle trafficking, potentially important processes at the cleavage furrow. However, it remains unclear which, if any, of the phosphoinositides play a role during cytokinesis. A systematic analysis to determine if any of the phosphoinositides might be present or of functional importance at the cleavage furrow has not been published. Several studies hint at a possible role for one or more phosphoinositides at the cleavage furrow. The best of these are genetic data identifying mutations in phosphoinositide-modifying enzymes (a PtdIns(4)P-5-kinase in S. pombe and a PI-4-kinase in D. melanogaster) that interfere with cytokinesis. The genetic nature of these experiments leaves questions as to how direct may be their contribution to cytokinesis. Here we show that a single phosphoinositide, PtdIns(4,5)P2, specifically accumulates at the furrow. Interference with PtdIns(4,5)P2 interferes with adhesion of the plasma membrane to the contractile ring at the furrow. Finally, four distinct interventions to specifically interfere with PtdIns(4,5)P2 each impair cytokinesis. We conclude that PtdIns(4,5)P2 is present at the cleavage furrow and is required for normal cytokinesis at least in part because of a role in adhesion between the contractile ring and the plasma membrane.     

An ultrastructural study of cell plate modifications induced by 2,6-dichlorobenzonitrile in onion root meristems

Summary The effect of 2,6-dichlorobenzonitrile on cytokinesis of meristematic cells of onion root during both treatment and recovery has been studied by electron microscopic techniques. 2,6-dichlorobenzonitrile interferes with cell plate formation in such a way that Golgi apparatus vesicles of treated cells appear to be different than controls and seem to coalesce as anomalous partial cell plates. During recovery, an apparently normal progression of cytokinesis is observed and abnormal portions of the cell plate are retained. Nuclear constrictions are observed frequently during recovery as a result of temporal alterations in cytokinesis. Our results show that 2,6-dichlorobenzo-nitrile induces anomalous and/or incomplete cell plates, which might be caused by an altered function of Golgi apparatus.     

Roles of the DYRK kinase Pom2 in cytokinesis, mitochondrial morphology, and sporulation in fission yeast

Pom2 is predicted to be a dual-specificity tyrosine-phosphorylation regulated kinase (DYRK) related to Pom1 in Schizosaccharomyces pombe. DYRKs share a kinase domain capable of catalyzing autophosphorylation on tyrosine and exogenous phosphorylation on serine/threonine residues. Here we show that Pom2 is functionally different from the well-characterized Pom1, although they share 55% identity in the kinase domain and the Pom2 kinase domain functionally complements that of Pom1. Pom2 localizes to mitochondria throughout the cell cycle and to the contractile ring during late stages of cytokinesis. Overexpression but not deletion of pom2 results in severe defects in cytokinesis, indicating that Pom2 might share an overlapping function with other proteins in regulating cytokinesis. Gain and loss of function analyses reveal that Pom2 is required for maintaining mitochondrial morphology independently of microtubules. Intriguingly, most meiotic pom2Δ cells form aberrant asci with meiotic and/or forespore membrane formation defects. Taken together, Pom2 is a novel DYRK kinase involved in regulating cytokinesis, mitochondrial morphology, meiosis, and sporulation in fission yeast.     

Cytokinesis: thinking outside the cell

How might the extracellular matrix contribute to cytokinesis? In a recent report, evidence is presented that the conserved extracellular matrix protein hemicentin(HIM-4) is required for cytokinesis in worms and mice.     

Divide and ProsPer: the emerging role of PtdIns3P in cytokinesis

Cytokinesis is the final step of cell division whereby the dividing cells separate physically. Failure of this process has been proposed to cause tumourigenesis. Several specific lipids are essential for cytokinesis, and recent evidence has revealed that phosphatidylinositol 3-phosphate (PtdIns3P) - a well-known regulator of endosomal trafficking, receptor signaling, nutrient sensing and autophagy - plays an evolutionarily conserved role during cytokinesis. The emerging picture is that PtdIns3P and its regulators and effectors constitute a novel regulatory mechanism for cytokinesis. Elucidating the role of PtdIns3P in cytokinesis might contribute to insight into mechanisms of tumour development and suppression.     

CHO1, a mammalian kinesin-like protein, interacts with F-actin and is involved in the terminal phase of cytokinesis

CHO1 is a kinesin-like protein of the mitotic kinesin-like protein (MKLP)1 subfamily present in central spindles and midbodies in mammalian cells. It is different from other subfamily members in that it contains an extra approximately 300 bp in the COOH-terminal tail. Analysis of the chicken genomic sequence showed that heterogeneity is derived from alternative splicing, and exon 18 is expressed in only the CHO1 isoform. CHO1 and its truncated isoform MKLP1 are coexpressed in a single cell. Surprisingly, the sequence encoded by exon 18 possesses a capability to interact with F-actin, suggesting that CHO1 can associate with both microtubule and actin cytoskeletons. Microinjection of exon 18-specific antibodies did not result in any inhibitory effects on karyokinesis and early stages of cytokinesis. However, almost completely separated daughter cells became reunited to form a binulceate cell, suggesting that the exon 18 protein may not have a role in the formation and ingression of the contractile ring in the cortex. Rather, it might be involved directly or indirectly in the membrane events necessary for completion of the terminal phase of cytokinesis.     

Cellularisation in the endosperm of Arabidopsis thaliana is coupled to mitosis and shares multiple components with cytokinesis

Distinct forms of cytokinesis characterise specific phases of development in plants. In Arabidopsis, as in many other species, the endosperm that nurtures the embryo in the seed initially develops as a syncytium. This syncytial phase ends with simultaneous partitioning of the multinucleate cytoplasm into individual cells, a process referred to as cellularisation. Our in vivo observations show that, as in cytokinesis, cellularisation of the Arabidopsis endosperm is coupled to nuclear division. A genetic analysis reveals that most Arabidopsis mutations affecting cytokinesis in the embryo also impair endosperm cellularisation. These results imply that cellularisation and cytokinesis share multiple components of the same basic machinery. We further report the identification of mutations in a novel gene, SPATZLE, that specifically interfere with cellularisation of the endosperm, but not with cytokinesis in the embryo. The analysis of this mutant might identify a specific checkpoint for the onset of cellularisation.     

Regulation of the actin cytoskeleton by an interaction of IQGAP related protein GAPA with filamin and cortexillin I

Filamin and Cortexillin are F-actin crosslinking proteins in Dictyostelium discoideum allowing actin filaments to form three-dimensional networks. GAPA, an IQGAP related protein, is required for cytokinesis and localizes to the cleavage furrow during cytokinesis. Here we describe a novel interaction with Filamin which is required for cytokinesis and regulation of the F-actin content. The interaction occurs through the actin binding domain of Filamin and the GRD domain of GAPA. A similar interaction takes place with Cortexillin I. We further report that Filamin associates with Rac1a implying that filamin might act as a scaffold for small GTPases. Filamin and activated Rac associate with GAPA to regulate actin remodelling. Overexpression of filamin and GAPA in the various strains suggests that GAPA regulates the actin cytoskeleton through interaction with Filamin and that it controls cytokinesis through association with Filamin and Cortexillin.     

Anther wall formation, microsporogenesis and male gametogenesis of four closely related species in Astereae (Asteraceae): description, comparison and systematic implications

The embryological features of microsporangiate wall formation, microsporogenesis and male gametogenesis in Aster subulatus , Kalimeris indica , Heteropappus arenarius and Erigeron annuus are described for the first time. Although external morphology might suggest that these four species differ from each another, we found no distinctly different embryological features. They all share similar characteristics, such as a tetrasporangiate anther, coexisting amoeboid and glandular tapeta, simultaneous cytokinesis (except for 12% occurrence of successive cytokinesis in E. annuus ), 3-celled mature pollen grains and endothecium of anther wall that does not develop fibrous thickenings. Based on the embryological data, we suggest that Kalimeris and Heteropappus may be included within Aster , and that Erigeron is closely related to Aster sensu lato.     

The RhoGEF Pebble is required for cell shape changes during cell migration triggered by the Drosophila FGF receptor Heartless

The FGF receptor Heartless (HTL) is required for mesodermal cell migration in the Drosophila gastrula. We show that mesoderm cells undergo different phases of specific cell shape changes during mesoderm migration. During the migratory phase, the cells adhere to the basal surface of the ectoderm and exhibit extensive protrusive activity. HTL is required for the protrusive activity of the mesoderm cells. Moreover, the early phenotype of htl mutants suggests that HTL is required for the adhesion of mesoderm cells to the ectoderm. In a genetic screen we identified pebble (pbl) as a novel gene required for mesoderm migration. pbl encodes a guanyl nucleotide exchange factor (GEF) for RHO1 and is known as an essential regulator of cytokinesis. We show that the function of PBL in cell migration is independent of the function of PBL in cytokinesis. Although RHO1 acts as a substrate for PBL in cytokinesis, compromising RHO1 function in the mesoderm does not block cell migration. These data suggest that the function of PBL in cell migration might be mediated through a pathway distinct from RHO1. This idea is supported by allele-specific differences in the expressivity of the cytokinesis and cell migration phenotypes of different pbl mutants. We show that PBL is autonomously required in the mesoderm for cell migration. Like HTL, PBL is required for early cell shape changes during mesoderm migration. Expression of a constitutively active form of HTL is unable to rescue the early cellular defects in pbl mutants, suggesting that PBL is required for the ability of HTL to trigger these cell shape changes. These results provide evidence for a novel function of the Rho-GEF PBL in HTL-dependent mesodermal cell migration.     

Dolastatin 11 connects two long-pitch strands in F-actin to stabilize microfilaments

Dolastatin 11, a drug isolated from the Indian Ocean sea hare Dolabella auricularia, arrests cytokinesis in vivo and increases the amount of F-actin to stabilize F-actin in vitro, like phalloidin and jasplakinolide. However, according to the previous biochemical study, the binding of dolastatin 11 to F-actin does not compete with that of phalloidin, suggesting that the binding sites are different. To understand the mechanism of F-actin stabilization by dolastatin 11, we determined the position of bound dolastatin 11 in F-actin using the X-ray fiber diffraction from oriented filament sols. Our analysis shows that the position of dolastatin 11 is clearly different from that of phalloidin. However, these bound drugs are present in the gap between the two long-pitch F-actin strands in a similar way. The result suggests that the connection between the two long-pitch F-actin strands might be a key for the control of F-actin stabilization.     

Control of plant cytokinesis by an NPK1-mediated mitogen-activated protein kinase cascade

Cytokinesis is the last essential step in the distribution of genetic information to daughter cells and partition of the cytoplasm. In plant cells, various proteins have been found in the phragmoplast, which corresponds to the cytokinetic apparatus, and in the cell plate, which corresponds to a new cross wall, but our understanding of the functions of these proteins in cytokinesis remains incomplete. Reverse genetic analysis of NPK1 MAPKKK (nucleus- and phragmoplast-localized protein kinase 1 mitogen-activated protein kinase kinase kinase) and investigations of factors that might be functionally related to NPK1 have helped to clarify new aspects of the mechanisms of cytokinesis in plant cells. In this review, we summarize the evidence for the involvement of NPK1 in cytokinesis. We also describe the characteristics of a kinesin-like protein and the homologue of a mitogen-activated protein kinase that we identified recently, and we discuss possible relationships among these proteins in cytokinesis.     

Regulation of multiple cell cycle events by Cdc14 homologues in vertebrates

Whereas early cytokinesis events have been relatively well studied, little is known about its final stage, abscission. The Cdc14 phosphatase is involved in the regulation of multiple cell cycle events, and in all systems studied Cdc14 misexpression leads to cytokinesis defects. In this work, we have cloned two CDC14 cDNA from Xenopus, including a previously unreported CDC14B homologue. We use Xenopus and human cell lines and demonstrate that localization of Cdc14 proteins is independent of both cell-type and species specificity. Ectopically expressed XCdc14A is centrosomal in interphase and localizes to the midbody in cytokinesis. By using XCdc14A misregulation, we confirm its control over different cell cycle events and unravel new functions during abscission. XCdc14A regulates the G1/S and G2/M transitions. We show that Cdc25 is an in vitro substrate for XCdc14A and might be its target at the G2/M transition. Upregulated wild-type or phosphatase-dead XCdc14A arrest cells in a late stage of cytokinesis, connected by thin cytoplasmic bridges. It does not interfere with central spindle formation, nor with the relocalization of passenger protein and centralspindlin complexes to the midbody. We demonstrate that XCdc14A upregulation prevents targeting of exocyst and SNARE complexes to the midbody, both essential for abscission to occur.     

Mutagenic activity of estradiol evaluated by an in vitro micronucleus assay. Short communication

The objective of the present study was to evaluate possible genetic changes in cultured human lymphocytes treated with estradiol, using the cytokinesis block micronucleus assay. Eight experimental concentrations of estradiol were used (range from 10(-10) M to 0.7 x 10(-4) M). The obtained results indicate that estradiol exhibits aneugenic and/or clastogenic effects, expressed as increased frequency of micronucleated lymphocytes at two highest experimental concentrations used in this investigation. In addition to genotoxic effects, these concentrations decreased the cytokinesis block proliferation index (CBPI) and percentage of binucleated cells, indicating the cell cycle delay and possible cytotoxic effects. In conclusion, estradiol treatment might represent a human health risk, especially if overdosed or used for a prolonged period of time.     

Identification of proteins involved in cytokinesis of Dictyostelium

Dictyostelium is one of the model systems of choice for studying the cytokinesis of animal-type cells. Two types of cytokinesis mutants have been used to identify proteins involved in the cytokinesis of Dictyostelium: (1) type I, the mutant cells grow on substrates to produce giant multinucleate cells; (2) type II, the mutant cells divide nearly normally on substrates, but are unable to divide at all and get highly multinucleate in suspension culture. These two mutant types might correspond to the myosin II-independent and myosin II-including cytokinesis mechanisms, respectively.     

Inhibition of cytokinesis by lead (Pb2+) in a centric diatom.

Effect of inorganic and organo lead has been studied on the mitosis of a centric diatom Cyclotella meneghiniana f. unipunctata. Binucleate cells were formed in the presence of different concentrations of Pb2+ (1.0, 2.0, 3.0 and 5.0 mM) due to inhibition of cell plate formation. Lead at 5.0 mM concentration was more inhibitory than the other concentrations. Organo lead was a powerful depressant of cytokinesis than inorganic lead. Failure of cytokinesis might be due to disruption of microtubules. Formation of distinct nuclei delayed post incubation cell divisions suggest partial damage of mitotic spindles.     

Centromere localization of INCENP-Aurora B is sufficient to support spindle checkpoint function

During mitosis, the chromosomal passenger complex (CPC) comprising the Aurora B kinase, INCENP, survivin and borealin is essential for correcting non-bipolar chromosome attachments and for cytokinesis. In addition, the CPC might fullfil a role in the mitotic spindle assembly checkpoint (SAC), but this activity might be related to its role in correcting non-bipolar chromosome attachments. Here, we demonstrate that treatment of mitotic cells with the antibiotic actinomycin D causes a displacement of an intact and active CPC from centromeres onto chromosome arms, which results in chromosome misalignment, cytokinesis failure and SAC override, but still preserves histone H3 phosphorylation on chromosome arms. This surprising and unique scenario allows the reconstitution of endogenous Aurora B at centromeres/inner kinetochores by expressing a Cenp-B-INCENP fusion protein. We find that although the selective recruitment of endogenous Aurora B to centromeres/inner kinetochores is not sufficient to restore chromosome alignment and cytokinesis, it can restore Cenp-A phosphorylation at kinetochores, BubR1 recruitment to kinetochores and SAC activity after spindle disruption. These results indicate that INCENP-Aurora B localized at centromeres/inner kinetochores is sufficient to mediate SAC activity upon spindle disruption.     

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.     

Direct evidence for a critical role of myosin II in budding yeast cytokinesis and the evolvability of new cytokinetic mechanisms in the absence of myosin II

In the budding yeast Saccharomyces cerevisiae, an actomyosin-based contractile ring is present during cytokinesis, as occurs in animal cells. However, the precise requirement for this structure during budding yeast cytokinesis has been controversial. Here we show that deletion of MYO1, the single myosin II gene, is lethal in a commonly used strain background. The terminal phenotype of myo1Delta is interconnected chains of cells, suggestive of a cytokinesis defect. To further investigate the role of Myo1p in cytokinesis, we conditionally disrupted Myo1 function by using either a dominant negative Myo1p construct or a strain where expression of Myo1p can be shut-off. Both ways of disruption of Myo1 function result in a failure in cytokinesis. Additionally, we show that a myo1Delta strain previously reported to grow nearly as well as the wild type contains a single genetic suppressor that alleviates the severe cytokinesis defects of myo1Delta. Using fluorescence time-lapse imaging and electron microscopy techniques, we show that cytokinesis in this strain is achieved through formation of multiple aberrant septa. Taken together, these results strongly suggest that the actomyosin ring is crucial for successful cytokinesis in budding yeast, but new cytokinetic mechanisms can evolve through genetic changes when myosin II function is impaired.     

Microspore development in Podostemaceae-Podostemoideae, with implications on the characterization of the subfamilies

Subfamilies Podostemoideae and Tristichoideae of the aquatic flowering plant family Podostemaceae are conventionally characterized by a different mode of microsporogenesis. Simultaneous meiotic division into the four microspores is found in Tristichoideae, successive meiotic division is said to be typical of Podostemoideae. In contrast, the results of the present study reveal that in subfamily Podostemoideae both modes of microsporogenesis occur. This is exemplified by the early pollen development of two neotropical species: Apinagia latifolia and Marathrum rubrum. Successive versus simultaneous meiotic cytokinesis are thus not differential characters of the two subfamilies. It is worthy to note that successive cytokinesis occurs in a family (Podostemaceae) of the Eudicots which are characterized by simultaneous cytokinesis. The occurrence of Ubisch bodies (orbicules) in several species of Apinagia and Marathrum parallels the echinate ornamentation of the pollen grains.