Genetic dissection of cytokinesis

Higher plants have evolved specific mechanisms for partitioning the cytoplasm of dividing cells. In the predominant mode of phragmoplast-assisted cytokinesis, a cell wall and flanking plasma membranes are made de novo from a transient membrane compartment, the cell plate, which in turn forms by vesicle fusion from the centre to the periphery of the dividing cell. Other modes of cytokinesis appear to occur in meiotic cells and developing gametophytes. Here we review recent progress in the analysis of plant cytokinesis, focusing on genetic studies in Arabidopsis which are beginning to identify structural and regulatory components of phragmoplast-assisted cytokinesis. Two classes of mutations have been described. In one class, the defects appear to be confined to cell plate formation, suggesting that the execution of cytokinesis is specifically affected. Mutations in the other class display more general defects in cell division. We also discuss possible roles of proteins that have been localised in cytokinetic cells but not characterised genetically. Finally, mutations affecting meiotic or gametophytic cell divisions suggest that mechanistically different modes of cytokinesis occur in higher plants.     

Fission yeast synaptobrevin is involved in cytokinesis and cell elongation

Synaptobrevin is a vesicle-associated membrane protein playing an essential role in regulated vesicle transport. In this study, we characterized Syb1, synaptobrevin of Schizosaccharomyces pombe. Syb1 was located on various sizes of vesicle-like structures in the cytoplasm and enriched in the medial region and cell ends. Transport of Syb1 to the medial region was mainly dependent on F-actin and Myo52/Myo4. Syb1 is essential for cell viability and most of the syb1-null cells showed a round or short cylindrical form. These results suggest that Syb1 is involved in membrane trafficking of cytokinesis and cell elongation.     

Endocytosis restricts Arabidopsis KNOLLE syntaxin to the cell division plane during late cytokinesis

Cytokinesis represents the final stage of eukaryotic cell division during which the cytoplasm becomes partitioned between daughter cells. The process differs to some extent between animal and plant cells, but proteins of the syntaxin family mediate membrane fusion in the plane of cell division in diverse organisms. How syntaxin localization is kept in check remains elusive. Here, we report that localization of the Arabidopsis KNOLLE syntaxin in the plane of cell division is maintained by sterol-dependent endocytosis involving a clathrin- and DYNAMIN-RELATED PROTEIN1A-dependent mechanism. On genetic or pharmacological interference with endocytosis, KNOLLE mis-localizes to lateral plasma membranes after cell-plate fusion. Fluorescence-loss-in-photo-bleaching and fluorescence-recovery-after-photo-bleaching experiments reveal lateral diffusion of GFP-KNOLLE from the plane of division to lateral membranes. In an endocytosis-defective sterol biosynthesis mutant displaying lateral KNOLLE diffusion, KNOLLE secretory trafficking remains unaffected. Thus, restriction of lateral diffusion by endocytosis may serve to maintain specificity of syntaxin localization during late cytokinesis.     

The cytokinesis gene KEULE encodes a Sec1 protein that binds the syntaxin KNOLLE

KEULE is required for cytokinesis in Arabidopsis thaliana. We have positionally cloned the KEULE gene and shown that it encodes a Sec1 protein. KEULE is expressed throughout the plant, yet appears enriched in dividing tissues. Cytokinesis-defective mutant sectors were observed in all somatic tissues upon transformation of wild-type plants with a KEULE-green fluorescent protein gene fusion, suggesting that KEULE is required not only during embryogenesis, but at all stages of the plant's life cycle. KEULE is characteristic of a Sec1 protein in that it appears to exist in two forms: soluble or peripherally associated with membranes. More importantly, KEULE binds the cytokinesis-specific syntaxin KNOLLE. Sec1 proteins are key regulators of vesicle trafficking, capable of integrating a large number of intra- and/or intercellular signals. As a cytokinesis-related Sec1 protein, KEULE appears to represent a novel link between cell cycle progression and the membrane fusion apparatus.     

An S-locus receptor-like kinase plays a role as a negative regulator in plant defense responses

Plant cells often use cell surface receptors to sense environmental changes and then transduce external signals via activated signaling pathways to trigger adaptive responses. In Arabidopsis, the receptor-like protein kinase (RLK) gene family contains more than 600 members, and some of these are induced by pathogen infection, suggesting a possible role in plant defense responses. We previously characterized an S-locus RLK (CBRLK1) at the biochemical level. In this study, we examined the physiological function of CBRLK1 in defense responses. CBRLK1 mutant and CBRLK1-overexpressing transgenic plants showed enhanced and reduced resistance against a virulent bacterial pathogen, respectively. The altered pathogen resistances of the mutant and overexpressing transgenic plants were associated with increased and reduced induction of the pathogenesis-related gene PR1, respectively. These results suggest that CBRLK1 plays a negative role in the disease resistance signaling pathway in Arabidopsis.     

Mid2p stabilizes septin rings during cytokinesis in fission yeast

Septins are filament-forming proteins with a conserved role in cytokinesis. In the fission yeast Schizosaccharomyces pombe, septin rings appear to be involved primarily in cell-cell separation, a late stage in cytokinesis. Here, we identified a protein Mid2p on the basis of its sequence similarity to S. pombe Mid1p, Saccharomyces cerevisiae Bud4p, and Candida albicans Int1p. Like septin mutants, mid2delta mutants had delays in cell-cell separation. mid2delta mutants were defective in septin organization but not contractile ring closure or septum formation. In wild-type cells, septins assembled first during mitosis in a single ring and during septation developed into double rings that did not contract. In mid2delta cells, septins initially assembled in a single ring but during septation appeared in the cleavage furrow, forming a washer or disc structure. FRAP studies showed that septins are stable in wild-type cells but exchange 30-fold more rapidly in mid2delta cells. Mid2p colocalized with septins and required septins for its localization. A COOH-terminal pleckstrin homology domain of Mid2p was required for its localization and function. No genetic interactions were found between mid2 and the related gene mid1. Thus, these studies identify a new factor responsible for the proper stability and function of septins during cytokinesis.     

Furrow-specific endocytosis during cytokinesis of zebrafish blastomeres

Mutations affecting endocytosis, such as those in clathrin and dynamin, unexpectedly cause defects in cytokinesis in a number of organisms. To explore the relationship between endocytosis and cytokinesis, we used the relatively large cells of the transparent zebrafish embryo. Using fluorescent markers for fluid-phase as well as plasma membrane uptake, we demonstrate that cytokinesis involves furrow-specific endocytosis. Clathrin-coated pits are visible near the furrow in ultrathin sections, while immunolabeling demonstrates that clathrin and caveolin are localized to the cleavage furrow. Hence, it is likely that both clathrin- and caveolae-mediated endocytosis occurs at the furrow during cytokinesis. Dynamin II is also localized to the furrow and may mediate furrow-specific endocytosis. Treatment of embryos with chlorpromazine or with methyl-beta-cyclodextrin, both of which inhibit endocytosis, prevents the normal completion of cytokinesis. These data suggest that furrow-specific endocytosis is an integral part of cytokinesis.     

Emerging role of SUMOylation in plant development

Post-translational attachment of small ubiquitin-like modifier (SUMO), defined as SUMOylation, has emerged as a new mechanism of protein regulation in plant biology. In plant, SUMOylation has been shown to play crucial roles in a variety of biotic and abiotic stress responses. Recent work using viable mutants with defective SUMOylation have indicated an important role for SUMOylation in a wide range of developmental processes, such as cell division, expansion, survival and differentiation, vegetative growth and reproductive development. This review will summarize the currently emerging information regarding the function of SUMOylation in plant development.     

Macronuclear division and cytokinesis in Tetrahymena

Tetrahymena contains a micronucleus and a macronucleus. The micronucleus divides with typical mitosis, while the macronucleus divides amitotically. Although the mechanism responsible for macronuclear division was previously unknown, we clarified the organization of microtubules during macronuclear division. The macronuclear microtubules dynamically changed their distribution in an organized way throughout the macronuclear division. The macronuclear microtubules and the cytoplasmic microtubules cooperatively carried out the macronuclear division. When the micronuclear division was finished, p85 appeared at the presumptive division plane prior to the cytokinesis. The p85 directly interacted with calmodulin in a Ca(2+)-dependent manner, and p85 and CaM colocalized to the division furrow during cytokinesis. Moreover, the Ca(2+)/CaM inhibitor, W7, inhibited the direct interaction between p85 and CaM, the localization of both proteins to the division plane, and the formation of the division furrow. Thus, Ca(2+)/CaM and p85 have important roles in initiation and progression of cytokinesis in Tetrahymena.     

Sid2p, a spindle pole body kinase that regulates the onset of cytokinesis.

The fission yeast Schizosaccharomyces pombe divides by medial fission through the use of an actomyosin contractile ring. Precisely at the end of anaphase, the ring begins to constrict and the septum forms. Proper coordination of cell division with mitosis is crucial to ensure proper segregation of chromosomes to daughter cells. The Sid2p kinase is one of several proteins that function as part of a novel signaling pathway required for initiation of medial ring constriction and septation. Here, we show that Sid2p is a component of the spindle pole body at all stages of the cell cycle and localizes transiently to the cell division site during medial ring constriction and septation. A medial ring and an intact microtubule cytoskeleton are required for the localization of Sid2p to the division site. We have established an in vitro assay for measuring Sid2p kinase activity, and found that Sid2p kinase activity peaks during medial ring constriction and septation. Both Sid2p localization to the division site and activity depend on the function of all of the other septation initiation genes: cdc7, cdc11, cdc14, sid1, spg1, and sid4. Thus, Sid2p, a component of the spindle pole body, by virtue of its transient localization to the division site, appears to determine the timing of ring constriction and septum delivery in response to activating signals from other Sid gene products.     

Cytoskeleton organization during the cell cycle in two stramenopile microalgae, Ochromonas danica (Chrysophyceae) and Heterosigma akashiwo (Raphidophyceae), with special reference to F-actin organization and its role in cytokinesis

F-actin organization during the cell cycle was investigated in two stramenopile microalgae, Ochromonas danica (Chrysophyceae; UTEX LB1298) and Heterosigma akashiwo (Raphidophyceae; NIES-6) using FITC-phalloidin. In the interphase cell of O. danica, F-actin bundles were localized forming a network structure in the cortical region, which converged from the anterior region to the posterior, whereas in the interphase cell of H. akashiwo, F-actin bundles were observed forming a network structure in the cortical region without any polarity. In both O. danica and H. akashiwo, at the initial stage of mitosis the cortical F-actin disappeared, and then during cytokinesis assembly of an actin-based ring-like structure occurred in the cell cortex in the plane of cytokinesis. The ring-like structure initiated from aster-like structures was composed of F-actin in both O. danica and H. akashiwo. Different from animal cells, later stages of cytokinesis of O. danica seemed to be promoted by microtubules, although the early stages of cytokinesis progressed with a constriction of the ring-like structure, whereas cytokinesis of H. akashiwo was apparently completed by constriction of the cell mediated by the F-actin ring, as in animal cells.     

An anillin homologue,Mid2p,acts during fission yeast cytokinesis to organize the septin ring and promote cell separation

Anillin is a conserved protein required for cell division (Field, C.M., and B.M. Alberts. 1995. J. Cell Biol. 131:165-178; Oegema, K., M.S. Savoian, T.J. Mitchison, and C.M. Field. 2000. J. Cell Biol. 150:539-552). One fission yeast homologue of anillin, Mid1p, is necessary for the proper placement of the division site within the cell (Chang, F., A. Woollard, and P. Nurse. 1996. J. Cell Sci. 109(Pt 1):131-142; Sohrmann, M., C. Fankhauser, C. Brodbeck, and V. Simanis. 1996. Genes Dev. 10:2707-2719). Here, we identify and characterize a second fission yeast anillin homologue, Mid2p, which is not orthologous with Mid1p. Mid2p localizes as a single ring in the middle of the cell after anaphase in a septin- and actin-dependent manner and splits into two rings during septation. Mid2p colocalizes with septins, and mid2 Delta cells display disorganized, diffuse septin rings and a cell separation defect similar to septin deletion strains. mid2 gene expression and protein levels fluctuate during the cell cycle in a sep1- and Skp1/Cdc53/F-box (SCF)-dependent manner, respectively, implying that Mid2p activity must be carefully regulated. Overproduction of Mid2p depolarizes cell growth and affects the organization of both the septin and actin cytoskeletons. In the presence of a nondegradable Mid2p fragment, the septin ring is stabilized and cell cycle progression is delayed. These results suggest that Mid2p influences septin ring organization at the site of cell division and its turnover might normally be required to permit septin ring disassembly.     

Dynacortin contributes to cortical viscoelasticity and helps define the shape changes of cytokinesis

During cytokinesis, global and equatorial pathways deform the cell cortex in a stereotypical manner, which leads to daughter cell separation. Equatorial forces are largely generated by myosin-II and the actin crosslinker, cortexillin-I. In contrast, global mechanics are determined by the cortical cytoskeleton, including the actin crosslinker, dynacortin. We used direct morphometric characterization and laser-tracking microrheology to quantify cortical mechanical properties of wild-type and cortexillin-I and dynacortin mutant Dictyostelium cells. Both cortexillin-I and dynacortin influence cytokinesis and interphase cortical viscoelasticity as predicted from genetics and biochemical data using purified dynacortin proteins. Our studies suggest that the regulation of cytokinesis ultimately requires modulation of proteins that control the cortical mechanical properties that establish the force-balance that specifies the shapes of cytokinesis. The combination of genetic, biochemical, and biophysical observations suggests that the cell's cortical mechanical properties control how the cortex is remodeled during cytokinesis.     

Mitosis in Giardia lamblia: multiple modes of cytokinesis

Mitosis in Giardia is poorly understood. Until today, it is still controversial whether Giardia divides with a mirror-image symmetry (ventral-ventral or dorsal-dorsal) or in a dorsal-ventral mode. Here, we report the different modes by which cytokinesis takes place in Giardia lamblia. To determine how Giardia divides, video microscopy, scanning electron microscopy, semi-thick sections and freeze-fracture replicas were analyzed by transmission electron microscopy. Between 12 and 15% of the cells cultivated for 24-48 h were found in the process of division. Three types of cytokinesis were found: (1) ventral-ventral, where the discs face each other; (2) dorsal-dorsal, where the discs are in opposite directions; and (3) ventral-dorsal. Giardia divides with mirror-image symmetry either in ventral-ventral or dorsal-dorsal modes. During ventral-ventral type of division, Giardia becomes detached and swims freely in the culture medium, whereas, in the other modes of division, the cells can be found either adhered or swimming.     

Insights into the role and structure of plant ureases

The broad distribution of ureases in leguminous seeds, as well as the accumulation pattern of the protein during seed maturation, are suggestive of an important physiological role for this enzyme. Since the isolation and characterization of jack bean urease by Sumner in 1926, many investigations have been dedicated to the structural and biological features of this enzyme; nevertheless, many questions still remain. It has been reported that ureases from plants (jack bean and soybean seeds) display biological properties unrelated to their ureolytic activity, notably a high insecticidal activity against Coleoptera (beetles) and Hemiptera (bugs), suggesting that ureases might be involved in plant defense. Besides the insecticidal activity, canatoxin, a jack bean urease isoform, causes convulsions and death in mice and rats, induces indirect hemagglutination (hemilectin activity) and promotes exocytosis in several cell types. Not only plant ureases but also some microbial ureases (found in Bacillus pasteurii and Helicobacter pylori) are able to induce activation of platelets in a process mediated by lipoxygenase-derived metabolites. This review summarizes the biological and structural properties of plant ureases, compares them with those displayed by bacterial ureases, and discusses the significance of these findings.