Formation of septum-like structures at locations remote from the budding sites in cytokinesis-defective mutants of Saccharomyces cerevisiae.

Cell wall structures that partition membrane-bound portions of cytoplasm were formed at sites along the peripheral wall when a cytokinesis-defective cell division cycle mutant (cdc3) of Saccharomyces cerevisiae was grown at a restrictive temperature. The appearance of these structures, as observed in electron micrographs, was similar to that of normal septa. Aberrant septa were also detected in cytokinesis mutants harboring mutations cdc10, cdc11, and cdc12, after growth at 37 degrees C. Formation of the abnormal septa was abolished by the introduction, in a cdc3-containing strain, of additional cell cycle mutations that precluded events leading to cytokinesis and cell division. These results showed that septum formation can occur in the absence of cytokinesis. Formation of the abnormal structures was controlled by the same sequences of cell cycle events as formation of normal septa but was not subject to the spatial controls that ensure association of the septum with the budding site.     

Characterization of a cytokinesis defective (cyd1) mutant of Arabidopsis.

Although several mutations and genes affecting plant cytokinesis have been identified, mutant screens are not yet saturated and knowledge about gene function is still limited. A novel Arabidopsis mutation, cytokinesis defective1 (cyd1), was identified by partial or missing cell walls in stomata. Stomata with incomplete or no cytokinesis still differentiate and some contain swellings of the outer wall not found in the wild type. The incomplete walls are correctly placed opposite stomatal wall thickenings suggesting that the mutation interferes with the execution of cytokinesis rather than with the placement of the division site. Cytokinesis defects are also detectable in other cell types throughout the plant, defects which include cell wall protrusions, two or more nuclei in one cell, and reduced cell number. The extent of cytokinetic partitioning correlates with nuclear number in abnormal stomata. Many cyd1 epidermal cells, stomata and pollen are larger, and trichomes have more branches. cyd1 is partially lethal with poor seed set and some defective ovules, but many plants are fertile despite abnormalities in vegetative and reproductive development such as missing, reduced, fused or misshapen leaves and floral organs. cyd1 appears to be the only cytokinesis mutant described where defects are known to occur in both mature vegetative and reproductive organs. Thus, the CYD1 gene product appears to be necessary for the execution of cytokinesis throughout the shoot. The examination of stomata by microscopy may be a useful screen for the directed isolation of additional cytokinesis mutations that are not embryo or seedling lethal     

Its8, a fission yeast homolog of Mcd4 and Pig-n, is involved in GPI anchor synthesis and shares an essential function with calcineurin in cytokinesis

In fission yeast, calcineurin is required for cytokinesis and ion homeostasis; however, most of its physiological roles remain obscure. To identify genes that share an essential function with calcineurin, we screened for mutations that confer sensitivity to the calcineurin inhibitor FK506 and high temperature and isolated the mutant its8-1. its8(+) encodes a homolog of the budding yeast MCD4 and human Pig-n that are involved in glycosylphosphatidylinositol (GPI) anchor synthesis. Consistently, reduced inositol labeling of proteins suggested impaired GPI anchor synthesis in its8-1 mutants. The temperature upshift induced a further decrease in inositol labeling and caused dramatic increases in the frequency of septation in its8-1 mutants. BE49385A, an inhibitor of MCD4 and Pig-n, also increased the septation index of the wild-type cell. Osmotic stabilization suppressed these morphological defects, indicating that cell wall weakness caused by impaired GPI anchor synthesis resulted in abnormal cytokinesis. Furthermore, calcineurin-deleted cells exhibited hypersensitivity to BE49385A, and FK506 exacerbated the cytokinesis defects of the its8-1 mutant. Thus, calcineurin and Its8 may share an essential function in cytokinesis and cell viability through the regulation of cell wall integrity.     

Polyoxin D inhibits colloidal gold-wheat germ agglutinin labelling of chitin in dimorphic forms of Candida albicans

Yeasts and mycelia of the pathogen Candida albicans grown in the presence of polyoxin D, a competitive inhibitor of chitin synthase, formed chains of swollen bulbous cells as observed by fluorescence microscopy. Wheat germ agglutinin (WGA) complexed to colloidal gold (Au) was used as a specific label at the ultrastructural level to visualize chitin in walls of control and polyoxin-treated cells. In control cells, Au-WGA labelling was preferentially localized in the innermost wall layers and was predominant at bud scars and septa. After 4.5 h in 4 mM-polyoxin D, budding in yeasts and lateral wall growth in mycelia continued, but primary septa failed to form and no Au-WGA labelling was detected in the walls. These results demonstrated that the morphological alterations caused by polyoxin D were due to the absence of chitin, a wall component important for formation of primary septa and for maintenance of structural integrity during morphogenesis.     

The function of chitin synthases 2 and 3 in the Saccharomyces cerevisiae cell cycle

The morphology of three Saccharomyces cerevisiae strains, all lacking chitin synthase 1 (Chs1) and two of them deficient in either Chs3 (calR1 mutation) or Chs2 was observed by light and electron microscopy. Cells deficient in Chs2 showed clumpy growth and aberrant shape and size. Their septa were very thick; the primary septum was absent. Staining with WGA-gold complexes revealed a diffuse distribution of chitin in the septum, whereas chitin was normally located at the neck between mother cell and bud and in the wall of mother cells. Strains deficient in Chs3 exhibited minor abnormalities in budding pattern and shape. Their septa were thin and trilaminar. Staining for chitin revealed a thin line of the polysaccharide along the primary septum; no chitin was present elsewhere in the wall. Therefore, Chs2 is specific for primary septum formation, whereas Chs3 is responsible for chitin in the ring at bud emergence and in the cell wall. Chs3 is also required for chitin synthesized in the presence of alpha-pheromone or deposited in the cell wall of cdc mutants at nonpermissive temperature, and for chitosan in spore walls. Genetic evidence indicated that a mutant lacking all three chitin synthases was inviable; this was confirmed by constructing a triple mutant rescued by a plasmid carrying a CHS2 gene under control of a GAL1 promoter. Transfer of the mutant from galactose to glucose resulted in cell division arrest followed by cell death. We conclude that some chitin synthesis is essential for viability of yeast cells.     

Individual chitin synthase enzymes synthesize microfibrils of differing structure at specific locations in the Candida albicans cell wall

The shape and integrity of fungal cells is dependent on the skeletal polysaccharides in their cell walls of which beta(1,3)-glucan and chitin are of principle importance. The human pathogenic fungus Candida albicans has four genes, CHS1, CHS2, CHS3 and CHS8, which encode chitin synthase isoenzymes with different biochemical properties and physiological functions. Analysis of the morphology of chitin in cell wall ghosts revealed two distinct forms of chitin microfibrils: short microcrystalline rodlets that comprised the bulk of the cell wall; and a network of longer interlaced microfibrils in the bud scars and primary septa. Analysis of chitin ghosts of chs mutant strains by shadow-cast transmission electron microscopy showed that the long-chitin microfibrils were absent in chs8 mutants and the short-chitin rodlets were absent in chs3 mutants. The inferred site of chitin microfibril synthesis of these Chs enzymes was corroborated by their localization determined in Chsp-YFP-expressing strains. These results suggest that Chs8p synthesizes the long-chitin microfibrils, and Chs3p synthesizes the short-chitin rodlets at the same cellular location. Therefore the architecture of the chitin skeleton of C. albicans is shaped by the action of more than one chitin synthase at the site of cell wall synthesis.     

Spm1, a stress-activated MAP kinase that regulates morphogenesis in S.pombe.

A gene encoding a novel MAP kinase family member, Spm1, was isolated from the fission yeast Schizosaccharomyces pombe. Overproduction of Spm1 inhibits proliferation. Disruption of the spm1+ gene interferes with cell separation and morphogenesis. Under conditions of nutrient limitation, hypertonic stress or elevated temperature, spm1 delta cells grow as short branched filaments in which the cell walls and septa are thickened, suggesting defects in polarized growth and cell wall remodeling. At high osmolarity, spm1 delta cells fail to form colonies. The Spm1 protein is tyrosine phosphorylated and activated in response to osmotic and heat stress, consistent with a role for Spm1 in adaptation to these conditions. Two other S.pombe MAP kinases are known, Spk1, required for sexual differentiation and sporulation, and Spc1/Sty1/Phh1, which is activated in hypertonic conditions. However, the distinctive features of the spm1 delta mutant phenotype and direct biochemical assays suggest that Spm1 does not lie on other known MAP kinase pathways. Our results demonstrate the existence of a new MAP kinase pathway that regulates cell wall remodeling and cytokinesis in response to environmental stresses.     

KORRIGAN, an Arabidopsis endo-1,4-beta-glucanase, localizes to the cell plate by polarized targeting and is essential for cytokinesis

The formation of the cell plate, a unique structure in dividing plant cells, is pivotal for cytokinesis. A mutation in the Arabidopsis KORRIGAN (KOR) gene causes the formation of aberrant cell plates, incomplete cell walls, and multinucleated cells, leading to severely abnormal seedling morphology. The mutant, designed kor1-2, was identified as a stronger allele than the previously identified kor1-1, which appears to be defective only in cell elongation. KOR1 encodes an endo-1,4-beta-d-glucanase with a transmembrane domain and two putative polarized targeting signals in the cytosolic tail. When expressed in tobacco BY2 cells, a KOR1-GFP (green fluorescence protein) fusion protein was localized to growing cell plates. Substitution mutations in the polarized targeting motifs of KOR1 caused the fusion proteins to localize to the plasma membrane as well. Expression of these mutant genes in kor1-2 plants complemented only the cell elongation defect but not the cytokinesis defect, indicating that polarized targeting of KOR1 to forming cell plates is essential for cytokinesis. Our results suggest that KOR1 plays a critical role during cytokinesis.     

Cytokinesis-defective mutants of Arabidopsis

We have identified mutations in six previously uncharacterized genes of Arabidopsis, named club, bublina, massue, rod, bloated, and bims, that are required for cytokinesis. The mutants are seedling lethal, have morphological abnormalities, and are characterized by cell wall stubs, gapped walls, and multinucleate cells. In these and other respects, the new mutants are phenotypically similar to knolle, keule, hinkel, and pleiade mutants. The mutants display a gradient of stomatal phenotypes, correlating roughly with the severity of their cytokinesis defect. Similarly, the extent to which the different mutant lines were capable of growing in tissue culture correlated well with the severity of the cytokinesis defect. Phenotypic analysis of the novel and previously characterized loci indicated that the secondary consequences of a primary defect in cytokinesis include anomalies in body organization, organ number, and cellular differentiation, as well as organ fusions and perturbations of the nuclear cycle. Two of the 10 loci are required for both cytokinesis and root hair morphogenesis. The results have implications for the identification of novel cytokinesis genes and highlight the mechanistic similarity between cytokinesis and root hair morphogenesis, two processes that result in a rapid deposition of new cell walls via polarized secretion.     

The Arabidopsis KNOLLE and KEULE genes interact to promote vesicle fusion during cytokinesis

Partitioning of the cytoplasm during cytokinesis or cellularisation requires syntaxin-mediated membrane fusion [1-3]. Whereas in animals, membrane fusion promotes ingression of a cleavage furrow from the plasma membrane [4,5], somatic cells of higher plants form de novo a transient membrane compartment, the cell plate, which is initiated in the centre of the division plane and matures into a new cell wall and its flanking plasma membranes [6,7]. Cell plate formation results from the fusion of Golgi-derived vesicles delivered by a dynamic cytoskeletal array, the phragmoplast. Mutations in two Arabidopsis genes, KNOLLE (KN) and KEULE (KEU), cause abnormal seedlings with multinucleate cells and incomplete cell walls [1,8]. The KN gene encodes a cytokinesis-specific syntaxin which localises to the cell plate [9]. Here, we show that KN protein localisation is unaffected in keu mutant cells, which, like kn, display phragmoplast microtubules and accumulate ADL1 protein in the plane of cell division but vesicles fail to fuse with one another. Genetic interactions between KN and KEU were analysed in double mutant embryos. Whereas the haploid gametophytes gave rise to functional gametes, the embryos behaved like single cells displaying multiple, synchronously cycling nuclei, cell cycle-dependent microtubule arrays and ADL1 accumulation between pairs of daughter nuclei. This complete inhibition of cytokinesis from fertilisation indicates that KN and KEU, have partially redundant functions and interact specifically in vesicle fusion during cytokinesis of somatic cells.     

Yapsins are a family of aspartyl proteases required for cell wall integrity in Saccharomyces cerevisiae

The yeast cell wall is a crucial extracellular organelle that protects the cell from lysis during environmental stress and morphogenesis. Here, we demonstrate that the yapsin family of five glycosylphosphatidylinositol-linked aspartyl proteases is required for cell wall integrity in Saccharomyces cerevisiae. Yapsin null mutants show hypersensitivity to cell wall perturbation, and both the yps1Delta2Delta mutant and the quintuple yapsin mutant (5ypsDelta) undergo osmoremedial cell lysis at 37 degrees C. The cell walls of both 5ypsDelta and yps1Delta2Delta mutants have decreased amounts of 1,3- and 1,6-beta-glucan. Although there is decreased incorporation of both 1,3- and 1,6-beta-glucan in the 5ypsDelta mutant in vivo, in vitro specific activity of both 1,3- and 1,6-beta-glucan synthesis is similar to wild type, indicating that the yapsins affect processes downstream of glucan synthesis and that the yapsins may be involved in the incorporation or retention of cell wall glucan. Presumably as a response to the significant alterations in cell wall composition, the cell wall integrity mitogen-activated kinase signaling cascade (PKC1-MPK pathway) is basally active in 5ypsDelta. YPS1 expression is induced during cell wall stress and remodeling in a PKC1-MPK1-dependent manner, indicating that Yps1p is a direct, and important, output of the cell wall integrity response. The Candida albicans (SAP9) and Candida glabrata (CgYPS1) homologues of YPS1 complement the phenotypes of the yps1Delta mutant. Taken together, these data indicate that the yapsins play an important role in glucan homeostasis in S. cerevisiae and that yapsin homologues may play a similar role in the pathogenic yeasts C. albicans and C. glabrata.     

Lipid droplets maintain lipid homeostasis during anaphase for efficient cell separation in budding yeast

The neutral lipids steryl ester and triacylglycerol (TAG) are stored in the membrane-bound organelle lipid droplet (LD) in essentially all eukaryotic cells. It is unclear what physiological conditions require the mobilization or storage of these lipids. Here, we study the budding yeast mutant are1Δ are2Δ dga1Δ lro1Δ, which cannot synthesize the neutral lipids and therefore lacks LDs. This quadruple mutant is delayed at cell separation upon release from mitotic arrest. The cells have abnormal septa, unstable septin assembly during cytokinesis, and prolonged exocytosis at the division site at the end of cytokinesis. Lipidomic analysis shows a marked increase of diacylglycerol (DAG) and phosphatidic acid, the precursors for TAG, in the mutant during mitotic exit. The cytokinesis and separation defects are rescued by adding phospholipid precursors or inhibiting fatty acid synthesis, which both reduce DAG levels. Our results suggest that converting excess lipids to neutral lipids for storage during mitotic exit is important for proper execution of cytokinesis and efficient cell separation.     

Division-site selection, cell separation, and formation of anucleate minicells inSchizosaccharomyces pombe mutants resistant to cell-wall lytic enzymes

Summary In most eukaryotic organisms that have cell walls, cell separation or cytokinesis is a degradative enzymatic process. In the fission yeastSchizosaccharomyces pombe, it is a post-M-phase event that includes the degradation of part of the cell wall and the primary septum. We describe the isolation of mutants partially defective in cytokinesis by enrichment of clones resistant to cell-wall lytic enzymes. The mutations confer mycelial morphology (chains of non-separated cells) and define four genes.Sep2-SA2 was subjected to detailed genetic and cytological analysis. Its cells frequently form complex septa composed of multiple layers, which appear as twin septa separated by anucleate minicells if the cell length is extended. This suggests that a polar signal-like mechanism may also operate inS. pombe during division-site selection andsep2 ⁺ takes part in it.Sep2 ⁺ seems to be involved in several cell cycle functions because its mutation can transiently block cell-cycle progression after nuclear division and provoke a transition from haploidy to diploidy in the double mutantsep2-SA2 cexl-SA2. Cexl-SA2 is another novel mutation which causes cell-length extension.Abbreviations DAPI 4,6-diamidino-2-phenylindole - YEA yeast extract agar - YEL yeast extract liquid - SMA synthetic minimal agar - MEA malt extract agar     

Complete glycosylphosphatidylinositol anchors are required in Candida albicans for full morphogenesis, virulence and resistance to macrophages

Glycosylphosphatidylinositol (GPI)-anchored proteins are involved in cell wall integrity and cell-cell interactions. We disrupted the Candida albicans homologue of the Saccharomyces cerevisiae GPI7/LAS21 gene, which encodes a GPI anchor-modifying activity. In the mutant and on solid media, the yeast-to-hyphae transition was blocked, whereas chlamydospore formation was enhanced. However, the morphogenetic switch was normal in liquid medium. Abnormal budding patterns, cytokinesis and cell shape were observed in both liquid and solid media. The cell wall structure was also modified in the mutants, as shown by hypersensitivity to Calcofluor white. In vitro and in vivo assays revealed that the mutant interacted with its host in a modified way, resulting in reduced virulence in mice and reduced survival in the gastrointestinal environment of mice. The mitogen-activated protein (MAP) kinase pathway of macrophages was downregulated by the wild-type cells but not by the DeltaCagpi7 null strains. In agreement with this abnormal behaviour, mutant cells were more sensitive to the lytic action of macrophages. Our results indicate that a functional GPI anchor is required for full hyphal formation in C. albicans, and that perturbation of the GPI biosynthesis results in hypersensitivity to host defences.     

Multiplication of the dictyosome during the formation of autospores in the green alga Chlorococcum infusionum

Numerical changes in dictyosomes during the formation of autospores in a green alga, Chlorococcum infusionum, were investigated by electron microscopy. Two dictyosomes were seen near the nucleus in young vegetative cells. Four dictyosomes were seen in large mononucleate cells which appeared to enter mitosis soon. Binucleate cells contained 4 or 8 dictyosomes, the latter number being found in the large binucleate cells. Large tetranucleate cells contained 16-25 dictyosomes in each cell. Dictyosomes consisted of about 4 cisternae with a diameter of 0.4-0.6 micron in mono- to tetranucleate cells. Cytokinesis began with the formation of the septa during the third nuclear division, and 16 cells were finally formed. Dictyosomes did not increase in number in 8- and 16-nucleate cells. In septum-forming cells, dictyosomes were 0.6-1.0 microns in diameter, with 6-9 cisternae. A single dictyosome was included in each of the 16 resultant cells. These observations suggest that the dictyosomes multiply in association with the multiplication of the nuclei without correlation with formation of the cell wall or cytokinesis.     

Cell wall-DNA association in Bacillus subtilis.

Autolysis of cell walls of Bacillus subtilis 168 resulted in solubilization of wall-associated DNA. Most of the DNA was solubilized only in the later stages of autolysis. Solubilization of up to 70% of the wall by autolysins resulted in only 25 to 30% solubilization of wall-associated DNA. When the wall fragments remaining after 70% autolysis were analyzed by electron microscopy, it was observed that the preparations were highly enriched for completed septa, or poles. Partial autolysis at pH 5.2 or pH 8.6, both of which reflect hydrogen ion levels that permit either N-acetylglucosaminidase or N-acetylmuramyl-L-alanine amidase, but not both, to act, gave rise to enrichment of cell poles. When walls were incubated with subtilisin, DNase, or RNase, release of DNA (or DNA fragments) was accelerated. Density gradient centrifugation patterns of lysates of cells pulse-labeled with N-[3H]acetylglucosamine and then chased revealed that a small, but significant, proportion of the radioactivity sedimented to a density position equivalent to that of DNA-membrane complexes. Because the pulse-chase sequence enriched for radioactivity in cell poles, the results suggest that at least some molecules from polar cell walls have an affinity for DNA-membrane complexes. We suggest that DNA binds strongly, possibly via a DNA-membrane complex, to cell poles of B. subtilis. The results provide support for a view offered previously (Koch et al., FEMS Microbiol. Lett. 12:201-208, 1981) that some special structure in or very near the poles of gram-positive bacilli is involved in the segregation of DNA during cell division.     

cyd,a mutant of pea that alters embryo morphology is defective in cytokinesis

Embryogenesis in higher plants requires the precise regulation of cell division, orientation of cell elongation and specification of cell differentiation. The division plane is determined by the position of a new cell plate at cytokinesis. A mutant of pea has been isolated in which both the embryo pattern and surface morphology is altered. The phenotype of the mutant is manifest primarily in the cotyledons where cell plates only partially form, generating cell wall stubs and multinucleate cells. Some cotyledonary cells of the mutant proceed through nine DNA replication cycles, including nuclear division, but not cytokinesis, producing nuclei with a DNA content of ca. 1000C. The cytological phenotype of the mutant could be mimicked by the treatment of wild-type cells with caffeine. We have termed this mutant cytokinesis-defective (cyd). © 1995 Wiley-Liss, Inc.     

Class I and class II chitin synthases are involved in septum formation in the filamentous fungus Aspergillus nidulans

The class II and class I chitin synthases of the filamentous fungus Aspergillus nidulans are encoded by chsA and chsC, respectively. Previously, we presented several lines of evidence suggesting that ChsA and ChsC have overlapping functions in maintaining cell wall integrity. In order to determine the functions of these chitin synthases, we employed electron and fluorescence microscopy and investigated in detail the cell wall of a DeltachsA DeltachsC double mutant (DeltaAC mutant) along with the localization of ChsA and ChsC. In the lateral cell wall of the DeltaAC mutant, electron-transparent regions were thickened. Septa of the DeltaAC mutant were aberrantly thick and had a large pore. Some septa were located abnormally close to adjacent septa. A functional hemagglutinin (HA)-tagged ChsA (HA-ChsA) and a functional FLAG-tagged ChsC (FLAG-ChsC) were each localized to a subset of septation sites. Comparison with the localization pattern of actin, which is known to localize at forming septa, suggested that ChsA and ChsC transiently exist at the septation sites during and shortly after septum formation. Double staining of HA-ChsA and FLAG-ChsC indicated that their localizations were not identical but partly overlapped at the septation sites. Fluorescence of FLAG-ChsC, but not of HA-ChsA, was also observed at hyphal tips. These data indicate that ChsA and ChsC share overlapping roles in septum formation.     

Isolation, Properties, Function, and Regulation of Endo-(1 → 3)-β-Glucanases in Schizosaccharomyces pombe

Cell-free extracts, membranous fractions, and cell wall preparations from Schizosaccharomyces pombe were examined for the presence of (1 → 3)-β-, (1 → 3)-α-, and (1 → 6)-β-glucanase activities. The various glucanases were assayed in cells at different growth stages. Only (1 → 3)-β-glucanase activity was found, and this was associated with the cell wall fraction. Chromatographic fractionation of the crude enzyme revealed two endo-(1 → 3)-β-glucanases, designated as glucanase I and glucanase II. Glucanase I consisted of two subunits of molecular weights 78,500 and 82,000, and glucanase II was a single polypeptide of 75,000. Although both enzymes had similar substrate specificities and similar hydrolytic action on laminarin, glucanase II had much higher hydrolytic activity on isolated cell walls of S. pombe. On the basis of differential lytic activity on cell walls, glucanase II was shown to be present in conjugating cells and highest in sporulating cells. Glucanase II appeared to be specifically involved in conjugation and sporulation since vegetative cells and nonconjugating and nonsporulating cells did not contain this enzyme. The appearance of glucanase II in conjugating cells may be due to de novo enzyme synthesis since no activation could be demonstrated by combining extracts from vegetative and conjugating cells. Increased glucanase activity occurred when walls from conjugating cells were combined with walls from sporulating cells. Studies with trypsin and proteolytic inhibitors suggest that glucanase II exists as a zymogen in conjugating cells. A temperature-sensitive mutant of S. pombe was isolated which lysed at 37°C. Glucanase activity was higher in vegetative cells held at 37°C than cells held at 25°C. Unlike the wild-type strain, this mutant contained glucanase II activity during vegetative growth and may be a regulatory mutant.