Septation

In fungal cells cytokinesis requires coordinated closure of a contractile actomyosin ring (CAR) and synthesis of a special cell wall structure known as the division septum. Many CAR proteins have been identified and characterized, but how these molecules interact with the septum synthesis enzymes to form the septum remains unclear. Our genetic study using fission yeast shows that cooperation between the paxillin homolog Pxl1, required for ring integrity, and Bgs1, the enzyme responsible for linear β(1,3)glucan synthesis and primary septum formation, is required for stable anchorage of the CAR to the plasma membrane before septation onset, and for cleavage furrow formation. Thus, lack of Pxl1 in combination with Bgs1 depletion, causes failure of ring contraction and lateral cell wall over-growth towards the cell lumen without septum formation. We also describe here that Pxl1 concentration at the CAR increases during cytokinesis and that this increase depends on the SH3 domain of the F-BAR protein Cdc15. In consequence, Bgs1 depletion in cells carrying a cdc15 ΔSH3 allele causes ring disassembly and septation blockage, as it does in cells lacking Pxl1. On the other hand, the absence of Pxl1 is lethal when Cdc15 function is affected, generating a large sliding of the CAR with deposition of septum wall material along the cell cortex, and suggesting additional functions for both Pxl1 and Cdc15 proteins. In conclusion, our findings indicate that CAR anchorage to the plasma membrane through Cdc15 and Pxl1, and concomitant Bgs1 activity, are necessary for CAR maintenance and septum formation in fission yeast.

Schizosaccharomyces pombe contains four putative (1,3)beta-D-glucan synthase (GS) catalytic subunits, Bgs1p-4p. In this work, we cloned bgs4+ and show that Bgs4p is the only subunit found to be a part of the GS enzyme and essential for maintaining cell integrity during cytokinesis and polarized growth. Here we show that bgs4+, cwg1+ (cwg1-1 shows reduced cell-wall beta-glucan and GS catalytic activity) and orb11+ (orb11-59 is defective in cell morphogenesis) are the same gene. bgs4+ is essential for spore germination and bgs4+ shut-off produces cell lysis at growing poles and mainly at the septum prior to cytokinesis, suggesting that Bgs4p is essential for cell wall growth and to compensate for an excess of cell wall degradation during cytokinesis. Shut-off and overexpression analysis suggest that Bgs4p forms part of a GS catalytic multiprotein complex and that Bgs4p-promoted cell-wall beta-glucan alterations induce compensatory mechanisms from other Bgs subunits and (1,3)alpha-D-glucan synthase. Physiological localization studies showed that Bgs4p localizes to the growing ends, the medial ring and septum, and at each stage of wall synthesis or remodeling that occurs during sexual differentiation: mating, zygote and spore formation, and spore germination. Bgs4p timing and requirements for proper positioning during cytokinesis and its localization pattern during spore maturation differ from those of Bgs1p. Bgs4p localizes overlapping the contractile ring once Bgs1p is present and a Calcofluor white-stained septum material is detected, suggesting that Bgs4p is involved in a late process of secondary or general septum synthesis. Unlike Bgs1p, Bgs4p needs the medial ring but not the septation initiation network proteins to localize with the other septation components. Furthermore, Bgs4p localization depends on the polarity establishment proteins. Finally, F-actin is necessary for Bgs4p delocalization from and relocalization to the growing regions, but it is not needed for the stable maintenance of Bgs4p at the growing sites, poles and septum. All these data show for the first time an essential role for a Bgs subunit in the synthesis of a (1,3)beta-D-glucan necessary to preserve cell integrity when cell wall synthesis or repair are needed.

Fungal cytokinesis requires the assembly of a dividing septum wall. In yeast, the septum has to be selectively digested during the critical cell separation process. Fission yeast cell wall α(1-3)glucan is essential, but nothing is known about its localization and function in the cell wall or about cooperation between the α- and β(1-3)glucan synthases Ags1 and Bgs for cell wall and septum assembly. Here, we generate a physiological Ags1-GFP variant and demonstrate a tight colocalization with Bgs1, suggesting a cooperation in the important early steps of septum construction. Moreover, we define the essential functions of α(1-3)glucan in septation and cell separation. We show that α(1-3)glucan is essential for both secondary septum formation and the primary septum structural strength needed to support the physical forces of the cell turgor pressure during cell separation. Consequently, the absence of Ags1 and therefore α(1-3)glucan generates a special and unique side-explosive cell separation due to an instantaneous primary septum tearing caused by the turgor pressure.

The cps5-138 fission yeast mutant shows an abnormal lemon-like morphology at 28°C in minimal medium and a lethal thermosensitive phenotype at 37°C. Cell growth is completely inhibited at 28°C in a Ca 2-free medium, in which the wild type is capable of growing normally. Under these conditions, actin patches become randomly distributed throughout the cell, and defects in septum formation and subsequent cytokinesis appear. The mutant cell is hypersensitive to the cell wall-digesting enzymatic complex Novozym234 even under permissive conditions. The gene SPBC31E1.02c, which complements all the mutant phenotypes described above, was cloned and codes for the Ca 2-ATPase homologue Pmr1p. The gene is not essential under optimal growth conditions but is required under conditions of low Ca 2 (<0.1 mM) or high temperature (>35°C). The green fluorescent protein-tagged Cps5 proteins, which are expressed under physiological conditions (an integrated single copy with its own promoter in the cps5 strain), display a localization pattern typical of endoplasmic reticulum proteins. Biochemical analyses show that 1,3-D-glucan synthase activity in the mutant is decreased to nearly half that of the wild type and that the mutant cell wall contains no detectable galactomannan when the cells are exposed to a Ca 2-free medium. The mutant acid phosphatase has an increased electrophoretic mobility, suggesting that incomplete protein glycosylation takes place in the mutant cells. These results indicate that S. pombe Pmr1p is essential for the maintenance of cell wall integrity and cytokinesis, possibly by allowing protein glycosylation and the polarized actin distribution to take place normally. Disruption and complementation analyses suggest that Pmr1p shares its function with a vacuolar Ca 2-ATPase homologue, Pmc1p (SPAPB2B4.04c), to prevent lethal activation of calcineurin for cell growth. A transient increase in the intracellular Ca 2 concentration ([Ca 2 ]) plays a key role in transmitting signals that regulate a variety of cellular functions in eukaryotes. A substantial body of knowledge has been accumulated concerning the roles of Ca 2 as a second messenger in various types of eukaryotic cells (5, 11). In yeasts, Ca 2 plays an essential role in the mating process (9, 26), and the Ca 2 calmodulin-dependent protein phosphatase calcineurin plays crucial roles in a variety of cellular functions, including ion homeostasis, cytokinesis, and transcriptional regulation (14, 55, 63). In the budding yeast Saccharomyces cerevisiae, FKS1, which codes for a putative catalytic subunit of 1,3-D-glucan synthase, is predominantly expressed under optimal growth conditions in a cell cycle-dependent manner, while the transcription of FKS2, an alternative gene for the putative glucan synthase, is completely dependent on calcineurin in the absence of a functional Fks1p and in the presence of mating pheromone or a high extracel-lular [Ca 2 ] (42, 74). In the fission yeast Schizosaccharomyces pombe, 1,3-D-and 1,3-D-glucan synthases, both of which contribute to the mechanical strength of the cell wall (12, 23, 29, 32), are presumed to be the downstream targets of Pck2p, a protein kinase C homologue (4, 8, 32). The overexpression of pck2 (OP-pck2) has been shown to increase 1,3-D-glucan synthase activity to a significant extent, as well as to induce an extremely high intracellular [Ca 2 ] (4, 8). The effects of OP-pck2 were reported to be abolished in the absence of Ehs1p, a homologue of the calcium channel component Mid1p of S. cerevisiae (9, 25). The ehs1-1 mutant displays several cell wall-related defective phenotypes, and these defects are suppressed by moderate OP-pck2 levels, suggesting that Pck2p contributes , along with the Ehs1p calcium channel, to the integrity of the cell wall (9). More recently, another component, Cta4p, a cation P 5-type ATPase that is also required for calcium ho-meostasis, has been identified (45). The null mutant displayed pleiotropic phenotypes, including defects in cytokinesis and microtubule dynamics, similar to the calcineurin null mutant phenotypes (73). These findings suggest that the regulation of intracellular [Ca 2 ] is critical to cell wall integrity, cytokinesis, and cytoskeletal organization, all of which are essential for fungal-cell morphogenesis. Transient and spatial changes in intracellular [Ca 2 ] are thus critical for generating signals that regulate cellular events or maintain ion homeostasis; the processes are mediated by Ca 2 channels, Ca 2 antiporters, and Ca 2-transporting ATPases, which are localized in the plasma or vesicle membranes to allow the transport of ions into or out of the cell or organelles via the membranes. However, at present, little is known concerning the molecular mechanisms of calcium signaling required for these cellular processes during the cell cycle. To address these issues, the molecular characterization of the mutants that show calcium-sensitive phenotypes might be useful.

Schizosaccharomyces pombe Bgs1p/Cps1p has been identified as a putative (1,3)beta-D-glucan synthase (GS) catalytic subunit with a possible function during cytokinesis and polarized growth. To study this possibility, double mutants of cps1-12 and cdc septation mutants were made. The double mutants displayed several hypersensitive phenotypes and altered actin distribution. Epistasis analysis showed mutations prior to septum synthesis were dominant over cps1-12, while cps1-12 was dominant over the end of septation mutant cdc16-116, suggesting Bgs1p is involved in septum cell-wall (1,3)beta-D-glucan synthesis at cytokinesis. We have studied the in vivo physiological localization of Bgs1p in a bgs1delta strain containing a functional GFP-bgs1(+) gene (integrated single copy and expressed under its own promoter). During vegetative growth, Bgs1p always localizes to the growing zones: one or both ends during cell growth and contractile ring and septum during cytokinesis. Bgs1p localization in cdc septation mutants indicates that Bgs1p needs the medial ring and septation initiation network (SIN) proteins to localize properly with the rest of septation components. Bgs1p localization in the actin mutant cps8-188 shows it depends on actin localization. In addition, Bgs1p remains polarized in the mislocalized growing poles and septa of tea1-1 and tea2-1 mutants. During the meiotic process of the life cycle, Bgs1p localizes to the mating projection, to the cell-to-cell contact zone during cell fusion and to the neck area during zygote formation. Also, Bgs1p localization suggests that it collaborates in forespore and spore wall synthesis. During spore germination, Bgs1p localizes first around the spore during isotropic growth, then to the zone of polarized growth and finally, to the medial ring and septum. At the end of spore-cell division, the Bgs1p displacement to the old end occurs only in the new cell. All these data show that Bgs1p is localized to the areas of polarized cell wall growth and so we propose that it might be involved in synthesizing the lineal (1,3)beta-D-glucan of the primary septum, as well as a similar lineal (1,3)beta-D-glucan when other processes of cell wall growth or repair are needed.

In animal cells, cytokinesis requires the formation of a cleavage furrow that divides the cell into two daughter cells. Furrow formation is achieved by constriction of an actomyosin ring that invaginates the plasma membrane. However, fungal cells contain a rigid extracellular cell wall surrounding the plasma membrane; thus, fungal cytokinesis also requires the formation of a special septum wall structure between the dividing cells. The septum biosynthesis must be strictly coordinated with the deposition of new plasma membrane material and actomyosin ring closure and must occur in such a way that no breach in the cell wall occurs at any time. Because of the high turgor pressure in the fungal cell, even a minor local defect might lead to cell lysis and death. Here we review our knowledge of the septum structure in the fission yeast Schizosaccharomyces pombe and of the recent advances in our understanding of the relationship between septum biosynthesis and actomyosin ring constriction and how the two collaborate to build a cross-walled septum able to support the high turgor pressure of the cell. In addition, we discuss the importance of the septum biosynthesis for the steady ingression of the cleavage furrow.

In animal cells cytokinesis relies on the contraction of an actomyosin ring that pulls the plasma membrane to create a cleavage furrow, whose ingression finally divides the mother cell into two daughter cells. Fungal cells are surrounded by a tough and flexible structure called cell wall, which is considered to be the functional equivalent of the extracellular matrix in animal cells. Therefore, in addition to cleavage furrow ingression, fungal cytokinesis also requires the centripetal formation of a septum wall structure that develops between the dividing cells, whose genesis must be strictly coordinated with both the actomyosin ring closure and plasma membrane ingression. Here we briefly review what is known about the septum structure and composition in the fission yeast Schizosaccharomyces pombe, the recent progress about the relationship between septum biosynthesis and actomyosin ring constriction, and the importance of the septum and ring in the steady progression of the cleavage furrow.

Fungal cleavage furrow formation during cytokinesis relays in the coordinated contraction of an actomyosinbased ring and the centripetal synthesis of both new plasma membrane and a special wall structure named division septum. Through transmission electron microscopy, the septum exhibits a three-layered structure
with a central primary septum, fl anked at both sides by the secondary septum. In contrast to the chitinous primary septum present in most of fungi, the fi ssion yeast Schizosaccharomyces pombe does not contain chitin, instead it divides through the formation of a linear β(1,3)glucan-rich primary septum, which has been shown to be specifi cally stained by the fl uorochrome Calcofl uor white. Recent fi ndings in S. pombe have revealed the importance of septum synthesis for the steady contraction of the ring during cytokinesis. Therefore, to study the molecular mechanisms that connect the extracellular septum wall with the other components of the cytokinetic machinery located in the plasma membrane and cytoplasm, new experimental
approaches are needed. Here we describe the methods developed to image the septum structure by fl uorescence microscopy, with a special focus in the analysis of septum progression by the use of time-lapse microscopy.

Normally, humans have only one urinary bladder but it’s not so in the case of bladder duplication because more than one bladder is present in this case and it occurs as a result of an abnormal development of the body specifically the abnormal development of the urinary system during the fourth week of embryological development. Bladder duplication was classified by Abrahamson into two forms based on the degree of duplication; complete and incomplete bladder duplication. And also, it can be classified into sagittal or coronal duplication based on the position of the accessory bladder in relation to the normal bladder.