Coordinating septum formation and the actomyosin ring during cytokinesis in Schizosaccharomyces pombe

During cytokinesis, animal and fungal cells form a membrane furrow via actomyosin ring constriction. Our understanding of actomyosin ring‐driven cytokinesis stems extensively from the fission yeast model system. However, unlike animal cells, actomyosin ring constriction occurs simultaneously with septum formation in fungi. While the formation of an actomyosin ring is essential for cytokinesis in fission yeast, proper furrow formation also requires septum deposition. The molecular mechanisms of spatiotemporal coordination of septum deposition with actomyosin ring constriction are poorly understood. Although the role of the actomyosin ring as a mechanical structure driving furrow formation is better understood, its role as a spatiotemporal landmark for septum deposition is not widely discussed. Here we review and discuss the recent advances describing how the actomyosin ring spatiotemporally regulates membrane traffic to promote septum‐driven cytokinesis in fission yeast. Finally, we explore emerging questions in cytokinesis, and discuss the role of extracellular matrix during cytokinesis in other organisms.     

Roles of Hof1p, Bni1p, Bnr1p, and myo1p in cytokinesis in Saccharomyces cerevisiae

Cytokinesis in Saccharomyces cerevisiae occurs by the concerted action of the actomyosin system and septum formation. Here we report on the roles of HOF1, BNI1, and BNR1 in cytokinesis, focusing on Hof1p. Deletion of HOF1 causes a temperature-sensitive defect in septum formation. A Hof1p ring forms on the mother side of the bud neck in G2/M, followed by the formation of a daughter-side ring. Around telophase, Hof1p is phosphorylated and the double rings merge into a single ring that contracts slightly and may colocalize with the actomyosin structure. Upon septum formation, Hof1p splits into two rings, disappearing upon cell separation. Hof1p localization is dependent on septins but not Myo1p. Synthetic lethality suggests that Bni1p and Myo1p belong to one functional pathway, whereas Hof1p and Bnr1p belong to another. These results suggest that Hof1p may function as an adapter linking the primary septum synthesis machinery to the actomyosin system. The formation of the actomyosin ring is not affected by bni1Delta, hof1Delta, or bnr1Delta. However, Myo1p contraction is affected by bni1Delta but not by hof1Delta or bnr1Delta. In bni1Delta cells that lack the actomyosin contraction, septum formation is often slow and asymmetric, suggesting that actomyosin contraction may provide directionality for efficient septum formation.     

Extracellular cell wall β(1,3)glucan is required to couple septation to actomyosin ring contraction

Cytokinesis has been extensively studied in different models, but the role of the extracellular cell wall is less understood. Here we studied this process in fission yeast. The essential protein Bgs4 synthesizes the main cell wall β(1,3)glucan. We show that Bgs4-derived β(1,3)glucan is required for correct and stable actomyosin ring positioning in the cell middle, before the start of septum formation and anchorage to the cell wall. Consequently, β(1,3)glucan loss generated ring sliding, oblique positioned rings and septa, misdirected septum synthesis indicative of relaxed rings, and uncoupling between a fast ring and membrane ingression and slow septum synthesis, suggesting that cytokinesis can progress with defective septum pushing and/or ring pulling forces. Moreover, Bgs4-derived β(1,3)glucan is essential for secondary septum formation and correct primary septum completion. Therefore, our results show that extracellular β(1,3)glucan is required for cytokinesis to connect the cell wall with the plasma membrane and for contractile ring function, as proposed for the equivalent extracellular matrix in animal cells.     

Involvement of an Actomyosin Contractile Ring in Saccharomyces cerevisiae Cytokinesis

In Saccharomyces cerevisiae, the mother cell and bud are connected by a narrow neck. The mechanism by which this neck is closed during cytokinesis has been unclear. Here we report on the role of a contractile actomyosin ring in this process. Myo1p (the only type II myosin in S. cerevisiae) forms a ring at the presumptive bud site shortly before bud emergence. Myo1p ring formation depends on the septins but not on F-actin, and preexisting Myo1p rings are stable when F-actin is depolymerized. The Myo1p ring remains in the mother–bud neck until the end of anaphase, when a ring of F-actin forms in association with it. The actomyosin ring then contracts to a point and disappears. In the absence of F-actin, the Myo1p ring does not contract. After ring contraction, cortical actin patches congregate at the mother–bud neck, and septum formation and cell separation rapidly ensue. Strains deleted for MYO1 are viable; they fail to form the actin ring but show apparently normal congregation of actin patches at the neck. Some myo1Δ strains divide nearly as efficiently as wild type; other myo1Δ strains divide less efficiently, but it is unclear whether the primary defect is in cytokinesis, septum formation, or cell separation. Even cells lacking F-actin can divide, although in this case division is considerably delayed. Thus, the contractile actomyosin ring is not essential for cytokinesis in S. cerevisiae. In its absence, cytokinesis can still be completed by a process (possibly localized cell–wall synthesis leading to septum formation) that appears to require septin function and to be facilitated by F-actin.     

Chitin synthase III activity,but not the chitin ring,is required for remedial septa formation in budding yeast

Chitin is a minor but essential component of the Saccharomyces cerevisiae cell wall. In wild-type, chitin synthase II is required for the formation of primary septa and chitin synthase III (CSIII) is not essential. However, in chs2 mutants CSIII becomes essential for the formation of aberrant septa. We examined which of two CSIII functions, the formation of a chitin ring at bud emergence or of chitin in the remedial septa, was required for viability. By using cell cycle synchronization in combination with nikkomycin Z, a specific inhibitor of CSIII, we inhibited chitin synthesis in a chs2 mutant, during formation of either the ring or the remedial septa. The results show that only synthesis of the chitin during aberrant septa formation is essential for viability. Thus, the unique function of the chitin ring seems to be maintenance of the integrity of the mother-bud neck, as we recently found, and the importance of chitin in septum closure, both in normal and abnormal situations, is underlined.     

A Highlights from MBoC Selection: Inhibition of cell membrane ingression at the division site by cell walls in fission yeast

Eukaryotic cells assemble actomyosin rings during cytokinesis to function as force-generating machines to drive membrane invagination and to counteract the intracellular pressure and the cell surface tension. How the extracellular matrix affects actomyosin ring contraction has not been fully explored. While studying the Schizosaccharomyces pombe 1,3-β-glucan-synthase mutant cps1-191, which is defective in division septum synthesis and arrests with a stable actomyosin ring, we found that weakening of the extracellular glycan matrix caused the generated spheroplasts to divide under the nonpermissive condition. This nonmedial slow division was dependent on a functional actomyosin ring and vesicular trafficking, but independent of normal septum synthesis. Interestingly, the high intracellular turgor pressure appears to play a minimal role in inhibiting ring contraction in the absence of cell wall remodeling in cps1-191 mutants, as decreasing the turgor pressure alone did not enable spheroplast division. We propose that during cytokinesis, the extracellular glycan matrix restricts actomyosin ring contraction and membrane ingression, and remodeling of the extracellular components through division septum synthesis relieves the inhibition and facilitates actomyosin ring contraction.     

The localization of the integral membrane protein Cps1p to the cell division site is dependent on the actomyosin ring and the septation-inducing network in Schizosaccharomyces pombe

Schizosaccharomyces pombe cells divide by medial fission through the use of an actomyosin-based contractile ring. Constriction of the actomyosin ring is accompanied by the centripetal addition of new membranes and cell wall material. In this article, we characterize the mechanism responsible for the localization of Cps1p, a septum-synthesizing 1,3-beta-glucan synthase, to the division site during cytokinesis. We show that Cps1p is an integral membrane protein that localizes to the cell division site late in anaphase. Neither F-actin nor microtubules are essential for the initial assembly of Cps1p to the medial division site. F-actin, but not microtubules, is however important for the eventual incorporation of Cps1p into the actomyosin ring. Assembly of Cps1p into the cell division ring is also dependent on the septation-inducing network (SIN) proteins that regulate division septum formation after assembly of the actomyosin ring. Fluorescence-recovery after-photobleaching experiments reveal that Cps1p does not diffuse appreciably within the plasma membrane and is retained at the division site by a mechanism that does not depend on an intact F-actin cytoskeleton. We conclude that the actomyosin ring serves as a spatial cue for Cps1p localization, whereas the maintenance of Cps1p at the division site occurs by a novel F-actin- and microtubule-independent mechanism. Furthermore, we propose that the SIN proteins ensure localization of Cps1p at the appropriate point in the cell cycle.     

The multiprotein exocyst complex is essential for cell separation in Schizosaccharomyces pombe

Schizosaccharomyces pombe cells divide by medial fission through the use of an actomyosin-based contractile ring. A mulitlayered division septum is assembled in concert with ring constriction. Finally, cleavage of the inner layer of the division septum results in the liberation of daughter cells. Although numerous studies have focused on actomyosin ring and division septum assembly, little information is available on the mechanism of cell separation. Here we describe a mutant, sec8-1, that is defective in cell separation but not in other aspects of cytokinesis. sec8-1 mutants accumulate about 100-nm vesicles and have reduced secretion of acid phosphatase, suggesting that they are defective in exocytosis. Sec8p is a component of the exocyst complex. Using biochemical methods, we show that Sec8p physically interacts with other members of the exocyst complex, including Sec6p, Sec10p, and Exo70p. These exocyst proteins localize to regions of active exocytosis-at the growing ends of interphase cells and in the medial region of cells undergoing cytokinesis-in an F-actin-dependent and exocytosis-independent manner. Analysis of a number of mutations in various exocyst components has established that these components are essential for cell viability. Interestingly, all exocyst mutants analyzed appear to be able to elongate and to assemble division septa but are defective for cell separation. We therefore propose that the fission yeast exocyst is involved in targeting of enzymes responsible for septum cleavage. We further propose that cell elongation and division septum assembly can continue with minimal levels of exocyst function.     

The Saccharomyces cerevisiae actin cytoskeletal component Bsp1p has an auxiliary role in actomyosin ring function and in the maintenance of bud-neck structure

Iqg1p is a component of the actomyosin contractile ring that is required for actin recruitment and septum deposition. Cells lacking Iqg1p function have an altered bud-neck structure and fail to form a functional actomyosin contractile ring resulting in a block to cytokinesis and septation. Here it is demonstrated that increased expression of the actin cytoskeleton associated protein Bsp1p bypasses the requirement for contractile ring function. This also correlates with reduced bud-neck width and remedial septum formation. Increased expression of this protein in a temperature-sensitive iqg1-1 background causes remedial septum formation at the bud neck that is reliant upon chitin synthase III activity and restores cell separation. The observed suppression correlates with a restoration of normal bud-neck structure. While Bsp1p is a component of the contractile ring, its recruitment to the bud neck is not required for the observed suppression. Loss of Bsp1p causes a brief delay in the redistribution of the actin cytoskeleton normally observed at the end of actin ring contraction. Compromise of Iqg1p function, in the absence of Bsp1p function, leads to a profound change in the distribution of actin and the pattern of cell growth accompanied by a failure to complete cytokinesis and cell separation.     

Saccharomyces cerevisiae Dma proteins participate in cytokinesis by controlling two different pathways

Cytokinesis completion in the budding yeast S. cerevisiae is driven by tightly regulated pathways, leading to actomyosin ring contraction coupled to plasma membrane constriction and to centripetal growth of the primary septum, respectively. These pathways can partially substitute for each other, but their concomitant inactivation leads to cytokinesis block and cell death. Here we show that both the lack of the functionally redundant FHA-RING ubiquitin ligases Dma1 and Dma2 and moderate Dma2 overproduction affect actomyosin ring contraction as well as primary septum deposition, although they do not apparently alter cell cycle progression of otherwise wild-type cells. In addition, overproduction of Dma2 impairs the interaction between Tem1 and Iqg1, which is thought to be required for AMR contraction, and causes asymmetric primary septum deposition as well as mislocalization of the Cyk3-positive regulator of this process. In agreement with these multiple inhibitory effects, a Dma2 excess that does not cause any apparent defect in wild-type cells leads to lethal cytokinesis block in cells lacking the Hof1 protein, which is essential for primary septum formation in the absence of Cyk3. Altogether, these findings suggest that the Dma proteins act as negative regulators of cytokinesis.     

The use of morphomutants to investigate septum formation and cell separation in Schizosaccharomyces pombe

Cytokinesis in the fission yeast Schizosaccharomyces begins by formation of a medially placed actomyosin ring, continues by progressive development of a septum, and is completed by cleavage of the mature septum to bring about cell separation. The cytological analysis of the Schizosaccharomyces pombe morphomutants sph2-3 and sep1-1 presented here demonstrates that the medial actomyosin ring colocalizes to the leading edge of the centripetally growing septum, and cleavage of the septum is triggered by rupture of the mother cell wall at the septal basis.     

The (1,3)beta-D-glucan synthase subunit Bgs1p is responsible for the fission yeast primary septum formation

Cytokinesis is a crucial event in the cell cycle of all living cells. In fungal cells, it requires co-ordinated contraction of an actomyosin ring and synthesis of both plasmatic membrane and a septum structure that will constitute the new cell wall end. Schizosaccharomyces pombe contains four essential putative (1,3)beta-d-glucan synthase catalytic subunits, Bgs1p to Bgs4p. Here we examined the function of Bgs1p in septation by studying the lethal phenotypes of bgs1(+) shut-off and bgs1Delta cells and demonstrated that Bgs1p is responsible and essential for linear (1,3)beta-d-glucan and primary septum formation. bgs1(+) shut-off generates a more than 300-fold Bgs1p reduction, but the septa still present large amounts of disorganized linear (1,3)beta-d-glucan and partial primary septa. Conversely, both structures are absent in bgs1Delta cells, where there is no Bgs1p. The septum analysis of bgs1(+)-repressed cells indicates that linear (1,3)beta-d-glucan is necessary but not sufficient for primary septum formation. Linear (1,3)beta-d-glucan is the polysaccharide that specifically interacts with the fluorochrome Calcofluor white in fission yeast. We also show that in the absence of Bgs1p abnormal septa are formed, but the cells cannot separate and eventually die.     

The Budding Yeast Amphiphysin Complex Is Required for Contractile Actin Ring (CAR) Assembly and Post-Contraction GEF-Independent Accumulation of Rho1-GTP

The late events of the budding yeast cell division cycle, cytokinesis and cell separation, require the assembly of a contractile actomyosin ring (CAR), primary and secondary septum formation followed by enzymatic degradation of the primary septum. Here we present evidence that demonstrates a role for the budding yeast amphiphysin complex, a heterodimer comprising Rvs167 and Rvs161, in CAR assembly and cell separation. The iqg1-1 allele is synthetically lethal with both rvs167 and rvs161 null mutations. We show that both Iqg1 and the amphiphysin complex are required for CAR assembly in early anaphase but cells are able to complete assembly in late anaphase when these activities are, respectively, either compromised or absent. Amphiphysin dependent CAR assembly is dependent upon the Rvs167 SH3 domain, but this function is insufficient to explain the observed synthetic lethality. Dosage suppression of the iqg1-1 allele demonstrates that endocytosis is required for the default cell separation pathway in the absence of CAR contraction but is unlikely to be required to maintain viability. The amphiphysin complex is required for normal, post-mitotic, localization of Chs3 and the Rho1 GEF, Rom2, which are responsible for secondary septum deposition and the accumulation of GTP bound Rho1 at the bud neck. It is concluded that a failure of polarity establishment in the absence of CAR contraction and amphiphysin function leads to loss of viability as a result of the consequent cell separation defect.     

Drc1p/Cps1p, a 1,3-beta-glucan synthase subunit, is essential for division septum assembly in Schizosaccharomyces pombe.

Schizosaccharomyces pombe divides by medial fission through the use of an actomyosin-based contractile ring. A division septum is formed centripetally, concomitant with ring constriction. Although several genes essential for cytokinesis have been described previously, enzymes that participate in the assembly of the division septum have not been identified. Here we describe a temperature-sensitive mutation, drc1-191, that prevents division septum assembly and causes mutant cells to arrest with a stable actomyosin ring. Unlike the previously characterized cytokinesis mutants, which undergo multiple mitotic cycles, drc1-191 is the first cytokinesis mutant that arrests with two interphase nuclei. Interestingly, unlike drc1-191, drc1-null mutants proceed through multiple mitotic cycles, leading to the formation of large cells with many nuclei. drc1 is allelic to cps1, which encodes a 1,3-beta-glucan synthase subunit. We conclude that Drc1p/Cps1p is not required for cell elongation and cell growth, but plays an essential role in assembly of the division septum. Furthermore, it appears that constriction of the actomyosin ring might depend on assembly of the division septum. We discuss possible mechanisms that account for the differences in the phenotypes of the drc1-191 and the drc1-null mutants and also reflect the potential links between Drc1p and other cytokinesis regulators.     

Cell cycle-dependent roles for the FCH-domain protein Cdc15p in formation of the actomyosin ring in Schizosaccharomyces pombe

Cell division in the fission yeast Schizosaccharomyces pombe requires the formation and constriction of an actomyosin ring at the division site. The actomyosin ring is assembled in metaphase and anaphase A, is maintained throughout mitosis, and constricts after completion of anaphase. Maintenance of the actomyosin ring during late stages of mitosis depends on the septation initiation network (SIN), a signaling cascade that also regulates the deposition of the division septum. However, SIN is not active in metaphase and is not required for the initial assembly of the actomyosin ring early in mitosis. The FER/CIP4-homology (FCH) domain protein Cdc15p is a component of the actomyosin ring. Mutations in cdc15 lead to failure in cytokinesis and result in the formation of elongated, multinucleate cells without a division septum. Here we present evidence that the requirement of Cdc15p for actomyosin ring formation is dependent on the stage of mitosis. Although cdc15 mutants are competent to assemble actomyosin rings in metaphase, they are unable to maintain actomyosin rings late in mitosis when SIN is active. In the absence of functional Cdc15p, ring formation upon metaphase arrest depends on the anillin-like Mid1p. Interestingly, when cytokinesis is delayed due to perturbations to the division machinery, Cdc15p is maintained in a hypophosphorylated form. The dephosphorylation of Cdc15p, which occurs transiently in unperturbed cytokinesis, is partially dependent on the phosphatase Clp1p/Flp1p. This suggests a mechanism where both SIN and Clp1p/Flp1p contribute to maintenance of the actomyosin ring in late mitosis through Cdc15p, possibly by regulating its phosphorylation status.     

Separate roles of IQGAP Rng2p in forming and constricting the Schizosaccharomyces pombe cytokinetic contractile ring

Eukaryotic cells require IQGAP family multidomain adapter proteins for cytokinesis, but many questions remain about how IQGAPs contribute to the process. Here we show that fission yeast IQGAP Rng2p is required for both the normal process of contractile ring formation from precursor nodes and an alternative mechanism by which rings form from strands of actin filaments. Our work adds to previous studies suggesting a role for Rng2p in node and ring formation. We demonstrate that Rng2p is also required for normal ring constriction and septum formation. Systematic analysis of domain-deletion mutants established how the four domains of Rng2p contribute to cytokinesis. Contrary to a previous report, the actin-binding calponin homology domain of Rng2p is not required for viability, ring formation, or ring constriction. The IQ motifs are not required for ring formation but are important for ring constriction and septum formation. The GTPase-activating protein (GAP)–related domain is required for node-based ring formation. The Rng2p C-terminal domain is the only domain essential for viability. Our studies identified several distinct functions of Rng2 at multiple stages of cytokinesis.     

Importance of a myosin II-containing progenitor for actomyosin ring assembly in fission yeast

An actomyosin-based contractile ring provides the forces necessary for cell cleavage in several organisms [1-3]. Myosin II is an essential component of the actomyosin ring and has also been detected as a "spot" in interphase Schizosaccharomyces pombe cells [4-5]. It is currently unknown if this myosin II-containing spot is important for cytokinesis. In this study, we characterize this myosin II-containing spot using a combination of genetic and cell biological analyses. Whereas myosin II at the actomyosin ring undergoes rapid turnover, myosin II at the spot does not. Maintenance of the myosin II-containing spot is independent of F-actin function. Interestingly, maintenance of this myosin II spot in interphase requires the function of Rng3p, a UCS domain-containing protein, the Caenorhabditis elegans homolog of which has recently been shown to be a cochaperone for myosin II assembly [6]. Disassembly of the spot in interphase prevents actomyosin ring formation in the subsequent mitosis, implying that the spot might represent a progenitor that is important for assembly of the actomyosin ring. Given that mitosis represents a short period of the fission yeast cell cycle, organization of this progenitor structure in interphase might ensure proper assembly of the actomyosin ring and successful cell division.     

The price of independence: cell separation in fission yeast

The ultimate goal of cell division is to give rise to two viable independent daughter cells. A tight spatial and temporal regulation between chromosome segregation and cytokinesis ensures the viability of the daughter cells. Schizosaccharomyces pombe, commonly known as fission yeast, has become a leading model organism for studying essential and conserved mechanisms of the eukaryotic cell division process. Like many other eukaryotic cells it divides by binary fission and the cleavage furrow undergoes ingression due to the contraction of an actomyosin ring. In contrast to mammalian cells, yeasts as cell-walled organisms, also need to form a division septum made of cell wall material to complete the process of cytokinesis. The division septum is deposited behind the constricting ring and it will constitute the new ends of the daughter cells. Cell separation also involves cell wall degradation and this process should be precisely regulated to avoid cell lysis. In this review, we will give a brief overview of the whole cytokinesis process in fission yeast, from the positioning and assembly of the contractile ring to the final step of cell separation, and the problems generated when these processes are not precise.     

Gef1p and Scd1p,the Two GDP-GTP exchange factors for Cdc42p,form a ring structure that shrinks during cytokinesis in Schizosaccharomyces pombe

Fission yeast Cdc42p, a small GTPase of the Rho family, is essential for cell proliferation and maintenance of the rod-like cell morphology. Scd1/Ral1p is a GDP-GTP exchange factor (GEF) for Cdc42p. This study and a parallel study by others establish that Gef1p is another GEF for Cdc42p. Deletions of gef1 and scd1 are synthetically lethal, generating round dead cells, and hence mimic the phenotype of cdc42 deletion. Gef1p is localized mainly to the cell division site. Scd1p is also there, but it is also detectable in other parts of the cell, including the nucleus, growing ends, and the tips of conjugation tubes. Gef1p and Scd1p form a ring structure at the cell division site, which shrinks during cytokinesis following the contraction of the actomyosin ring. Formation of the Gef1p/Scd1p ring apparently depends on the integrity of the actomyosin ring. In turn, recruitment of Cdc42p to the cell division site follows the shrinking Gef1p/Scd1p ring; the Cdc42p accumulates like a closing iris. These observations suggest that Gef1p and Scd1p may have a role in mediating between contraction of the actomyosin ring and formation of the septum, by recruiting active Cdc42p to the septation site.     

Scw1p antagonizes the septation initiation network to regulate septum formation and cell separation in the fission yeast Schizosaccharomyces pombe

Cytokinesis in the fission yeast Schizosaccharomyces pombe is regulated by a signaling pathway termed the septation initiation network (SIN). The SIN is essential for initiation of actomyosin ring constriction and septum formation. In a screen to search for mutations that can rescue the sid2-250 SIN mutant, we obtained scw1-18. Both the scw1-18 mutant and the scw1 deletion mutant (scw1Δ mutant), have defects in cell separation. Both the scw1-18 and scw1Δ mutations rescue the growth defects of not just the sid2-250 mutant but also the other temperature-sensitive SIN mutants. Other cytokinesis mutants, such as those defective for actomyosin ring formation, are not rescued by scw1Δ. scw1Δ does not seem to rescue the SIN by restoring SIN signaling defects. However, scw1Δ may function downstream of the SIN to promote septum formation, since scw1Δ can rescue the septum formation defects of the cps1-191β-1,3-glucan synthase mutant, which is required for synthesis of the primary septum.