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.     

The Putative Exchange Factor Gef3p Interacts with Rho3p GTPase and the Septin Ring during Cytokinesis in Fission Yeast

The small GTP-binding proteins of the Rho family and its regulatory proteins play a central role in cytokinetic actomyosin ring assembly and cytokinesis. Here we show that the fission yeast guanine nucleotide exchange factor Gef3p interacts with Rho3p at the division site. Gef3p contains a putative DH homology domain and a BAR/IMD-like domain. The protein localized to the division site late in mitosis, where it formed a ring that did not constrict with actomyosin ring (cytokinetic actomyosin ring) invagination; instead, it split into a double ring that resembled the septin ring. Gef3p co-localized with septins and Mid2p and required septins and Mid2p for its localization. Gef3p interacts physically with the GTP-bound form of Rho3p. Although Gef3p is not essential for cell separation, the simultaneous disruption of gef3⁺ and Rho3p-interacting proteins, such as Sec8p, an exocyst component, Apm1p, a subunit of the clathrin adaptor complex or For3p, an actin-polymerizing protein, yielded cells with strong defects in septation and polarity respectively. Our results suggest that interactions between septins and Rho-GEFs provide a new targeting mechanism for GTPases in cytokinesis, in this case probably contributing to Rho3p function in vesicle tethering and vesicle trafficking in the later steps of cell separation.     

Role of septins and the exocyst complex in the function of hydrolytic enzymes responsible for fission yeast cell separation

Cell separation in Schizosaccharomyces pombe is achieved by the concerted action of the Eng1 endo-beta-1,3-glucanase and the Agn1 endo-alpha-1,3-glucanase, which are transported to the septum and localize to a ringlike structure that surrounds the septum. The requirements for the correct localization of both hydrolases as a ring were analyzed using green fluorescent protein fusion proteins. Targeting to the septum required a functional exocyst, because both proteins failed to localize correctly in sec8-1 or exo70delta mutants, suggesting that Agn1 and Eng1 might be two of the cargo proteins present in the vesicles that accumulate in exocyst mutants. Septins and Mid2 were also required for correct formation of a ring. In their absence, Eng1 and Agn1 were found in a disk-like structure that spanned the septum, rather than in a ring. Even though septin and mid2delta mutants have a cell separation defect, the septum and the distribution of linear beta-1,3-glucans were normal in these cells, suggesting that mislocalization of Eng1 and Agn1 might be the reason underlying the failure to separate efficiently. Thus, one of the functions of the septin ring would be to act as a positional marker for the localization of hydrolytic proteins to the medial region.     

Dual Function of Cyk2, a cdc15/PSTPIP Family Protein,in Regulating Actomyosin Ring Dynamics and Septin Distribution

We previously showed that the budding yeast Saccharomyces cerevisiae assembles an actomyosin-based ring that undergoes a contraction-like size change during cytokinesis. To learn more about the biochemical composition and activity of this ring, we have characterized the in vivo distribution and function of Cyk2p, a budding yeast protein that exhibits significant sequence similarity to the cdc15/PSTPIP family of cleavage furrow proteins. Video microscopy of cells expressing green fluorescent protein (GFP)-tagged Cyk2p revealed that Cyk2p forms a double ring that coincides with the septins through most of the cell cycle. During cytokinesis, however, the Cyk2 double ring merges with the actomyosin ring and exhibits a contraction-like size change that is dependent on Myo1p. The septin double ring, in contrast, does not undergo the contraction-like size change but the separation between the two rings increases during cytokinesis. These observations suggest that the septin-containing ring is dynamically distinct from the actomyosin ring and that Cyk2p transits between the two types of structures. Gene disruption of CYK2 does not affect the assembly of the actomyosin ring but results in rapid disassembly of the ring during the contraction phase, leading to incomplete cytokinesis, suggesting that Cyk2p has an important function in modulating the stability of the actomyosin ring during contraction. Overexpression of Cyk2p also blocks cytokinesis, most likely due to a loss of the septins from the bud neck, indicating that Cyk2p may also play a role in regulating the localization of the septins.     

Aim44p regulates phosphorylation of Hof1p to promote contractile ring closure during cytokinesis in budding yeast

Whereas actomyosin and septin ring organization and function in cytokinesis are thoroughly described, little is known regarding the mechanisms by which the actomyosin ring interacts with septins and associated proteins to coordinate cell division. Here we show that the protein product of YPL158C, Aim44p, undergoes septin-dependent recruitment to the site of cell division. Aim44p colocalizes with Myo1p, the type II myosin of the contractile ring, throughout most of the cell cycle. The Aim44p ring does not contract when the actomyosin ring closes. Instead, it forms a double ring that associates with septin rings on mother and daughter cells after cell separation. Deletion of AIM44 results in defects in contractile ring closure. Aim44p coimmunoprecipitates with Hof1p, a conserved F-BAR protein that binds both septins and type II myosins and promotes contractile ring closure. Deletion of AIM44 results in a delay in Hof1p phosphorylation and altered Hof1p localization. Finally, overexpression of Dbf2p, a kinase that phosphorylates Hof1p and is required for relocalization of Hof1p from septin rings to the contractile ring and for Hof1p-triggered contractile ring closure, rescues the cytokinesis defect observed in aim44∆ cells. Our studies reveal a novel role for Aim44p in regulating contractile ring closure through effects on Hof1p.     

Septin ring assembly requires concerted action of polarisome components, a PAK kinase Cla4p, and the actin cytoskeleton in Saccharomyces cerevisiae

Septins are filament-forming proteins that function in cytokinesis in a wide variety of organisms. In budding yeast, the small GTPase Cdc42p triggers the recruitment of septins to the incipient budding site and the assembly of septins into a ring. We herein report that Bni1p and Cla4p, effectors of Cdc42p, are required for the assembly of the septin ring during the initiation of budding but not for its maintenance after the ring converts to a septin collar. In bni1Delta cla4-75-td mutant, septins were recruited to the incipient budding site. However, the septin ring was not assembled, and septins remained at the polarized growing sites. Bni1p, a formin family protein, is a member of the polarisome complex with Spa2p, Bud6p, and Pea2p. All spa2Delta cla4-75-td, bud6Delta cla4-75-td, and pea2Delta cla4-75-td mutants showed defects in septin ring assembly. Bni1p stimulates actin polymerization for the formation of actin cables. Point mutants of BNI1 that are specifically defective in actin cable formation also exhibited septin ring assembly defects in the absence of Cla4p. Consistently, treatment of cla4Delta mutant with the actin inhibitor latrunculin A inhibited septin ring assembly. Our results suggest that polarisome components and Cla4p are required for the initial assembly of the septin ring and that the actin cytoskeleton is involved in this process.     

Organization of a sterol-rich membrane domain by cdc15p during cytokinesis in fission yeast

Many membrane processes occur in discrete membrane domains containing lipid rafts, but little is known about how these domains are organized and positioned. In the fission yeast Schizosaccharomyces pombe, a sterol-rich membrane domain forms at the cell-division site. Here, we show that formation of this membrane domain is independent of the contractile actin ring, septation, mid1p and the septins, and also requires cdc15p, an essential contractile ring protein that associates with lipid rafts. cdc15 mutants have membrane domains in the shape of spirals. Overexpression of cdc15p in interphase cells induces abnormal membrane domain formation in an actin-independent manner. We propose that cdc15p functions to organize lipid rafts at the cleavage site for cytokinesis.     

Requirements of fission yeast septins for complex formation, localization, and function

Septins are GTP binding proteins important for cytokinesis in many eukaryotes. The Schizosaccaromyces pombe genome sequence predicts orthologues of four of five Saccharomyces cerevisiae septins involved in cytokinesis and these are named Spns1-4p. That spns1-4 are not essential genes permitted the application of a combined genetic and proteomics approach to determine their functional relationships. Our findings indicate that Spns1-4p are present throughout interphase as a diffusely localized approximately 8.5S complex containing two copies of each septin linked together as a chain in the order Spn3p-Spn4p-Spn1p-Spn2p. Septin recruitment to the medial region of the cell is genetically separable from ring formation, and whereas it is normally restricted to mitosis, it can be promoted without activation of the mitotic cell cycle machinery. Coalescence into ring structures requires Spn1p and Spn4p associate with at least one other septin subunit and the expression of Mid2p that is normally restricted to mitosis. This study establishes the functional requirements for septin complex organization in vivo.     

Role of Polo Kinase and Mid1p in Determining the Site of Cell Division in Fission Yeast

The fission yeast Schizosaccharomyces pombe divides symmetrically using a medial F-actin– based contractile ring to produce equal-sized daughter cells. Mutants defective in two previously described genes, mid1 and pom1, frequently divide asymmetrically. Here we present the identification of three new temperature-sensitive mutants defective in localization of the division plane. All three mutants have mutations in the polo kinase gene, plo1, and show defects very similar to those of mid1 mutants in both the placement and organization of the medial ring. In both cases, ring formation is frequently initiated near the cell poles, indicating that Mid1p and Plo1p function in recruiting medial ring components to the cell center. It has been reported previously that during mitosis Mid1p becomes hyperphosphorylated and relocates from the nucleus to a medial ring. Here we show that Mid1p first forms a diffuse cortical band during spindle formation and then coalesces into a ring before anaphase. Plo1p is required for Mid1p to exit the nucleus and form a ring, and Pom1p is required for proper placement of the Mid1p ring. Upon overexpression of Plo1p, Mid1p exits the nucleus prematurely and displays a reduced mobility on gels similar to that of the hyperphosphorylated form observed previously in mitotic cells. Genetic and two-hybrid analyses suggest that Plo1p and Mid1p act in a common pathway distinct from that involving Pom1p. Plo1p localizes to the spindle pole bodies and spindles of mitotic cells and also to the medial ring at the time of its formation. Taken together, the data indicate that Plo1p plays a role in the positioning of division sites by regulating Mid1p. Given its previously known functions in mitosis and the timing of cytokinesis, Plo1p is thus implicated as a key molecule in the spatial and temporal coordination of cytokinesis with mitosis.     

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.     

Septin collar formation in budding yeast requires GTP binding and direct phosphorylation by the PAK, Cla4

Assembly at the mother-bud neck of a filamentous collar containing five septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1) is necessary for proper morphogenesis and cytokinesis. We show that Cdc10 and Cdc12 possess GTPase activity and appropriate mutations in conserved nucleotide-binding residues abrogate GTP binding and/or hydrolysis in vitro. In vivo, mutants unable to bind GTP prevent septin collar formation, whereas mutants that block GTP hydrolysis do not. GTP binding-defective Cdc10 and Cdc12 form soluble heteromeric complexes with other septins both in yeast and in bacteria; yet, unlike wild-type, mutant complexes do not bind GTP and do not assemble into filaments in vitro. Absence of a p21-activated protein kinase (Cla4) perturbs septin collar formation. This defect is greatly exacerbated when combined with GTP binding-defective septins; conversely, the septin collar assembly defect of such mutants is suppressed efficiently by CLA4 overexpression. Cla4 interacts directly with and phosphorylates certain septins in vitro and in vivo. Thus, septin collar formation may correspond to septin filament assembly, and requires both GTP binding and Cla4-mediated phosphorylation of septins.     

Evidence that a septin diffusion barrier is dispensable for cytokinesis in budding yeast

Septins are essential for cytokinesis in Saccharomyces cerevisiae, but their precise roles remain elusive. Currently, it is thought that before cytokinesis, the hourglass-shaped septin structure at the mother-bud neck acts as a scaffold for assembly of the actomyosin ring (AMR) and other cytokinesis factors. At the onset of cytokinesis, the septin hourglass splits to form a double ring that sandwiches the AMR and may function as diffusion barriers to restrict diffusible cytokinesis factors to the division site. Here, we show that in cells lacking the septin Cdc10 or the septin-associated protein Bud4, the septins form a ring-like structure at the mother-bud neck that fails to re-arrange into a double ring early in cytokinesis. Strikingly, AMR assembly and constriction, the localization of membrane-trafficking and extracellular-matrix-remodeling factors, cytokinesis, and cell-wall-septum formation all occur efficiently in cdc10Δ and bud4Δ mutants. Thus, diffusion barriers formed by the septin double ring do not appear to be critical for S. cerevisiae cytokinesis. However, an AMR mutation and a septin mutation have synergistic effects on cytokinesis and the localization of cytokinesis proteins, suggesting that tethering to the AMR and a septin diffusion barrier may function redundantly to localize proteins to the division site.     

Septins,under Cla4p regulation,and the chitin ring are required for neck integrity in budding yeast

CLA4, encoding a protein kinase of the PAK type, and CDC11, encoding a septin, were isolated in a screen for synthetic lethality with CHS3, which encodes the chitin synthase III catalytic moiety. Although Ste20p shares some essential function with Cla4p, it did not show synthetic lethality with Chs3p. cla4 and cdc11 mutants exhibited similar morphological and septin localization defects, including aberrant and ectopic septa. Myo1p, which requires septins for localization, formed abnormally wide rings in cla4 mutants. In cultures started with unbudded cells, an inhibitor of Chs3p activity, nikkomycin Z, aggravated the abnormalities of cla4 and cdc11 mutants and gave rise to enlarged necks at the mother-bud junction, leading to cell death. It is concluded that Cla4p is required for the correct localization and/or assembly of the septin ring and that both the septin ring and the Chs3p-requiring chitin ring at the mother-bud neck cooperate in maintaining the neck constricted throughout the cell cycle, a vital function in budding yeast.     

Schizosaccharomyces pombe Pxl1 is a paxillin homologue that modulates Rho1 activity and participates in cytokinesis

Schizosaccharomyces pombe Rho GTPases regulate actin cytoskeleton organization and cell integrity. We studied the fission yeast gene SPBC4F6.12 based on its ability to suppress the thermosensitivity of cdc42-1625 mutant strain. This gene, named pxl1(+), encodes a protein with three LIM domains that is similar to paxillin. Pxl1 does not interact with Cdc42 but it interacts with Rho1, and it negatively regulates this GTPase. Fission yeast Pxl1 forms a contractile ring in the cell division region and deletion of pxl1(+) causes a delay in cell-cell separation, suggesting that it has a function in cytokinesis. Pxl1 N-terminal region is required and sufficient for its localization to the medial ring, whereas the LIM domains are necessary for its function. Pxl1 localization requires actin polymerization and the actomyosin ring, but it is independent of the septation initiation network (SIN) function. Moreover, Pxl1 colocalizes and interacts with Myo2, and Cdc15, suggesting that it is part of the actomyosin ring. Here, we show that in cells lacking Pxl1, the myosin ring is not correctly assembled and that actomyosin ring contraction is delayed. Together, these data suggest that Pxl1 modulates Rho1 GTPase signaling and plays a role in the formation and contraction of the actomyosin ring during cytokinesis.     

Role of a Cdc42p effector pathway in recruitment of the yeast septins to the presumptive bud site

The septins are GTP-binding, filament-forming proteins that are involved in cytokinesis and other processes. In the yeast Saccharomyces cerevisiae, the septins are recruited to the presumptive bud site at the cell cortex, where they form a ring through which the bud emerges. We report here that in wild-type cells, the septins typically become detectable in the vicinity of the bud site several minutes before ring formation, but the ring itself is the first distinct structure that forms. Septin recruitment depends on activated Cdc42p but not on the normal pathway for bud-site selection. Recruitment occurs in the absence of F-actin, but ring formation is delayed. Mutant phenotypes and suppression data suggest that the Cdc42p effectors Gic1p and Gic2p, previously implicated in polarization of the actin cytoskeleton, also function in septin recruitment. Two-hybrid, in vitro protein binding, and coimmunoprecipitation data indicate that this role involves a direct interaction of the Gic proteins with the septin Cdc12p.     

A Rho-type GTPase, rho-4, is required for septation in Neurospora crassa

Proteins in the Rho family are small monomeric GTPases primarily involved in polarization, control of cell division, and reorganization of cytoskeletal elements. Phylogenetic analysis of predicted fungal Rho proteins suggests that a new Rho-type GTPase family, whose founding member is Rho4 from the archiascomycete Schizosaccharomyces pombe, is involved in septation. S. pombe rho4Delta mutants have multiple, abnormal septa. In contrast to S. pombe rho4Delta mutants, we show that strains containing rho-4 loss-of-function mutations in the filamentous fungus Neurospora crassa lead to a loss of septation. Epitope-tagged RHO-4 localized to septa and to the plasma membrane. In other fungi, the steps required for septation include formin, septin, and actin localization followed by cell wall synthesis and the completion of septation. rho-4 mutants were unable to form actin rings, showing that RHO-4 is required for actin ring formation. Characterization of strains containing activated alleles of rho-4 showed that RHO-4-GTP is likely to initiate new septum formation in N. crassa.     

SUMO modification of septin-interacting proteins in Candida albicans

The initiation of bud and hyphal growth in the opportunistic fungal pathogen Candida albicans both involve polarized morphogenesis. However, there are many differences including the function of the septin proteins, a family of proteins involved in membrane organization in a wide range of organisms. Septins form a characteristic ring on the inner surface of the plasma membrane at the bud neck, whereas the septins are diffusely localized across emerging hyphal tips. In addition, septin rings are maintained at sites of septum formation in hyphae rather than being disassembled immediately after cytokinesis. The possibility that C. albicans septins are regulated by the small ubiquitin-like protein SUMO was examined in this study because the Saccharomyces cerevisiae septins were shown previously to be modified by SUMO (Smt3p). However, SUMO conjugation to septins was not detected during budding or hyphal morphogenesis in C. albicans. These results are supported by the lack of conserved SUMO consensus motifs between septins from the two organisms even after adjusting the predicted Cdc3p and Cdc12p septin sequences to account for mRNA splicing in C. albicans. Interestingly, a homolog of the Smt3p SUMO was identified in the C. albicans genome, and an epitope tagged version of Smt3p was conjugated to a variety of proteins. Immunofluorescence analysis showed prominent Smt3p SUMO localization at bud necks and sites of septum formation in hyphae similar to the septins. However, Smt3p was primarily detected on the mother cell side of the septin ring. A subset of these Smt3p-modified proteins co-immunoprecipitated with the septin Cdc11p. These results indicate that septin-associated proteins and not the septins themselves are the key target of SUMO modification at the bud neck in C. albicans.     

Phosphorylation-dependent regulation of septin dynamics during the cell cycle

Septins are GTPases involved in cytokinesis. In yeast, they form a ring at the cleavage site. Using FRAP, we show that septins are mobile within the ring at bud emergence and telophase and are immobile during S, G2, and M phases. Immobilization of the septins is dependent on both Cla4, a PAK-like kinase, and Gin4, a septin-dependent kinase that can phosphorylate the septin Shs1/Sep7. Induction of septin ring dynamics in telophase is triggered by the translocation of Rts1, a kinetochore-associated regulatory subunit of PP2A phosphatase, to the bud neck and correlates with Rts1-dependent dephosphorylation of Shs1. In rts1-Delta cells, the actomyosin ring contracts properly but cytokinesis fails. Together our results implicate septins in a late step of cytokinesis and indicate that proper regulation of septin dynamics, possibly through the control of their phosphorylation state, is required for the completion of cytokinesis.     

Role of the septin ring in the asymmetric localization of proteins at the mother-bud neck in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, septins form a scaffold in the shape of a ring at the future budding site that rearranges into a collar at the mother-bud neck. Many proteins bind asymmetrically to the septin collar. We found that the protein Bni4-CFP was located on the exterior of the septin ring before budding and on the mother side of the collar after budding, whereas the protein kinase Kcc4-YFP was located on the interior of the septin ring before budding and moved into the bud during the formation of the septin collar. Unbudded cells treated with the actin inhibitor latrunculin-A assembled cortical caps of septins on which Bni4-CFP and Kcc4-YFP colocalized. Bni4-CFP and Kcc4-YFP also colocalized on cortical caps of septins found in strains deleted for the genes encoding the GTPase activating proteins of Cdc42 (RGA1, RGA2, and BEM3). However, Bni4-CFP and Kcc4-YFP were still partially separated in mutants (gin4, elm1, cla4, and cdc3-1) in which septin morphology was severely disrupted in other ways. These observations provide clues to the mechanisms for the asymmetric localization of septin-associated proteins.     

The Cdc42p GTPase is targeted to the site of cell division in the fission yeast Schizosaccharomyces pombe

The Rho-family GTPase Cdc42p regulates many aspects of cell polarity and growth in eukaryotic cells, including the organization of the actin cytoskeleton. To further examine Cdc42p function in the fission yeast Schizosaccharomyces pombe, a functional green fluorescent protein (GFP)-Cdc42p fusion protein was generated. GFP-Cdc42p was observed at the medial region of the cell at the cell-division site early in cytokinesis and remained there through cell separation, and was also localized to the periphery of the cell and to internal membranes. Unexpectedly, treatment with the actin-depolymerizing drug latrunculin-A disrupted the medial region targeting pattern, and cells deficient in the actin-binding proteins tropomyosin and profilin also did not exhibit medial GFP-Cdc42p staining. In addition, medial GFP-Cdc42p localization was eliminated in a number of cytokinesis mutants, including strains defective in assembling the medial actinomyosin ring, medial ring contraction, and septum assembly. GFP-Cdc42p targeting was less affected in mutants that formed misplaced or multiple septa. These results suggest that the localization of Cdc42p at the cell-division site was dependent upon the actin cytoskeleton and that Cdc42p may function in the interdependent processes of cytokinesis and septation.