Fission yeast paxillin contains two Cdc15 binding motifs for robust recruitment to the cytokinetic ring

The F-BAR protein Cdc15 mediates attachment of the cytokinetic ring (CR) to the plasma membrane and is essential for cytokinesis in Schizosaccharomyces pombe. While its N-terminal F-BAR domain is responsible for oligomerization and membrane binding, its C-terminal SH3 domain binds other partners at a distance from the membrane. We previously demonstrated that the essential cytokinetic formin Cdc12, through an N-terminal motif, directly binds the cytosolic face of the F-BAR domain. Here, we show that paxillin-like Pxl1, which is important for CR stability, contains a motif highly related to that in formin Cdc12, and also binds the Cdc15 F-BAR domain directly. Interestingly, Pxl1 has a second site for binding the Cdc15 SH3 domain. To understand the importance of these two Pxl1-Cdc15 interactions, we mapped and disrupted both. Disrupting the Pxl1-Cdc15 F-BAR domain interaction reduced Pxl1 levels in the CR, whereas disrupting Pxl1’s interaction with the Cdc15 SH3 domain, did not. Unexpectedly, abolishing Pxl1-Cdc15 interaction greatly reduced but did not eliminate CR Pxl1 and did not significantly affect cytokinesis. These data point to another mechanism of Pxl1 CR recruitment and show that very little CR Pxl1 is sufficient for its cytokinetic function.     

The formins Cdc12 and For3 cooperate during contractile ring assembly in cytokinesis

Both de novo–assembled actin filaments at the division site and existing filaments recruited by directional cortical transport contribute to contractile ring formation during cytokinesis. However, it is unknown which source is more important. Here, we show that fission yeast formin For3 is responsible for node condensation into clumps in the absence of formin Cdc12. For3 localization at the division site depended on the F-BAR protein Cdc15, and for3 deletion was synthetic lethal with mutations that cause defects in contractile ring formation. For3 became essential in cells expressing N-terminal truncations of Cdc12, which were more active in actin assembly but depended on actin filaments for localization to the division site. In tetrad fluorescence microscopy, double mutants of for3 deletion and cdc12 truncations were severely defective in contractile ring assembly and constriction, although cortical transport of actin filaments was normal. Together, these data indicate that different formins cooperate in cytokinesis and that de novo actin assembly at the division site is predominant for contractile ring formation.     

Formin Cdc12’s specific actin assembly properties are tailored for cytokinesis in fission yeast

Formins generate unbranched actin filaments by a conserved, processive actin assembly mechanism. Most organisms express multiple formin isoforms that mediate distinct cellular processes and facilitate actin filament polymerization by significantly different rates, but how these actin assembly differences correlate to cellular activity is unclear. We used a computational model of fission yeast cytokinetic ring assembly to test the hypothesis that particular actin assembly properties help tailor formins for specific cellular roles. Simulations run in different actin filament nucleation and elongation conditions revealed that variations in formin’s nucleation efficiency critically impact both the probability and timing of contractile ring formation. To probe the physiological importance of nucleation efficiency, we engineered fission yeast formin chimera strains in which the FH1-FH2 actin assembly domains of full-length cytokinesis formin Cdc12 were replaced with the FH1-FH2 domains from functionally and evolutionarily diverse formins with significantly different actin assembly properties. Although Cdc12 chimeras generally support life in fission yeast, quantitative live-cell imaging revealed a range of cytokinesis defects from mild to severe. In agreement with the computational model, chimeras whose nucleation efficiencies are least similar to Cdc12 exhibit more severe cytokinesis defects, specifically in the rate of contractile ring assembly. Together, our computational and experimental results suggest that fission yeast cytokinesis is ideally mediated by a formin with properly tailored actin assembly parameters.     

Dephosphorylation of Iqg1 by Cdc14 regulates cytokinesis in budding yeast

Cytokinesis separates cells by contraction of a ring composed of filamentous actin (F-actin) and type II myosin. Iqg1, an IQGAP family member, is an essential protein in Saccharomyces cerevisiae required for assembly and contraction of the actomyosin ring. Localization of F-actin to the ring occurs only after anaphase and is mediated by the calponin homology domain (CHD) of Iqg1, but the regulatory mechanisms that temporally restrict actin ring assembly are not well defined. We tested the hypothesis that dephosphorylation of four perfect cyclin-dependent kinase (Cdk) sites flanking the CHD promotes actin ring formation, using site-specific alanine mutants. Cells expressing the nonphosphorylatable iqg1-4A allele formed actin rings before anaphase and exhibited defects in myosin contraction and cytokinesis. The Cdc14 phosphatase is required for normal cytokinesis and acts on specific Cdk phosphorylation sites. Overexpression of Cdc14 resulted in premature actin ring assembly, whereas inhibition of Cdc14 function prevented actin ring formation. Cdc14 associated with Iqg1, dependent on several CHD-flanking Cdk sites, and efficiently dephosphorylated these sites in vitro. Of importance, the iqg1-4A mutant rescued the inability of cdc14-1 cells to form actin rings. Our data support a model in which dephosphorylation of Cdk sites around the Iqg1 CHD by Cdc14 is both necessary and sufficient to promote actin ring formation. Temporal control of actin ring assembly by Cdk and Cdc14 may help to ensure that cytokinesis onset occurs after nuclear division is complete.     

Formin Cdc12’s specific actin assembly properties are tailored for cytokinesis in fission yeast

Formins generate unbranched actin filaments by a conserved, processive actin assembly mechanism. Most organisms express multiple formin isoforms that mediate distinct cellular processes and facilitate actin filament polymerization by significantly different rates, but how these actin assembly differences correlate to cellular activity is unclear. We used a computational model of fission yeast cytokinetic ring assembly to test the hypothesis that particular actin assembly properties help tailor formins for specific cellular roles. Simulations run in different actin filament nucleation and elongation conditions revealed that variations in formin’s nucleation efficiency critically impact both the probability and timing of contractile ring formation. To probe the physiological importance of nucleation efficiency, we engineered fission yeast formin chimera strains in which the FH1-FH2 actin assembly domains of full-length cytokinesis formin Cdc12 were replaced with the FH1-FH2 domains from functionally and evolutionarily diverse formins with significantly different actin assembly properties. Although Cdc12 chimeras generally support life in fission yeast, quantitative live-cell imaging revealed a range of cytokinesis defects from mild to severe. In agreement with the computational model, chimeras whose nucleation efficiencies are least similar to Cdc12 exhibit more severe cytokinesis defects, specifically in the rate of contractile ring assembly. Together, our computational and experimental results suggest that fission yeast cytokinesis is ideally mediated by a formin with properly tailored actin assembly parameters.     

The DYRK-family kinase Pom1 phosphorylates the F-BAR protein Cdc15 to prevent division at cell poles

Division site positioning is critical for both symmetric and asymmetric cell divisions. In many organisms, positive and negative signals cooperate to position the contractile actin ring for cytokinesis. In rod-shaped fission yeast Schizosaccharomyces pombe cells, division at midcell is achieved through positive Mid1/anillin-dependent signaling emanating from the central nucleus and negative signals from the dual-specificity tyrosine phosphorylation-regulated kinase family kinase Pom1 at the cell poles. In this study, we show that Pom1 directly phosphorylates the F-BAR protein Cdc15, a central component of the cytokinetic ring. Pom1-dependent phosphorylation blocks Cdc15 binding to paxillin Pxl1 and C2 domain protein Fic1 and enhances Cdc15 dynamics. This promotes ring sliding from cell poles, which prevents septum assembly at the ends of cells with a displaced nucleus or lacking Mid1. Pom1 also slows down ring constriction. These results indicate that a strong negative signal from the Pom1 kinase at cell poles converts Cdc15 to its closed state, destabilizes the actomyosin ring, and thus promotes medial septation.     

Assembly and architecture of precursor nodes during fission yeast cytokinesis

The contractile ring is essential for cytokinesis in most fungal and animal cells. In fission yeast, cytokinesis nodes are precursors of the contractile ring and mark the future cleavage site. However, their assembly and architecture have not been well described. We found that nodes are assembled stoichiometrically in a hierarchical order with two modules linked by the positional marker anillin Mid1. Mid1 first recruits Cdc4 and IQGAP Rng2 to form module I. Rng2 subsequently recruits the myosin-II subunits Myo2 and Rlc1. Mid1 then independently recruits the F-BAR protein Cdc15 to form module II. Mid1, Rng2, Cdc4, and Cdc15 are stable node components that accumulate close to the plasma membrane. Both modules recruit the formin Cdc12 to nucleate actin filaments. Myo2 heads point into the cell interior, where they efficiently capture actin filaments to condense nodes into the contractile ring. Collectively, our work characterizing the assembly and architecture of precursor nodes defines important steps and molecular players for contractile ring assembly.     

Cdk1 phosphorylation of fission yeast paxillin inhibits its cytokinetic ring localization

Divisions of the genetic material and cytoplasm are coordinated spatially and temporally to ensure genome integrity. This coordination is mediated in part by the major cell cycle regulator cyclin-dependent kinase (Cdk1). Cdk1 activity peaks during mitosis, but during mitotic exit/cytokinesis Cdk1 activity is reduced, and phosphorylation of its substrates is reversed by various phosphatases including Cdc14, PP1, PP2A, and PP2B. Cdk1 is known to phosphorylate several components of the actin- and myosin-based cytokinetic ring (CR) that mediates division of yeast and animal cells. Here we show that Cdk1 also phosphorylates the Schizosaccharomyces pombe CR component paxillin Pxl1. We determined that both the Cdc14 phosphatase Clp1 and the PP1 phosphatase Dis2 contribute to Pxl1 dephosphorylation at mitotic exit, but PP2B/calcineurin does not. Preventing Pxl1 phosphorylation by Cdk1 results in increased Pxl1 levels, precocious Pxl1 recruitment to the division site, and increased duration of CR constriction. In vitro Cdk1-mediated phosphorylation of Pxl1 inhibits its interaction with the F-BAR domain of the cytokinetic scaffold Cdc15, thereby disrupting a major mechanism of Pxl1 recruitment. Thus, Pxl1 is a novel substrate through which S. pombe Cdk1 and opposing phosphatases coordinate mitosis and cytokinesis.     

The fission yeast cytokinesis formin Cdc12p is a barbed end actin filament capping protein gated by profilin

Cytokinesis in most eukaryotes requires the assembly and contraction of a ring of actin filaments and myosin II. The fission yeast Schizosaccharomyces pombe requires the formin Cdc12p and profilin (Cdc3p) early in the assembly of the contractile ring. The proline-rich formin homology (FH) 1 domain binds profilin, and the FH2 domain binds actin. Expression of a construct consisting of the Cdc12 FH1 and FH2 domains complements a conditional mutant of Cdc12 at the restrictive temperature, but arrests cells at the permissive temperature. Cells overexpressing Cdc12(FH1FH2)p stop growing with excessive actin cables but no contractile rings. Like capping protein, purified Cdc12(FH1FH2)p caps the barbed end of actin filaments, preventing subunit addition and dissociation, inhibits end to end annealing of filaments, and nucleates filaments that grow exclusively from their pointed ends. The maximum yield is one filament pointed end per six formin polypeptides. Profilins that bind both actin and poly-l-proline inhibit nucleation by Cdc12(FH1FH2)p, but polymerization of monomeric actin is faster, because the filaments grow from their barbed ends at the same rate as uncapped filaments. On the other hand, Cdc12(FH1FH2)p blocks annealing even in the presence of profilin. Thus, formins are profilin-gated barbed end capping proteins with the ability to initiate actin filaments from actin monomers bound to profilin. These properties explain why contractile ring assembly requires both formin and profilin and why viability depends on the ability of profilin to bind both actin and poly-l-proline.     

Hof1 and Rvs167 Have Redundant Roles in Actomyosin Ring Function during Cytokinesis in Budding Yeast

The Hof1 protein (Homologue of Fifteen) regulates formation of the primary septum during cytokinesis in the budding yeast Saccharomyces cerevisiae, whereas the orthologous Cdc15 protein in fission yeast regulates the actomyosin ring by using its F-BAR domain to recruit actin nucleators to the cleavage site. Here we show that budding yeast Hof1 also contributes to actin ring assembly in parallel with the Rvs167 protein. Simultaneous deletion of the HOF1 and RVS167 genes is lethal, and cells fail to assemble the actomyosin ring as they progress through mitosis. Although Hof1 and Rvs167 are not orthologues, they both share an analogous structure, with an F-BAR or BAR domain at the amino terminus, capable of inducing membrane curvature, and SH3 domains at the carboxyl terminus that bind to specific proline-rich targets. The SH3 domain of Rvs167 becomes essential for assembly of the actomyosin ring in cells lacking Hof1, suggesting that it helps to recruit a regulator of the actin cytoskeleton. This new function of Rvs167 appears to be independent of its known role as a regulator of the Arp2/3 actin nucleator, as actin ring assembly is not abolished by the simultaneous inactivation of Hof1 and Arp2/3. Instead we find that recruitment to the bud-neck of the Iqg1 actin regulator is defective in cells lacking Hof1 and Rvs167, though future studies will be needed to determine if this reflects a direct interaction between these factors. The redundant role of Hof1 in actin ring assembly suggests that the mechanism of actin ring assembly has been conserved to a greater extent across evolution than anticipated previously.     

The PCH family protein,Cdc15p,recruits two F-actin nucleation pathways to coordinate cytokinetic actin ring formation in Schizosaccharomyces pombe

Cytokinetic actin ring (CAR) formation in Schizosaccharomyces pombe requires two independent actin nucleation pathways, one dependent on the Arp2/3 complex and another involving the formin Cdc12p. Here we investigate the role of the S. pombe Cdc15 homology family protein, Cdc15p, in CAR assembly and find that it interacts with proteins from both of these nucleation pathways. Cdc15p binds directly to the Arp2/3 complex activator Myo1p, which likely explains why actin patches and the Arp2/3 complex fail to be medially recruited during mitosis in cdc15 mutants. Cdc15p also binds directly to Cdc12p. Cdc15p and Cdc12p not only display mutual dependence for CAR localization, but also exist together in a ring-nucleating structure before CAR formation. The disruption of these interactions in cdc15 null cells is likely to be the reason for their complete lack of CARs. We propose a model in which Cdc15p plays a critical role in recruiting and coordinating the pathways essential for the assembly of medially located F-actin filaments and construction of the CAR.     

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.     

Formin differentially utilizes profilin isoforms to rapidly assemble actin filaments

Cells contain multiple formin isoforms that drive the assembly of profilin-actin for diverse processes. Given that many organisms also contain several profilin isoforms, specific formin/profilin pairs might be matched to optimally stimulate actin polymerization. We utilized a combination of bulk actin polymerization and single filament total internal reflection fluorescence microscopy assays to measure the effect of different profilin isoforms on the actin assembly properties of the cytokinesis formins from fission yeast (Cdc12p) and the nematode worm (CYK-1). We discovered that Cdc12p only effectively utilizes the single fission yeast profilin isoform SpPRF. Conversely, CYK-1 prefers the essential worm cytokinesis profilin CePFN-1 to the two non-essential worm profilin isoforms (SpPRF = CePFN-1 > CePFN-2 > CePFN-3). Chimeras containing the profilin-binding formin homology 1 (FH1) domain from one formin and the barbed-end associated FH2 domain from the other formin, revealed that both the FH1 and FH2 domains help confer profilin isoform specialization. Although the Cdc12p and CYK-1 FH1 domains cannot differentiate between profilin isoforms in the absence of actin, formin FH1 domains appear to preferentially select specific isoforms of profilin-actin. Surprisingly, analysis of profilin point mutants revealed that differences in highly conserved residues in both the poly-L-proline and actin binding regions of profilin do not explain their differential utilization by formin. Therefore, rapid formin-mediated elongation of profilin-actin depends upon favorable interactions of profilin-actin with the FH1 domain as well as the barbed-end associated FH2 domain. Specific formin FH1FH2 domains are tailored to optimally utilize actin bound to particular profilin isoforms.     

Time-varying mobility and turnover of actomyosin ring components during cytokinesis in Schizosaccharomyces pombe

Cytokinesis in many eukaryotes is dependent on a contractile actomyosin ring (AMR), composed of F-actin, myosin II, and other actin and myosin II regulators. Through fluorescence recovery after photobleaching experiments, many components of the AMR have been shown to be mobile and to undergo constant exchange with the cytosolic pools. However, how the mobility of its components changes at distinct stages of mitosis and cytokinesis has not been addressed. Here, we describe the mobility of eight Schizosaccharomyces pombe AMR proteins at different stages of mitosis and cytokinesis using an approach we have developed. We identified three classes of proteins, which showed 1) high (Ain1, Myo2, Myo51), 2) low (Rng2, Mid1, Myp2, Cdc12), and 3) cell cycle–dependent (Cdc15) mobile fractions. We observed that the F-BAR protein Cdc15 undergoes a 20–30% reduction in its mobile fraction after spindle breakdown and initiation of AMR contraction. Moreover, our data indicate that this change in Cdc15 mobility is dependent on the septation initiation network (SIN). Our work offers a novel strategy for estimating cell cycle–dependent mobile protein fractions in cellular structures and provides a valuable dataset, that is of interest to researchers working on cytokinesis.     

The SH3 domains of two PCH family members cooperate in assembly of the Schizosaccharomyces pombe contractile ring

Schizosaccharomyces pombe cdc15 homology (PCH) family members participate in many cellular processes by bridging the plasma membrane and cytoskeleton. Their F-BAR domains bind and curve membranes, whereas other domains, typically SH3 domains, are expected to provide cytoskeletal links. We tested this prevailing model of functional division in the founding member of the family, Cdc15, which is essential for cytokinesis in S. pombe, and in the related PCH protein, Imp2. We find that the distinct functions of Imp2 and Cdc15 are SH3 domain independent. However, the Cdc15 and Imp2 SH3 domains share an essential role in recruiting proteins to the contractile ring, including Pxl1 and Fic1. Together, Pxl1 and Fic1, a previously uncharacterized C2 domain protein, add structural integrity to the contractile ring and prevent it from fragmenting during division. Our data indicate that the F-BAR proteins Cdc15 and Imp2 contribute to a single biological process with both distinct and overlapping functions.     

Investigation of the Interaction between Cdc42 and Its Effector TOCA1: HANDOVER OF Cdc42 TO THE ACTIN REGULATOR N-WASP IS FACILITATED BY DIFFERENTIAL BINDING AFFINITIES*

Transducer of Cdc42-dependent actin assembly protein 1 (TOCA1) is an effector of the Rho family small G protein Cdc42. It contains a membrane-deforming F-BAR domain as well as a Src homology 3 (SH3) domain and a G protein-binding homology region 1 (HR1) domain. TOCA1 binding to Cdc42 leads to actin rearrangements, which are thought to be involved in processes such as endocytosis, filopodia formation, and cell migration. We have solved the structure of the HR1 domain of TOCA1, providing the first structural data for this protein. We have found that the TOCA1 HR1, like the closely related CIP4 HR1, has interesting structural features that are not observed in other HR1 domains. We have also investigated the binding of the TOCA HR1 domain to Cdc42 and the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and a member of the Wiskott-Aldrich syndrome protein family, N-WASP. TOCA1 binds Cdc42 with micromolar affinity, in contrast to the nanomolar affinity of the N-WASP G protein-binding region for Cdc42. NMR experiments show that the Cdc42-binding domain from N-WASP is able to displace TOCA1 HR1 from Cdc42, whereas the N-WASP domain but not the TOCA1 HR1 domain inhibits actin polymerization. This suggests that TOCA1 binding to Cdc42 is an early step in the Cdc42-dependent pathways that govern actin dynamics, and the differential binding affinities of the effectors facilitate a handover from TOCA1 to N-WASP, which can then drive recruitment of the actin-modifying machinery.     

A Highlights from MBoC Selection: Fission yeast profilin is tailored to facilitate actin assembly by the cytokinesis formin Cdc12

The evolutionarily conserved small actin-monomer binding protein profilin is believed to be a housekeeping factor that maintains a general pool of unassembled actin. However, despite similar primary sequences, structural folds, and affinities for G-actin and poly-l-proline, budding yeast profilin ScPFY fails to complement fission yeast profilin SpPRF temperature-sensitive mutant cdc3-124 cells. To identify profilin''s essential properties, we built a combinatorial library of ScPFY variants containing either WT or SpPRF residues at multiple positions and carried out a genetic selection to isolate variants that support life in fission yeast. We subsequently engineered ScPFY(9-Mut), a variant containing nine substitutions in the actin-binding region, which complements cdc3-124 cells. ScPFY(9-Mut), but not WT ScPFY, suppresses severe cytokinesis defects in cdc3-124 cells. Furthermore, the major activity rescued by ScPFY(9-Mut) is the ability to enhance cytokinesis formin Cdc12-mediated actin assembly in vitro, which allows cells to assemble functional contractile rings. Therefore an essential role of profilin is to specifically facilitate formin-mediated actin assembly for cytokinesis in fission yeast.     

Cooperation between Paxillin-like Protein Pxl1 and Glucan Synthase Bgs1 Is Essential for Actomyosin Ring Stability and Septum Formation in Fission Yeast

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 overgrowth 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.     

Nonmedially assembled F-actin cables incorporate into the actomyosin ring in fission yeast

In many eukaryotes, cytokinesis requires the assembly and constriction of an actomyosin-based contractile ring. Despite the central role of this ring in cytokinesis, the mechanism of F-actin assembly and accumulation in the ring is not fully understood. In this paper, we investigate the mechanism of F-actin assembly during cytokinesis in Schizosaccharomyces pombe using lifeact as a probe to monitor actin dynamics. Previous work has shown that F-actin in the actomyosin ring is assembled de novo at the division site. Surprisingly, we find that a significant fraction of F-actin in the ring was recruited from formin-Cdc12p nucleated long actin cables that were generated at multiple nonmedial locations and incorporated into the ring by a combination of myosin II and myosin V activities. Our results, together with findings in animal cells, suggest that de novo F-actin assembly at the division site and directed transport of F-actin cables assembled elsewhere can contribute to ring assembly.