Interactions among a Fimbrin, a Capping Protein, and an Actin-depolymerizing Factor in Organization of the Fission Yeast Actin Cytoskeleton

We report studies of the fission yeast fimbrin-like protein Fim1, which contains two EF-hand domains and two actin-binding domains (ABD1 and ABD2). Fim1 is a component of both F-actin patches and the F-actin ring, but not of F-actin cables. Fim1 cross-links F-actin in vitro, but a Fim1 protein lacking either EF-hand domains (Fim1A12) or both the EF-hand domains and ABD1 (Fim1A2) has no actin cross-linking activity. Overexpression of Fim1 induced the formation of F-actin patches throughout the cell cortex, whereas the F-actin patches disappear in cells overexpressing Fim1A12 or Fim1A2. Thus, the actin cross-linking activity of Fim1 is probably important for the formation of F-actin patches. The overexpression of Fim1 also excluded the actin-depolymerizing factor Adf1 from the F-actin patches and inhibited the turnover of actin in these structures. Thus, Fim1 may function in stabilizing the F-actin patches. We also isolated the gene encoding Acp1, a subunit of the heterodimeric F-actin capping protein. fim1 acp1 double null cells showed more severe defects in the organization of the actin cytoskeleton than those seen in each single mutant. Thus, Fim1 and Acp1 may function in a similar manner in the organization of the actin cytoskeleton. Finally, genetic studies suggested that Fim1 may function in cytokinesis in cooperation with Cdc15 (PSTPIP) and Rng2 (IQGAP), respectively.     

α-Actinin and fimbrin cooperate with myosin II to organize actomyosin bundles during contractile-ring assembly

The actomyosin contractile ring assembles through the condensation of a broad band of nodes that forms at the cell equator in fission yeast cytokinesis. The condensation process depends on actin filaments that interconnect nodes. By mutating or titrating actin cross-linkers α-actinin Ain1 and fimbrin Fim1 in live cells, we reveal that both proteins are involved in node condensation. Ain1 and Fim1 stabilize the actin cytoskeleton and modulate node movement, which prevents nodes and linear structures from aggregating into clumps and allows normal ring formation. Our computer simulations modeling actin filaments as semiflexible polymers reproduce the experimental observations and provide a model of how actin cross-linkers work with other proteins to regulate actin-filament orientations inside actin bundles and organize the actin network. As predicted by the simulations, doubling myosin II Myo2 level rescues the node condensation defects caused by Ain1 overexpression. Taken together, our work supports a cooperative process of ring self-organization driven by the interaction between actin filaments and myosin II, which is progressively stabilized by the cross-linking proteins.     

Actin filament bundling by fimbrin is important for endocytosis, cytokinesis, and polarization in fission yeast

Through the coordinated action of diverse actin-binding proteins, cells simultaneously assemble actin filaments with distinct architectures and dynamics to drive different processes. Actin filament cross-linking proteins organize filaments into higher order networks, although the requirement of cross-linking activity in cells has largely been assumed rather than directly tested. Fission yeast Schizosaccharomyces pombe assembles actin into three discrete structures: endocytic actin patches, polarizing actin cables, and the cytokinetic contractile ring. The fission yeast filament cross-linker fimbrin Fim1 primarily localizes to Arp2/3 complex-nucleated branched filaments of the actin patch and by a lesser amount to bundles of linear antiparallel filaments in the contractile ring. It is unclear whether Fim1 associates with bundles of parallel filaments in actin cables. We previously discovered that a principal role of Fim1 is to control localization of tropomyosin Cdc8, thereby facilitating cofilin-mediated filament turnover. Therefore, we hypothesized that the bundling ability of Fim1 is dispensable for actin patches but is important for the contractile ring and possibly actin cables. By directly visualizing actin filament assembly using total internal reflection fluorescence microscopy, we determined that Fim1 bundles filaments in both parallel and antiparallel orientations and efficiently bundles Arp2/3 complex-branched filaments in the absence but not the presence of actin capping protein. Examination of cells exclusively expressing a truncated version of Fim1 that can bind but not bundle actin filaments revealed that bundling activity of Fim1 is in fact important for all three actin structures. Therefore, fimbrin Fim1 has diverse roles as both a filament "gatekeeper" and as a filament cross-linker.     

Movement of a cytokinesis factor cdc12p to the site of cell division.

A key question in cytokinesis is how the plane of cell division is positioned within the cell. Although a number of cytokinesis factors involved in formation of the actomyosin contractile ring have been identified, little is known about how these factors are localized and assembled at the cell-division site. Cells of the fission yeast Schizosaccharomyces pombe divide using a medial actomyosin ring that assembles in early mitosis [1]. The S. pombe cdc12 gene encodes a formin, a member of a family of proteins that have functions in cytokinesis and cell polarity and that may bind Rho/Cdc42 GTPases, profilin and other actin-associated proteins [1] [2] [3] [4]. The cdc12 protein (cdc12p) is required specifically for medial-ring assembly during cytokinesis and is a component of this ring [2] [5]. In this study, cdc12p was found, during interphase, in a discrete, motile cytoplasmic spot that moved to the future site of cell division at the onset of mitosis. Three lines of evidence indicated that this cdc12p spot moved on both actin and microtubule networks: movement required either actin or microtubules; the spot was associated with actin and microtubule structures; and individual spots were seen to move along both microtubule and non-microtubule tracks. These findings demonstrate that a cytokinesis factor may travel on both microtubule and actin networks to the future site of cell division.     

Cytokinesis depends on the motor domains of myosin-II in fission yeast but not in budding yeast

Budding yeast possesses one myosin-II, Myo1p, whereas fission yeast has two, Myo2p and Myp2p, all of which contribute to cytokinesis. We find that chimeras consisting of Myo2p or Myp2p motor domains fused to the tail of Myo1p are fully functional in supporting budding yeast cytokinesis. Remarkably, the tail alone of budding yeast Myo1p localizes to the contractile ring, supporting both its constriction and cytokinesis. In contrast, fission yeast Myo2p and Myp2p require both the catalytic head domain as well as tail domains for function, with the tails providing distinct functions (Bezanilla and Pollard, 2000). Myo1p is the first example of a myosin whose cellular function does not require a catalytic motor domain revealing a novel mechanism of action for budding yeast myosin-II independent of actin binding and ATPase activity.     

Fission yeast myosin-I, Myo1p, stimulates actin assembly by Arp2/3 complex and shares functions with WASp

Fission yeast myo1(+) encodes a myosin-I with all three tail homology domains (TH1, 2, 3) found in typical long-tailed myosin-Is. Myo1p tail also contains a COOH-terminal acidic region similar to the A-domain of WASp/Scar proteins and other fungal myosin-Is. Our analysis shows that Myo1p and Wsp1p, the fission yeast WASp-like protein, share functions and cooperate in controlling actin assembly. First, Myo1p localizes to cortical patches enriched at tips of growing cells and at sites of cell division. Myo1p patches partially colocalize with actin patches and are dependent on an intact actin cytoskeleton. Second, although deletion of myo1(+) is not lethal, Deltamyo1 cells have actin cytoskeletal defects, including loss of polarized cell growth, delocalized actin patches, and mating defects. Third, additional disruption of wsp1(+) is synthetically lethal, suggesting that these genes may share functions. In mapping the domains of Myo1p tail that share function with Wsp1p, we discovered that a Myo1p construct with just the head and TH1 domains is sufficient for cortical localization and to rescue all Deltamyo1 defects. However, it fails to rescue the Deltamyo1 Deltawsp1 lethality. Additional tail domains, TH2 and TH3, are required to complement the double mutant. Fourth, we show that a recombinant Myo1p tail binds to Arp2/3 complex and activates its actin nucleation activity.     

Mto2p, a novel fission yeast protein required for cytoplasmic microtubule organization and anchoring of the cytokinetic actin ring

Microtubules regulate diverse cellular processes, including chromosome segregation, nuclear positioning, and cytokinesis. In many organisms, microtubule nucleation requires gamma-tubulin and associated proteins present at specific microtubule organizing centers (MTOCs). In fission yeast, interphase cytoplasmic microtubules originate from poorly characterized interphase MTOCs and spindle pole body (SPB), and during late anaphase from the equatorial MTOC (EMTOC). It has been previously shown that Mto1p (Mbo1p/Mod20p) function is important for the organization/nucleation of all cytoplasmic microtubules. Here, we show that Mto2p, a novel protein, interacts with Mto1p and is important for establishing a normal interphase cytoplasmic microtubule array. In addition, mto2Delta cells fail to establish a stable EMTOC and localize gamma-tubulin complex members to this medial structure. As predicted from these functions, Mto2p localizes to microtubules, the SPB, and the EMTOC in an Mto1p-dependent manner. mto2Delta cells fail to anchor the cytokinetic actin ring in the medial region of the cell and under conditions that mildly perturb actin structures, these rings unravel in mto2Delta cells. Our results suggest that the Mto2p and the EMTOC are critical for anchoring the cytokinetic actin ring to the medial region of the cell and for proper coordination of mitosis with cytokinesis.     

An InCytes from MBC Selection: Roles of Formin Nodes and Myosin Motor Activity in Mid1p-dependent Contractile-Ring Assembly during Fission Yeast Cytokinesis

Two prevailing models have emerged to explain the mechanism of contractile-ring assembly during cytokinesis in the fission yeast Schizosaccharomyces pombe: the spot/leading cable model and the search, capture, pull, and release (SCPR) model. We tested some of the basic assumptions of the two models. Monte Carlo simulations of the SCPR model require that the formin Cdc12p is present in >30 nodes from which actin filaments are nucleated and captured by myosin-II in neighboring nodes. The force produced by myosin motors pulls the nodes together to form a compact contractile ring. Live microscopy of cells expressing Cdc12p fluorescent fusion proteins shows for the first time that Cdc12p localizes to a broad band of 30–50 dynamic nodes, where actin filaments are nucleated in random directions. The proposed progenitor spot, essential for the spot/leading cable model, usually disappears without nucleating actin filaments. α-Actinin ain1 deletion cells form a normal contractile ring through nodes in the absence of the spot. Myosin motor activity is required to condense the nodes into a contractile ring, based on slower or absent node condensation in myo2-E1 and UCS rng3-65 mutants. Taken together, these data provide strong support for the SCPR model of contractile-ring formation in cytokinesis.     

The Schizosaccharomyces pombe cdc3+ gene encodes a profilin essential for cytokinesis

The fission yeast Schizosaccharomyces pombe divides by medial fission and, like many higher eukaryotic cells, requires the function of an F- actin contractile ring for cytokinesis. In S. pombe, a class of cdc- mutants defective for cytokinesis, but not for DNA replication, mitosis, or septum synthesis, have been identified. In this paper, we present the characterization of one of these mutants, cdc3-124. Temperature shift experiments reveal that mutants in cdc3 are incapable of forming an F-actin contractile ring. We have molecularly cloned cdc3 and used the cdc3+ genomic DNA to create a strain carrying a cdc3 null mutation by homologous recombination in vivo. Cells bearing a cdc3-null allele are inviable. They arrest the cell cycle at cytokinesis without forming a contractile ring. DNA sequence analysis of the cdc3+ gene reveals that it encodes profilin, an actin-monomer-binding protein. In light of recent studies with profilins, we propose that Cdc3-profilin plays an essential role in cytokinesis by catalyzing the formation of the F-actin contractile ring. Consistent with this proposal are our observations that Cdc3-profilin localizes to the medial region of the cell where the F-actin contractile ring forms, and that it is essential for F-actin ring formation. Cells overproducing Cdc3-profilin become elongated, dumbbell shaped, and arrest at cytokinesis without any detectable F-actin staining. This effect of Cdc3-profilin overproduction is relieved by introduction of a multicopy plasmid carrying the actin encoding gene, act1+. We attribute these effects to potential sequestration of actin monomers by profilin, when present in excess.     

Roles of Pdk1p, a fission yeast protein related to phosphoinositide-dependent protein kinase, in the regulation of mitosis and cytokinesis

Proteins related to the phosphoinositide-dependent protein kinase family have been identified in the majority of eukaryotes. Although much is known about upstream mechanisms that regulate the PDK1-family of kinases in metazoans, how these kinases regulate cell growth and division remains unclear. Here, we characterize a fission yeast protein related to members of this family, which we have termed Pdk1p. Pdk1p localizes to the spindle pole body and the actomyosin ring in early mitotic cells. Cells deleted for pdk1 display multiple defects in mitosis and cytokinesis, all of which are exacerbated when the function of fission yeast polo kinase, Plo1p, is partially compromised. We conclude that Pdk1p functions in concert with Plo1p to regulate multiple processes such as the establishment of a bipolar mitotic spindle, transition to anaphase, placement of the actomyosin ring and proper execution of cytokinesis. We also present evidence that the effects of Pdk1p on cytokinesis are likely mediated via the fission yeast anillin-related protein, Mid1p, and the septation initiation network.     

Isolation and characterization of new fission yeast cytokinesis mutants.

Schizosaccharomyces pombe is an excellent organism in which to study cytokinesis as it divides by medial fission using an F-actin contractile ring. To enhance our understanding of the cell division process, a large genetic screen was carried out in which 17 genetic loci essential for cytokinesis were identified, 5 of which are novel. Mutants identifying three genes, rng3(+), rng4(+), and rng5(+), were defective in organizing an actin contractile ring. Four mutants defective in septum deposition, septum initiation defective (sid)1, sid2, sid3, and sid4, were also identified and characterized. Genetic analyses revealed that the sid mutants display strong negative interactions with the previously described septation mutants cdc7-24, cdc11-123, and cdc14-118. The rng5(+), sid2(+), and sid3(+) genes were cloned and shown to encode Myo2p (a myosin heavy chain), a protein kinase related to budding yeast Dbf2p, and Spg1p, a GTP binding protein that is a member of the ras superfamily of GTPases, respectively. The ability of Spg1p to promote septum formation from any point in the cell cycle depends on the activity of Sid4p. In addition, we have characterized a phenotype that has not been described previously in cytokinesis mutants, namely the failure to reorganize actin patches to the medial region of the cell in preparation for septum 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.     

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.     

SEPH, a Cdc7p orthologue from Aspergillus nidulans, functions upstream of actin ring formation during cytokinesis

In the filamentous fungus, Aspergillus nidulans, multiple rounds of nuclear division occur before cytokinesis, allowing an unambiguous identification of genes required specifically for cytokinesis. As in animal cells, both an intact microtubule cytoskeleton and progression through mitosis are required for actin ring formation and contraction. The sepH gene from A. nidulans was discovered in a screen for temperature-sensitive cytokinesis mutants. Sequence analysis showed that SEPH is 42% identical to the serine-threonine kinase Cdc7p from fission yeast. Signalling through the Septation Initiation Network (SIN), which includes Cdc7p and the GTPase Spg1p, is emerging as a primary regulatory pathway used by fission yeast to control cytokinesis. A similar group of proteins comprise the Mitotic Exit Network (MEN) in budding yeast. This is the first direct evidence for the existence of a functional SIN-MEN pathway outside budding and fission yeast. In addition to SEPH, potential homologues were also identified in other fungi and plants but not in animal cells. Deletion of sepH resulted in a viable strain that failed to septate at any temperature. Interestingly, quantitative analysis of the actin cytoskeleton revealed that sepH is required for construction of the actin ring. Therefore, SEPH is distinct from its counterpart in fission yeast, in which SIN components operate downstream of actin ring formation and are necessary for ring contraction and later events of septation. We conclude that A. nidulans has components of a SIN-MEN pathway, one of which, SEPH, is required for early events during cytokinesis.     

HIV-1 Vpr induces defects in mitosis, cytokinesis, nuclear structure, and centrosomes

Human immunodeficiency virus type 1 (HIV-1) Vpr is a 15-kDa accessory protein that contributes to several steps in the viral replication cycle and promotes virus-associated pathology. Previous studies demonstrated that Vpr inhibits G2/M cell cycle progression in both human cells and in the fission yeast Schizosaccharomyces pombe. Here, we report that, upon induction of vpr expression, fission yeast exhibited numerous defects in the assembly and function of the mitotic spindle. In particular, two spindle pole body proteins, sad1p and the polo kinase plo1p, were delocalized in vpr-expressing yeast cells, suggesting that spindle pole body integrity was perturbed. In addition, nuclear envelope structure, contractile actin ring formation, and cytokinesis were also disrupted. Similar Vpr-induced defects in mitosis and cytokinesis were observed in human cells, including aberrant mitotic spindles, multiple centrosomes, and multinucleate cells. These defects in cell division and centrosomes might account for some of the pathological effects associated with HIV-1 infection.     

Stg 1 is a novel SM22/transgelin-like actin-modulating protein in fission yeast

We identified a novel actin-modulating protein Stg 1 in the fission yeast Schizosaccharomyces pombe. Stg 1 is similar to mammalian SM22/transgelin, and biochemical experiments showed that Stg 1 crosslinked F-actin. Microscopic observation suggested that Stg 1 was a component of actin patch. Overexpression of Stg 1 caused a defect in cytokinesis by suppressing the formation of a contractile ring and formation of abnormal aggregates of F-actin in the ends and mid-region of cells. Although distribution of the actin cytoskeleton was not affected by disrupting Stg 1(+), genetic interaction suggested that Stg 1 was likely involved in controlling the organization of the actin cytoskeleton in cell morphogenesis and cytokinesis in fission yeast.     

Fission yeast Aip3p (spAip3p) is required for an alternative actin-directed polarity program

Aip3p is an actin-interacting protein that regulates cell polarity in budding yeast. The Schizosaccharomyces pombe-sequencing project recently led to the identification of a homologue of Aip3p that we have named spAip3p. Our results confirm that spAip3p is a true functional homologue of Aip3p. When expressed in budding yeast, spAip3p localizes similarly to Aip3p during the cell cycle and complements the cell polarity defects of an aip3Delta strain. Two-hybrid analysis shows that spAip3p interacts with actin similarly to Aip3p. In fission yeast, spAip3p localizes to both cell ends during interphase and later organizes into two rings at the site of cytokinesis. spAip3p localization to cell ends is dependent on microtubule cytoskeleton, its localization to the cell middle is dependent on actin cytoskeleton, and both patterns of localization require an operative secretory pathway. Overexpression of spAip3p disrupts the actin cytoskeleton and cell polarity, leading to morphologically aberrant cells. Fission yeast, which normally rely on the microtubule cytoskeleton to establish their polarity axis, can use the actin cytoskeleton in the absence of microtubule function to establish a new polarity axis, leading to the formation of branched cells. spAip3p localizes to, and is required for, branch formation, confirming its role in actin-directed polarized cell growth in both Schizosaccharomyces pombe and Saccharomyces cerevisiae.     

Roles of the DYRK kinase Pom2 in cytokinesis, mitochondrial morphology, and sporulation in fission yeast

Pom2 is predicted to be a dual-specificity tyrosine-phosphorylation regulated kinase (DYRK) related to Pom1 in Schizosaccharomyces pombe. DYRKs share a kinase domain capable of catalyzing autophosphorylation on tyrosine and exogenous phosphorylation on serine/threonine residues. Here we show that Pom2 is functionally different from the well-characterized Pom1, although they share 55% identity in the kinase domain and the Pom2 kinase domain functionally complements that of Pom1. Pom2 localizes to mitochondria throughout the cell cycle and to the contractile ring during late stages of cytokinesis. Overexpression but not deletion of pom2 results in severe defects in cytokinesis, indicating that Pom2 might share an overlapping function with other proteins in regulating cytokinesis. Gain and loss of function analyses reveal that Pom2 is required for maintaining mitochondrial morphology independently of microtubules. Intriguingly, most meiotic pom2Δ cells form aberrant asci with meiotic and/or forespore membrane formation defects. Taken together, Pom2 is a novel DYRK kinase involved in regulating cytokinesis, mitochondrial morphology, meiosis, and sporulation in fission yeast.     

Bni5p,a septin-interacting protein,is required for normal septin function and cytokinesis in Saccharomyces cerevisiae

In the budding yeast Saccharomyces cerevisiae, the Cdc3p, Cdc10p, Cdc11p, Cdc12p, and Sep7p/Shs1p septins assemble early in the cell cycle in a ring that marks the future cytokinetic site. The septins appear to be major structural components of a set of filaments at the mother-bud neck and function as a scaffold for recruiting proteins involved in cytokinesis and other processes. We isolated a novel gene, BNI5, as a dosage suppressor of the cdc12-6 growth defect. Overexpression of BNI5 also suppressed the growth defects of cdc10-1, cdc11-6, and sep7Delta strains. Loss of BNI5 resulted in a cytokinesis defect, as evidenced by the formation of connected cells with shared cytoplasms, and deletion of BNI5 in a cdc3-6, cdc10-1, cdc11-6, cdc12-6, or sep7Delta mutant strain resulted in enhanced defects in septin localization and cytokinesis. Bni5p localizes to the mother-bud neck in a septin-dependent manner shortly after bud emergence and disappears from the neck approximately 2 to 3 min before spindle disassembly. Two-hybrid, in vitro binding, and protein-localization studies suggest that Bni5p interacts with the N-terminal domain of Cdc11p, which also appears to be sufficient for the localization of Cdc11p, its interaction with other septins, and other critical aspects of its function. Our data suggest that the Bni5p-septin interaction is important for septin ring stability and function, which is in turn critical for normal cytokinesis.     

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.