The Cdc42p GTPase and its regulators Nrf1p and Scd1p are involved in endocytic trafficking in the fission yeast Schizosaccharomyces pombe

Nrf1p was first identified in a screen for negative regulators of the Cdc42p GTPase. Overexpression of Nrf1p resulted in dose-dependent lethality, with cells exhibiting an ellipsoidal morphology and abnormal vacuolar phenotypes including an increase in vacuolar fusion. Green fluorescent protein (GFP)-Cdc42p and GFP-Nrf1p colocalized to vacuolar membranes and GFP-Nrf1p vacuolar localization depended on Scd1p, the Schizosaccharomyces pombe homolog of the Cdc24p guanine nucleotide exchange factor. In this study, site-directed mutagenesis was conducted on Nrf1p to determine its functional domains. Mutations in the three putative transmembrane domains resulted in mislocalization of GFP-Nrf1p and an inability to induce lethality, suggesting a loss of function. Mutations in the second extramembranous loop of Nrf1p also resulted in a loss of function and altered the ability of GFP-Nrf1p to localize to vacuolar membranes. Analysis of Deltanrf1 and Deltascd1 mutants revealed defects in endocytosis. In addition, overexpression of constitutively active Cdc42(G12V)p resulted in an increase in endocytosis and an ability to rescue the endocytic defects in Deltanrf1 and Deltascd1 cells. These data are consistent with Nrf1p and Scd1p being necessary for efficient endocytosis, possibly through the regulation of Cdc42p.     

Cdc42p GTPase is involved in controlling polarized cell growth in Schizosaccharomyces pombe.

Cdc42p is a highly conserved low-molecular-weight GTPase that is involved in controlling cellular morphogenesis. We have isolated the Cdc42p homolog from the fission yeast Schizosaccharomyces pombe by its ability to complement the Saccharomyces cerevisiae cdc42-1ts mutation. S. pombe Cdc42p is 85% identical in predicted amino acid sequence to S. cerevisiae Cdc42p and 83% identical to the human Cdc42p homolog. The Cdc42p protein fractionates to both soluble and particulate fractions, suggesting that it exists in two cellular pools. We have disrupted the cdc42+ gene and shown that it is essential for growth. The cdc42 null phenotype is an arrest as small, round, dense cells. In addition, we have generated three site-specific mutations, G12V, Q61L, and D118A, in the Cdc42p GTP-binding domains that correspond to dominant-lethal mutations in S. cerevisiae CDC42. In contrast to the S. cerevisiae cdc42 mutations, the S. pombe cdc42 mutant alleles were not lethal when overexpressed. However, the cdc42 mutants did exhibit an abnormal morphological phenotype of large, misshapen cells, suggesting that S. pombe Cdc42p is involved in controlling polarized cell growth.     

Gef1p, a new guanine nucleotide exchange factor for Cdc42p, regulates polarity in Schizosaccharomyces pombe

Schizosaccharomyces pombe cdc42(+) regulates cell morphology and polarization of the actin cytoskeleton. Scd1p/Ral1p is the only described guanine nucleotide exchange factor (GEF) for Cdc42p in S. pombe. We have identified a new GEF, named Gef1p, specifically regulating Cdc42p. Gef1p binds to inactive Cdc42p but not to other Rho GTPases in two-hybrid assays. Overexpression of gef1(+) increases specifically the GTP-bound Cdc42p, and Gef1p is capable of stimulating guanine nucleotide exchange of Cdc42p in vitro. Overexpression of gef1(+) causes changes in cell morphology similar to those caused by overexpression of the constitutively active cdc42G12V allele. Gef1p localizes to the septum. gef1(+) deletion is viable but causes a mild cell elongation and defects in bipolar growth and septum formation, suggesting a role for Gef1p in the control of cell polarity and cytokinesis. The double mutant gef1delta scd1delta is not viable, indicating that they share an essential function as Cdc42p activators. However, both deletion and overexpression of either gef1(+) or scd1(+) causes different morphological phenotypes, which suggest different functions. Genetic evidence revealed a link between Gef1p and the signaling pathway of Shk1/Orb2p and Orb6p. In contrast, no genetic interaction between Gef1p and Shk2p-Mkh1p pathway was observed.     

Cdc50p,a conserved endosomal membrane protein,controls polarized growth in Saccharomyces cerevisiae

During the cell cycle of the yeast Saccharomyces cerevisiae, the actin cytoskeleton and the growth of cell surface are polarized, mediating bud emergence, bud growth, and cytokinesis. We identified CDC50 as a multicopy suppressor of the myo3 myo5-360 temperature-sensitive mutant, which is defective in organization of cortical actin patches. The cdc50 null mutant showed cold-sensitive cell cycle arrest with a small bud as reported previously. Cortical actin patches and Myo5p, which are normally localized to polarization sites, were depolarized in the cdc50 mutant. Furthermore, actin cables disappeared, and Bni1p and Gic1p, effectors of the Cdc42p small GTPase, were mislocalized in the cdc50 mutant. As predicted by its amino acid sequence, Cdc50p appears to be a transmembrane protein because it was solubilized from the membranes by detergent treatment. Cdc50p colocalized with Vps21p in endosomal compartments and was also localized to the class E compartment in the vps27 mutant. The cdc50 mutant showed defects in a late stage of endocytosis but not in the internalization step. It showed, however, only modest defects in vacuolar protein sorting. Our results indicate that Cdc50p is a novel endosomal protein that regulates polarized cell growth.     

Characterization of Synthetic-Lethal Mutants Reveals a Role for the Saccharomyces Cerevisiae Guanine-Nucleotide Exchange Factor Cdc24p in Vacuole Function and Na(+) Tolerance

Cdc24p is the guanine-nucleotide exchange factor for the Cdc42p GTPase, which controls cell polarity in Saccharomyces cerevisiae. To identify new genes that may affect cell polarity, we characterized six UV-induced csl (CDC24 synthetic-lethal) mutants that exhibited synthetic-lethality with cdc24-4(ts) at 23°. Five mutants were not complemented by plasmid-borne CDC42, RSR1, BUD5, BEM1, BEM2, BEM3 or CLA4 genes, which are known to play a role in cell polarity. The csl3 mutant displayed phenotypes similar to those observed with calcium-sensitive, Pet(-) vma mutants defective in vacuole function. CSL5 was allelic to VMA5, the vacuolar H(+)-ATPase subunit C, and one third of csl5 cdc24-4(ts) cells were elongated or had misshapen buds. A cdc24-4(ts) Δvma5::LEU2 double mutant did not exhibit synthetic lethality, suggesting that the csl5/vma5 cdc24-4(ts) synthetic-lethality was not simply due to altered vacuole function. The cdc24-4(ts) mutant, like Δvma5::LEU2 and csl3 mutants, was sensitive to high levels of Ca(2+) as well as Na(+) in the growth media, which did not appear to be a result of a fragile cell wall because the phenotypes were not remedied by 1 M sorbitol. Our results indicated that Cdc24p was required in one V-ATPase mutant and another mutant affecting vacuole morphology, and also implicated Cdc24p in Na(+) tolerance.     

Identification of novel, evolutionarily conserved Cdc42p-interacting proteins and of redundant pathways linking Cdc24p and Cdc42p to actin polarization in yeast

In the yeast Saccharomyces cerevisiae, Cdc24p functions at least in part as a guanine-nucleotide-exchange factor for the Rho-family GTPase Cdc42p. A genetic screen designed to identify possible additional targets of Cdc24p instead identified two previously known genes, MSB1 and CLA4, and one novel gene, designated MSB3, all of which appear to function in the Cdc24p-Cdc42p pathway. Nonetheless, genetic evidence suggests that Cdc24p may have a function that is distinct from its Cdc42p guanine-nucleotide-exchange factor activity; in particular, overexpression of CDC42 in combination with MSB1 or a truncated CLA4 in cells depleted for Cdc24p allowed polarization of the actin cytoskeleton and polarized cell growth, but not successful cell proliferation. MSB3 has a close homologue (designated MSB4) and two more distant homologues (MDR1 and YPL249C) in S. cerevisiae and also has homologues in Schizosaccharomyces pombe, Drosophila (pollux), and humans (the oncogene tre17). Deletion of either MSB3 or MSB4 alone did not produce any obvious phenotype, and the msb3 msb4 double mutant was viable. However, the double mutant grew slowly and had a partial disorganization of the actin cytoskeleton, but not of the septins, in a fraction of cells that were larger and rounder than normal. Like Cdc42p, both Msb3p and Msb4p localized to the presumptive bud site, the bud tip, and the mother-bud neck, and this localization was Cdc42p dependent. Taken together, the data suggest that Msb3p and Msb4p may function redundantly downstream of Cdc42p, specifically in a pathway leading to actin organization. From previous work, the BNI1, GIC1, and GIC2 gene products also appear to be involved in linking Cdc42p to the actin cytoskeleton. Synthetic lethality and multicopy suppression analyses among these genes, MSB, and MSB4, suggest that the linkage is accomplished by two parallel pathways, one involving Msb3p, Msb4p, and Bni1p, and the other involving Gic1p and Gic2p. The former pathway appears to be more important in diploids and at low temperatures, whereas the latter pathway appears to be more important in haploids and at high temperatures.     

Saccharomyces cerevisiae cdc42p GTPase is involved in preventing the recurrence of bud emergence during the cell cycle

The Saccharomyces cerevisiae Cdc42p GTPase interacts with multiple regulators and downstream effectors through an approximately 25-amino-acid effector domain. Four effector domain mutations, Y32K, F37A, D38E, and Y40C, were introduced into Cdc42p and characterized for their effects on these interactions. Each mutant protein showed differential interactions with a number of downstream effectors and regulators and various levels of functionality. Specifically, Cdc42(D38E)p showed reduced interactions with the Cla4p p21-activated protein kinase and the Bem3p GTPase-activating protein and cdc42(D38E) was the only mutant allele able to complement the Deltacdc42 null mutant. However, the mutant protein was only partially functional, as indicated by a temperature-dependent multibudded phenotype seen in conjunction with defects in both septin ring localization and activation of the Swe1p-dependent morphogenetic checkpoint. Further analysis of this mutant suggested that the multiple buds emerged consecutively with a premature termination of bud enlargement preceding the appearance of the next bud. Cortical actin, the septin ring, Cla4p-green fluorescent protein (GFP), and GFP-Cdc24p all predominantly localized to one bud at a time per multibudded cell. These data suggest that Cdc42(D38E)p triggers a morphogenetic defect post-bud emergence, leading to cessation of bud growth and reorganization of the budding machinery to another random budding site, indicating that Cdc42p is involved in prevention of the initiation of supernumerary buds during the cell cycle.     

Cdc42p GDP/GTP cycling is necessary for efficient cell fusion during yeast mating

The highly conserved small Rho G-protein, Cdc42p plays a critical role in cell polarity and cytoskeleton organization in all eukaryotes. In the yeast Saccharomyces cerevisiae, Cdc42p is important for cell polarity establishment, septin ring assembly, and pheromone-dependent MAP-kinase signaling during the yeast mating process. In this study, we further investigated the role of Cdc42p in the mating process by screening for specific mating defective cdc42 alleles. We have identified and characterized novel mating defective cdc42 alleles that are unaffected in vegetative cell polarity. Replacement of the Cdc42p Val36 residue with Met resulted in a specific cell fusion defect. This cdc42[V36M] mutant responded to mating pheromone but was defective in cell fusion and in localization of the cell fusion protein Fus1p, similar to a previously isolated cdc24 (cdc24-m6) mutant. Overexpression of a fast cycling Cdc42p mutant suppressed the cdc24-m6 fusion defect and conversely, overexpression of Cdc24p suppressed the cdc42[V36M] fusion defect. Taken together, our results indicate that Cdc42p GDP-GTP cycling is critical for efficient cell fusion.     

The Cdc42p GTPase is involved in a G2/M morphogenetic checkpoint regulating the apical-isotropic switch and nuclear division in yeast.

The Cdc42p GTPase is involved in the signal transduction cascades controlling bud emergence and polarized cell growth in S. cerevisiae. Cells expressing the cdc42(V44A) effector domain mutant allele displayed morphological defects of highly elongated and multielongated budded cells indicative of a defect in the apical-isotropic switch in bud growth. In addition, these cells contained one, two, or multiple nuclei indicative of a G2/M delay in nuclear division and also a defect in cytokinesis and/or cell separation. Actin and chitin were delocalized, and septin ring structure was aberrant and partially delocalized to the tips of elongated cdc42(V44A) cells; however, Cdc42(V44A)p localization was normal. Two-hybrid protein analyses showed that the V44A mutation interfered with Cdc42p's interactions with Cla4p, a p21(Cdc42/Rac)-activated kinase (PAK)-like kinase, and the novel effectors Gic1p and Gic2p, but not with the Ste20p or Skm1p PAK-like kinases, the Bni1p formin, or the Iqg1p IQGAP homolog. Furthermore, the cdc42(V44A) morphological defects were suppressed by deletion of the Swe1p cyclin-dependent kinase inhibitory kinase and by overexpression of Cla4p, Ste20p, the Cdc12 septin protein, or the guanine nucleotide exchange factor Cdc24p. In sum, these results suggest that proper Cdc42p function is essential for timely progression through the apical-isotropic switch and G2/M transition and that Cdc42(V44A)p differentially interacts with a number of effectors and regulators.     

Myb-Related Fission Yeast cdc5p Is a Component of a 40S snRNP-Containing Complex and Is Essential for Pre-mRNA Splicing

Myb-related cdc5p is required for G(2)/M progression in the yeast Schizosaccharomyces pombe. We report here that all detectable cdc5p is stably associated with a multiprotein 40S complex. Immunoaffinity purification has allowed the identification of 10 cwf (complexed with cdc5p) proteins. Two (cwf6p and cwf10p) are members of the U5 snRNP; one (cwf9p) is a core snRNP protein. cwf8p is the apparent ortholog of the Saccharomyces cerevisiae splicing factor Prp19p. cwf1(+) is allelic to the prp5(+) gene defined by the S. pombe splicing mutant, prp5-1, and there is a strong negative genetic interaction between cdc5-120 and prp5-1. Five cwfs have not been recognized previously as important for either pre-mRNA splicing or cell cycle control. Further characterization of cwf1p, cwf2p, cwf3p, and cwf4p demonstrates that they are encoded by essential genes, cosediment with cdc5p at 40S, and coimmunoprecipitate with cdc5p. We further show that cdc5p associates with the U2, U5, and U6 snRNAs and that cells lacking cdc5(+) function are defective in pre-mRNA splicing. These data raise the possibility that the cdc5p complex is an intermediate in the assembly or disassembly of an active S. pombe spliceosome.     

Mutational Analysis of Cdc19p, a Schizosaccharomyces Pombe Mcm Protein

The cdc19(+) gene encodes an essential member of the MCM family of replication proteins in Schizosaccharomyces pombe. We have examined the structure and function of the Cdc19p protein using molecular and genetic approaches. We find that overproduction of wild-type Cdc19p in wild-type cells has no effect, but cdc19-P1 mutant cells do not tolerate elevated levels of other MCM proteins or overexpression of mutant forms of Cdc19p. We have found genetic interactions between cdc19(+) and genes encoding subunits of DNA polymerase {delta small} and the replication initiator cdc18(+). We have constructed a series of point mutations and sequence deletions throughout Cdc19p, which allow us to distinguish essential from nonessential regions of the protein. Not surprisingly, conserved residues in the MCM homology domain are required for protein function, but some residues outside the core homology domain are dispensable.     

S. pombe cdc11p, together with sid4p, provides an anchor for septation initiation network proteins on the spindle pole body.

BACKGROUND: The signal for the onset of septum formation in the fission yeast Schizosaccharomyces pombe is transduced by the septation initiation network (SIN). Many of the components of the SIN are located on the spindle pole body during mitosis, from where it is presumed that the signal for septum formation is delivered. Cdc11 mutants are defective in SIN signaling, but the role of cdc11 in the pathway has remained enigmatic. RESULTS: We have cloned the cdc11 gene by a combination of chromosome walking and transfection of cosmids into a cdc11 mutant. Cdc11p most closely resembles Saccharomyces cerevisiae Nud1p and is essential for septum formation. Cdc11p is a phosphoprotein, which becomes hyperphosphorylated during anaphase. It localizes to the spindle pole body at all stages of the cell cycle, in a sid4p-dependent manner, and cdc11p is required for the localization of all the known SIN components, except sid4p, to the SPB. Cdc11p and sid4p can be coimmunoprecipitated from cell extracts. Finally, like its S. cerevisiae ortholog Nud1p, cdc11p is involved in the proper organization of astral microtubules during mitosis. CONCLUSIONS: We propose that cdc11p acts as a bridge between sid4p and the other SIN proteins, mediating their association with the spindle pole body.     

Subcellular localization of Cdc42p, a Saccharomyces cerevisiae GTP-binding protein involved in the control of cell polarity.

The Saccharomyces cerevisiae Cdc42 protein, a member of the Ras superfamily of low-molecular-weight GTP-binding proteins, is involved in the control of cell polarity during the yeast cell cycle. This protein has a consensus sequence (CAAX) for geranylgeranyl modification and is likely to be associated, at least in part, with cell membranes. Using cell fractionation and immunolocalization techniques, we have investigated the subcellular localization of Cdc42p. Cdc42p was found in both soluble and particulate pools, and neither its abundance nor its distribution varied through the cell cycle. The particulate form of Cdc42p could be solubilized with detergents but not with NaCl or urea, suggesting that it is tightly associated with membranes. An increase in soluble Cdc42p was observed in a geranylgeranyltransferase mutant strain (cdc43-2ts) grown at the restrictive temperature. In addition, Cdc42p from a cdc42C188S mutant strain (that has an alteration at the prenylation consensus site) was almost exclusively in the soluble fraction, suggesting that membrane localization is dependent on geranylgeranyl modification at Cys-188. Immunofluorescence and immunoelectron microscopy experiments demonstrated that Cdc42p localizes to the plasma membrane in the vicinity of secretory vesicles that were found at the site of bud emergence, at the tips and sides of enlarging buds, and within mating projections (shmoo tips) in alpha-factor-arrested cells. These results indicate that Cdc42p is localized to the bud site early in the cell cycle and suggest that this localization is critical for the selection of the proper site for bud emergence and for polarized cell growth.     

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.     

Characterization of ypa1 and ypa2, the Schizosaccharomyces pombe orthologs of the peptidyl proyl isomerases that activate PP2A, reveals a role for Ypa2p in the regulation of cytokinesis

The Schizosaccharomyces pombe septation initiation network (SIN) regulates cytokinesis. Cdc7p is the first kinase in the core SIN; we have screened genetically for SIN regulators by isolating cold-sensitive suppressors of cdc7-24. Our screen yielded a mutant in SPAC1782.05, one of the two fission yeast orthologs of mammalian phosphotyrosyl phosphatase activator. We have characterized this gene and its ortholog SPAC4F10.04, which we have named ypa2 and ypa1, respectively. We find that Ypa2p is the major form of protein phosphatase type 2A activator in S. pombe. A double ypa1-Δ ypa2-Δ null mutant is inviable, indicating that the two gene products have at least one essential overlapping function. Individually, the ypa1 and ypa2 genes are essential for survival only at low temperatures. The ypa2-Δ mutant divides at a reduced cell size and displays aberrant cell morphology and cytokinesis. Genetic analysis implicates Ypa2p as an inhibitor of the septation initiation network. We also isolated a cold-sensitive allele of ppa2, the major protein phosphatase type 2A catalytic subunit, implicating this enzyme as a regulator of the septation initiation network.     

Functions and functional domains of the GTPase Cdc42p

Cdc42p, a Rho family GTPase of the Ras superfamily, is a key regulator of cell polarity and morphogenesis in eukaryotes. Using 37 site-directed cdc42 mutants, we explored the functions and interactions of Cdc42p in the budding yeast Saccharomyces cerevisiae. Cytological and genetic analyses of these cdc42 mutants revealed novel and diverse phenotypes, showing that Cdc42p possesses at least two distinct essential functions and acts as a nodal point of cell polarity regulation in vivo. In addition, mapping the functional data for each cdc42 mutation onto a structural model of the protein revealed as functionally important a surface of Cdc42p that is distinct from the canonical protein-interacting domains (switch I, switch II, and the C terminus) identified previously in members of the Ras superfamily. This region overlaps with a region (alpha5-helix) recently predicted by structural models to be a specificity determinant for Cdc42p-protein interactions.     

Cdc1p is an endoplasmic reticulum-localized putative lipid phosphatase that affects Golgi inheritance and actin polarization by activating Ca2+ signaling

In the budding yeast Saccharomyces cerevisiae, mutations in the essential gene CDC1 cause defects in Golgi inheritance and actin polarization. However, the biochemical function of Cdc1p is unknown. Previous work showed that cdc1 mutants accumulate intracellular Ca(2+) and display enhanced sensitivity to the extracellular Mn(2+) concentration, suggesting that Cdc1p might regulate divalent cation homeostasis. By contrast, our data indicate that Cdc1p is a Mn(2+)-dependent protein that can affect Ca(2+) levels. We identified a cdc1 allele that activates Ca(2+) signaling but does not show enhanced sensitivity to the Mn(2+) concentration. Furthermore, our studies show that Cdc1p is an endoplasmic reticulum-localized transmembrane protein with a putative phosphoesterase domain facing the lumen. cdc1 mutant cells accumulate an unidentified phospholipid, suggesting that Cdc1p may be a lipid phosphatase. Previous work showed that deletion of the plasma membrane Ca(2+) channel Cch1p partially suppressed the cdc1 growth phenotype, and we find that deletion of Cch1p also suppresses the Golgi inheritance and actin polarization phenotypes. The combined data fit a model in which the cdc1 mutant phenotypes result from accumulation of a phosphorylated lipid that activates Ca(2+) signaling.     

Cdc42p and Fus2p act together late in yeast cell fusion

Cell fusion is the key event of fertilization that gives rise to the diploid zygote and is a nearly universal aspect of eukaryotic biology. In the yeast Saccharomyces cerevisiae, several mutants have been identified that are defective for cell fusion, and yet the molecular mechanism of this process remains obscure. One obstacle has been that genetic screens have mainly focused on mating-specific factors, whereas the process likely involves housekeeping proteins as well. Here we implicate Cdc42p, an essential protein with roles in multiple aspects of morphogenesis, as a core component of the yeast cell fusion pathway. We identify a point mutant in the Rho-insert domain of CDC42, called cdc42-138, which is specifically defective in cell fusion. The cell fusion defect is not a secondary consequence of ineffective signaling or polarization. Genetic and morphological data show that Cdc42p acts at a late stage in cell fusion in concert with a key cell fusion regulator, Fus2p, which contains a Dbl-homology domain. We find that Fus2p binds specifically with activated Cdc42p, and binding is blocked by the cdc42-138 mutation. Thus, in addition to signaling and morphogenetic roles in mating, Cdc42p plays a role late in cell fusion via activation of Fus2p.     

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.