Genetic analysis of cell morphogenesis in fission yeast--a role for casein kinase II in the establishment of polarized growth.

We have initiated a study to identify genes regulating cell morphogenesis in the fission yeast Schizosaccharomyces pombe. Five genes have been identified, orb1-orb5, whose mutation gives rise to spherical cells, indicative of an inability to polarize growth. Two further genes have been identified, tea1 and ban1, whose mutant alleles have disturbed patterns of tip growth, leading to T-shaped and curved cells. In fission yeast, sites of cell wall deposition are defined by actin localization, with actin distributions and therefore growth patterns undergoing cell cycle stage-specific reorganization. Studies of double mutants constructed between orb5-19 and various cdc mutants blocked before and after cell division show that orb5 is required for the re-establishment of polar growth following cytokinesis. This indicates that the mutant allele orb5-19 is defective in the reinitiation of polarized growth, even though actin reorganization to the cell tips occurs normally. orb5 encodes a fission yeast homologue of casein kinase II alpha. We propose that this kinase plays a role in the translation of cell polarity into polarized growth, but not in the establishment of polarity itself.     

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.     

cdc12p, a Protein Required for Cytokinesis in Fission Yeast, Is a Component of the Cell Division Ring and Interacts with Profilin

As in many other eukaryotic cells, cell division in fission yeast depends on the assembly of an actin ring that circumscribes the middle of the cell. Schizosaccharomyces pombe cdc12 is an essential gene necessary for actin ring assembly and septum formation. Here we show that cdc12p is a member of a family of proteins including Drosophila diaphanous, Saccharomyces cerevisiae BNI1, and S. pombe fus1, which are involved in cytokinesis or other actin-mediated processes. Using indirect immunofluorescence, we show that cdc12p is located in the cell division ring and not in other actin structures. When overexpressed, cdc12p is located at a medial spot in interphase that anticipates the future ring site. cdc12p localization is altered in actin ring mutants. cdc8 (tropomyosin homologue), cdc3 (profilin homologue), and cdc15 mutants exhibit no specific cdc12p staining during mitosis. cdc4 mutant cells exhibit a medial cortical cdc12p spot in place of a ring. mid1 mutant cells generally exhibit a cdc12p spot with a single cdc12p strand extending in a random direction. Based on these patterns, we present a model in which ring assembly originates from a single point on the cortex and in which a molecular pathway for the functions of cytokinesis proteins is suggested. Finally, we found that cdc12 and cdc3 mutants show a syntheticlethal genetic interaction, and a proline-rich domain of cdc12p binds directly to profilin cdc3p in vitro, suggesting that one function of cdc12p in ring assembly is to bind profilin.     

Actin- and tubulin-dependent functions during Saccharomyces cerevisiae mating projection formation.

Several conditional-lethal mutant alleles of the single-copy Saccharomyces cerevisiae beta-tubulin and actin genes were used to evaluate the roles of microtubules and actin filaments in the pheromone-induced extension of mating projections. Mutants defective in tubulin assembly form projections indistinguishable in appearance from those formed by wild-type cells. However, the tubulin mutants are unable to move their nuclei into the projections and to orient the spindle pole body associated with each nucleus toward the projection tip. Actin mutants are defective in spatial orientation of cell-surface growth required for formation of normal mating projections. Migration of nuclei into mating projections and Spa2p segregation to projection tips are also defective in actin mutants. Studies with abp1 null mutants showed that the function of the Abp1p actin-binding protein is either not required for projection formation or there are other proteins in yeast with similar functions. Our findings demonstrate that actin is required to restrict cell-surface growth to a defined region for pheromone-induced morphogenesis and suggest that nuclear position and orientation in mating projections depend on direct or indirect interaction of microtubules with actin filaments.     

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.     

Tropomyosin and Myosin-II Cellular Levels Promote Actomyosin Ring Assembly in Fission Yeast

Myosin-II (Myo2p) and tropomyosin are essential for contractile ring formation and cytokinesis in fission yeast. Here we used a combination of in vivo and in vitro approaches to understand how these proteins function at contractile rings. We find that ring assembly is delayed in Myo2p motor and tropomyosin mutants, but occurs prematurely in cells engineered to express two copies of myo2. Thus, the timing of ring assembly responds to changes in Myo2p cellular levels and motor activity, and the emergence of tropomyosin-bound actin filaments. Doubling Myo2p levels suppresses defects in ring assembly associated with a tropomyosin mutant, suggesting a role for tropomyosin in maximizing Myo2p function. Correspondingly, tropomyosin increases Myo2p actin affinity and ATPase activity and promotes Myo2p-driven actin filament gliding in motility assays. Tropomyosin achieves this by favoring the strong actin-bound state of Myo2p. This mode of regulation reflects a role for tropomyosin in specifying and stabilizing actomyosin interactions, which facilitates contractile ring assembly in the fission yeast system.     

Role of the Rab GTP-binding protein Ypt3 in the fission yeast exocytic pathway and its connection to calcineurin function

A genetic screen for mutations synthetically lethal with fission yeast calcineurin deletion led to the identification of Ypt3, a homolog of mammalian Rab11 GTP-binding protein. A mutant with the temperature-sensitive ypt3-i5 allele showed pleiotropic phenotypes such as defects in cytokinesis, cell wall integrity, and vacuole fusion, and these were exacerbated by FK506-treatment, a specific inhibitor of calcineurin. Green fluorescent protein (GFP)-tagged Ypt3 showed cytoplasmic staining that was concentrated at growth sites, and this polarized localization required the actin cytoskeleton. It was also detected as a punctate staining in an actin-independent manner. Electron microscopy revealed that ypt3-i5 mutants accumulated aberrant Golgi-like structures and putative post-Golgi vesicles, which increased remarkably at the restrictive temperature. Consistently, the secretion of GFP fused with the pho1(+) leader peptide (SPL-GFP) was abolished at the restrictive temperature in ypt3-i5 mutants. FK506-treatment accentuated the accumulation of aberrant Golgi-like structures and caused a significant decrease of SPL-GFP secretion at a permissive temperature. These results suggest that Ypt3 is required at multiple steps of the exocytic pathway and its mutation affects diverse cellular processes and that calcineurin is functionally connected to these cellular processes.     

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.     

A Highlights from MBoC Selection: Initial Polarized Bud Growth by Endocytic Recycling in the Absence of Actin Cable–dependent Vesicle Transport in Yeast

The assembly of filamentous actin is essential for polarized bud growth in budding yeast. Actin cables, which are assembled by the formins Bni1p and Bnr1p, are thought to be the only actin structures that are essential for budding. However, we found that formin or tropomyosin mutants, which lack actin cables, are still able to form a small bud. Additional mutations in components for cortical actin patches, which are assembled by the Arp2/3 complex to play a pivotal role in endocytic vesicle formation, inhibited this budding. Genes involved in endocytic recycling were also required for small-bud formation in actin cable-less mutants. These results suggest that budding yeast possesses a mechanism that promotes polarized growth by local recycling of endocytic vesicles. Interestingly, the type V myosin Myo2p, which was thought to use only actin cables to track, also contributed to budding in the absence of actin cables. These results suggest that some actin network may serve as the track for Myo2p-driven vesicle transport in the absence of actin cables or that Myo2p can function independent of actin filaments. Our results also show that polarity regulators including Cdc42p were still polarized in mutants defective in both actin cables and cortical actin patches, suggesting that the actin cytoskeleton does not play a major role in cortical assembly of polarity regulators in budding yeast.     

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.     

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.     

Pxl1p, a paxillin-related protein, stabilizes the actomyosin ring during cytokinesis in fission yeast

Paxillins are a family of conserved LIM domain-containing proteins that play important roles in the function and integrity of the actin cytoskeleton. Although paxillins have been extensively characterized by cell biological and biochemical approaches, genetic studies are relatively scarce. Here, we identify and characterize a paxillin-related protein Pxl1p in the fission yeast Schizosaccharomyces pombe. Pxl1p is a component of the fission yeast actomyosin ring, a structure that is essential for cytokinesis. Cells deleted for pxl1 display a novel phenotype characterized by a splitting of the actomyosin ring in late anaphase, leading to the formation of two rings of which only one undergoes constriction. In addition, the rate of actomyosin ring constriction is slower in the absence of Pxl1p. pxl1Delta mutants display strong genetic interactions with mutants defective in IQGAP-related protein Rng2p and mutants defective in components of the fission yeast type II myosin machinery. Collectively, these results suggest that Pxl1p might cooperate with type II myosin and Rng2p-IQGAP to regulate actomyosin ring constriction as well as to maintain its integrity during constriction.     

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.     

Hsp90 protein in fission yeast Swo1p and UCS protein Rng3p facilitate myosin II assembly and function

The F-actin-based molecular motor myosin II is involved in a variety of cellular processes such as muscle contraction, cell motility, and cytokinesis. In recent years, a family of myosin II-specific cochaperones of the UCS family has been identified from work with yeasts, fungi, worms, and humans. Biochemical analyses have shown that a complex of Hsp90 and the Caenorhabditis elegans UCS domain protein UNC-45 prevent myosin head aggregation, thereby allowing it to assume a proper structure. Here we demonstrate that a temperature-sensitive mutant of the fission yeast Hsp90 (Swo1p), swo1-w1, is defective in actomyosin ring assembly at the restrictive temperature. Two alleles of swo1, swo1-w1 and swo1-26, showed synthetic lethality with a specific mutant allele of the fission yeast type II myosin head, myo2-E1, but not with two other mutant alleles of myo2 or with mutations affecting 14 other genes important for cytokinesis. swo1-w1 also showed a strong genetic interaction with rng3-65, a gene encoding a mutation in the fission yeast UCS domain protein Rng3p, which has previously been shown to be important for myosin II assembly. A similar deleterious effect was found when myo2-E1, swo1-w1, and rng3-65 were pharmacologically treated with geldanamycin to partially inhibit Hsp90 function. Interestingly, Swo1p-green fluorescent protein is detected at the improperly assembled actomyosin rings in myo2-E1 but not in a wild-type strain. Yeast two-hybrid and coimmunoprecipitation analyses verified interactions between Rng3p and the myosin head domain as well as interactions between Rng3p and Swo1p. Our analyses of Myo2p, Swo1p, and the UCS domain protein Rng3p establish that Swo1p and Rng3p collaborate in vivo to modulate myosin II function.     

Actin filament severing by cofilin is more important for assembly than constriction of the cytokinetic contractile ring

We created two new mutants of fission yeast cofilin to investigate why cytokinesis in many organisms depends on this small actin-binding protein. These mutant cofilins bound actin monomers normally, but bound and severed ADP-actin filaments much slower than wild-type cofilin. Cells depending on mutant cofilins condensed nodes, precursors of the contractile ring, into clumps rather than rings. Starting from clumped nodes, mutant cells slowly assembled rings from diverse intermediate structures including spiral strands containing actin filaments and other contractile ring proteins. This process in mutant cells depended on α-actinin. These slowly assembled contractile rings constricted at a normal rate but with more variability, indicating ring constriction is not very sensitive to defects in severing by cofilin. Computer simulations of the search-capture-pull and release model of contractile ring formation predicted that nodes clump when the release step is slow, so cofilin severing of actin filament connections between nodes likely contributes to the release step.     

Shaping Fission Yeast with Microtubules

For cell morphogenesis, the cell must establish distinct spatial domains at specified locations at the cell surface. Here, we review the molecular mechanisms of cell polarity in the fission yeast Schizosaccharomyces pombe. These are simple rod-shaped cells that form cortical domains at cell tips for cell growth and at the cell middle for cytokinesis. In both cases, microtubule-based systems help to shape the cell by breaking symmetry, providing endogenous spatial cues to position these sites. The plus ends of dynamic microtubules deliver polarity factors to the cell tips, leading to local activation of the GTPase cdc42p and the actin assembly machinery. Microtubule bundles contribute to positioning the division plane through the nucleus and the cytokinesis factor mid1p. Recent advances illustrate how the spatial and temporal regulation of cell polarization integrates many elements, including historical landmarks, positive and negative controls, and competition between pathways.One of the ultimate goals in cell biology is to understand how cells are assembled. As in the development of multicellular organisms, single cells need to form distinct spatial domains with specific form, structure, and functions. How do cells organize themselves in space to form a specific shape and size?The fission yeast Schizosaccharomyces pombe is an attractive, simple unicellular model organism for studying cell morphogenesis. These are nonmotile cells with highly invariant shape 8–14 µm long and 3 µm in diameter. The relative simplicity of the cells and the powers of genetic approaches and live cell imaging facilitate rigorous and quantitative studies.Here, we review the current understanding of spatial regulation in fission yeast. The cell defines distinct cortical domains at each of the cell tips, along the sides of cells, and at the cell division plane. Each cortical domain is characterized by different sets of molecules, which impart distinct functions. In particular, as it proceeds through its cell cycle, the cell delineates distinct actin-rich cortical regions at cell tips for polarized cell growth and at the middle for cell division. In both cases, a self-organizing network of microtubules directly or indirectly contributes to the proper localization of these markers. In cell polarity, microtubule ends transport polarity factors to the plasma membrane, where they function to recruit protein complexes involved in actin assembly. In cytokinesis, a medial cortical site is marked by an interacting system of microtubules, the nucleus, and cell tip factors, and functions to organize actin filaments into a cytokinetic ring. This reliance on microtubules contrasts with polarity mechanisms in budding yeast in which spatial cues are dependent on septins and actin, but not microtubules. As many of these processes involve conserved proteins, this work in fission yeast contributes toward understanding the more complex microtubule-based regulation of cell migration, cytokinesis, and cell shape regulation in animal cells. This work in fission yeast thus provides a paradigm for how a self-organizing system can shape a cell.     

Regulation of a formin complex by the microtubule plus end protein tea1p

The plus ends of microtubules have been speculated to regulate the actin cytoskeleton for the proper positioning of sites of cell polarization and cytokinesis. In the fission yeast Schizosaccharomyces pombe, interphase microtubules and the kelch repeat protein tea1p regulate polarized cell growth. Here, we show that tea1p is directly deposited at cell tips by microtubule plus ends. Tea1p associates in large "polarisome" complexes with bud6p and for3p, a formin that assembles actin cables. Tea1p also interacts in a separate complex with the CLIP-170 protein tip1p, a microtubule plus end-binding protein that anchors tea1p to the microtubule plus end. Localization experiments suggest that tea1p and bud6p regulate formin distribution and actin cable assembly. Although single mutants still polarize, for3Deltabud6Deltatea1Delta triple-mutant cells lack polarity, indicating that these proteins contribute overlapping functions in cell polarization. Thus, these experiments begin to elucidate how microtubules contribute to the proper spatial regulation of actin assembly and polarized cell growth.     

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.     

The Ran GTPase system in fission yeast affects microtubules and cytokinesis in cells that are competent for nucleocytoplasmic protein transport

Misregulation of the evolutionarily conserved GTPase Ran in fission yeast results in defects in several cellular processes in cells that are competent for nucleocytoplasmic protein transport. These results suggest that transport is neither the only nor the primary Ran-dependent process in living cells. The ability of Ran to independently regulate multiple cellular processes in vivo is demonstrated by showing that (i) eight different transport-competent RanGEF (guanine nucleotide exchange factor) mutants have defects in mitotic spindle formation; (ii) the RanGEF temperature-sensitive mutant pim1-d1 has abnormal actin ring structures at the septum. Overexpression of Imp2p, which specifically destabilizes these structures, restores viability. (iii) Ran-dependent processes differ in their requirements for active Ran in vivo. Microtubule function, cytokinesis, and nuclear envelope structure are the Ran-dependent processes most sensitive to the amount of Ran protein in the cell, whereas nucleocytoplasmic protein transport is the most robust. Therefore, the ability of Ran from Schizosaccharomyces pombe to independently regulate multiple cellular processes may reflect differences in its interactions with the binding proteins that mediate these functions and explain the complex phenotypic consequences of its misregulation in vivo.