Kentaro Nakano

Pom1 DYRK Regulates Localization of the Rga4 GAP to Ensure Bipolar Activation of Cdc42 in Fission Yeast

BACKGROUND In the fission yeast Schizosaccharomyces pombe, cell growth takes place exclusively at both ends of the cylindrical cell. During this highly polarized growth, microtubules are responsible for the placement of the cell-end marker proteins, the Tea1-Tea4/Wsh3 complex, which recruits the Pom1 DYRK-family protein kinase. Pom1 is required for proper positioning of growth sites, and the Deltapom1 mutation brings about monopolar cell growth. RESULTS Pom1 kinase physically interacts with Rga4, which has a GAP (GTPase-activating protein) domain for Rho-family GTPase. Genetic and biochemical evidence indicates that Rga4 functions as GAP for the Cdc42 GTPase, an evolutionarily conserved regulator of F-actin. CRIB (Cdc42/Rac interactive binding)-GFP microscopy has revealed that GTP-bound, active Cdc42 is concentrated to growing cell ends accompanied by developed F-actin structures, where the Rga4 GAP is excluded. The monopolar Deltapom1 mutant fails to eliminate Rga4 from the nongrowing cell end, resulting in monopolar distribution of GTP-Cdc42 to the growing cell end. However, mutational inactivation of Rga4 allows Cdc42 to be active at both ends of Deltapom1 cells, suggesting that mislocalization of Rga4 in the Deltapom1 mutant contributes to its monopolar phenotype. CONCLUSIONS Pom1 kinase recruited to cell ends by the Tea1-Tea4/Wsh3 complex is essential for proper localization of a GAP for Cdc42, Rga4, which ensures bipolar localization of GTP-bound, active Cdc42. Because of the established role of Cdc42 in F-actin formation, these observations provide a new insight into how the microtubule system achieves localized formation of F-actin to generate cell polarity.

The small GTPase Rho3 and the diaphanous/formin For3 function in polarized cell growth in fission yeast

We identified a novel Rho gene rho3+ and studied its interaction with diaphanous/formin for3+ in the fission yeast Schizosaccharomyces pombe. Both rho3 null cells and for3 null cells showed defects in organization of not only actin cytoskeleton but also cytoplasmic microtubules (MTs). rho3 for3 double null cells had defects that were more severe than each single null cell: polarized growth was deficient in the double null cells. Function of For3 needed the highly conserved FH1 and FH2 domains, an N-terminal region containing a Rho-binding domain, and the C-terminal region. For3 bound to active forms of both Rho3 and Cdc42 but not to that of Rho1. For3 was localized as dots to the ends of interphase cells and to the mid-region in dividing cells. This localization was probably dependent on its interaction with Rho proteins. Overexpression of For3 produced huge swollen cells containing depolarized F-actin patches and thick cytoplasmic MT bundles. In addition, overexpression of a constitutively active Rho3Q71L induced a strong defect in cytokinesis. In conclusion, we propose that the Rho3-For3 signaling system functions in the polarized cell growth of fission yeast by controlling both actin cytoskeleton and MTs.

Identification of Myo3, a second type-II myosin heavy chain in the fission yeast Schizosaccharomyces pombe

We cloned the myo3 + gene of Schizosaccharomyces pombe which encodes a type‐II myosin heavy chain. myo3 null cells showed a defect in cytokinesis under certain conditions. Overproduction of Myo3 also showed a defect in cytokinesis. Double mutant analysis indicated that Myo3 genetically interacts with Cdc8 tropomyosin and actin. Myo3 may be implicated in cytokinesis and stabilization of F‐actin cables. Moreover, the function of Myo2 can be replaced by overexpressed Myo3. We observed a modest synthetic interaction between Myo2 and Myo3. Thus, Myo2 and Myo3 seem to cooperate in the formation of the F‐actin ring in S. pombe.

The small GTP-binding protein Rho1 is a multifunctional protein that regulates actin localization, cell polarity, and septum formation in the fission yeast Schizosaccharomyces pombe.

The small GTP‐binding protein Rho has been shown to regulate the formation of the actin cytoskeleton in animal cells. We have previously isolated two rho genes, rho1+ and rho2+ , from the fission yeast Schizosaccharomyces pombe in order to investigate the function of Rho using genetic techniques. In this paper, we report the cellular function of Rho1.

Subcellular localization and possible function of actin, tropomyosin and actin-related protein 3 (Arp3) in the fission yeast Schizosaccharomyces pombe

We investigated subcellular localizations and interactions of actin and two actin cytoskeleton-related proteins, Cdc8 tropomyosin and actin-related protein 3, Arp3, in the fission yeast Schizosaccharomyces pombe, using specific antibodies and by gene disruption. Actin was localized to the medial microfilamentous ring in the region of the septum during cytokinesis and to cortical patches by immunoelectron microscopy. F-actin cables were detected throughout the cell cycle by fluorescent staining with Bodipy-phallacidin. Cables were often linked to the patches and to the medial ring during its formation. Tropomyosin was localized to the medial ring and the cables. It was also distributed in the cell as patches, although co-localization with F-actin was not frequent. In cdc8ts mutant cells, F-actin cables were not observed although the F-actin patches were detected and cell polarity was maintained. These observations suggest that the F-actin cables may be involved in the formation of the medial ring, and that tropomyosin plays an important role in organizing both the ring and the cable, but is not involved in the F-actin patch formation or maintenance of cell polarity. Binding of Arp3 to actin was revealed by immunoprecipitation as well as by DNase I column chromatography. Arp3 seemed to form a complex with several proteins in the cell extracts, as previously reported for other organisms. Contrary to a previous report (McCollum et al., EMBO J. 15, 6438-6446, 1996), Arp3 was found to be concentrated in the medial region from early anaphase to late cytokinesis. Following arp3 gene disruption, F-actin patches were delocalized throughout the cell and cells did not undergo polarized growth, suggesting that Arp3 influences the proper localization of the actin patches in the cell and thereby controls the polarized growth of the cell.

Dividing the spoils of growth and the cell cycle: The fission yeast as a model for the study of cytokinesis.

Cytokinesis is the final stage of the cell cycle, and ensures completion of both genome segregation and organelle distribution to the daughter cells. Cytokinesis requires the cell to solve a spatial problem (to divide in the correct place, orthogonally to the plane of chromosome segregation) and a temporal problem (to coordinate cytokinesis with mitosis). Defects in the spatiotemporal control of cytokinesis may cause cell death, or increase the risk of tumor formation [Fujiwara et al., 2005 (Fujiwara T, Bandi M, Nitta M, Ivanova EV, Bronson RT, Pellman D. 2005. Cytokinesis failure generating tetraploids promotes tumorigenesis in p53‐null cells. Nature 437:1043–1047); reviewed by Ganem et al., 2007 (Ganem NJ, Storchova Z, Pellman D. 2007. Tetraploidy, aneuploidy and cancer. Curr Opin Genet Dev 17:157–162.)]. Asymmetric cytokinesis, which permits the generation of two daughter cells that differ in their shape, size and properties, is important both during development, and for cellular homeostasis in multicellular organisms [reviewed by Li, 2007 (Li R. 2007. Cytokinesis in development and disease: variations on a common theme. Cell Mol Life Sci 64:3044–3058)]. The principal focus of this review will be the mechanisms of cytokinesis in the mitotic cycle of the yeast Schizosaccharomyces pombe. This simple model has contributed significantly to our understanding of how the cell cycle is regulated, and serves as an excellent model for studying aspects of cytokinesis. Here we will discuss the state of our knowledge of how the contractile ring is assembled and disassembled, how it contracts, and what we know of the regulatory mechanisms that control these events and assure their coordination with chromosome segregation.

The small GTPase Rho4 is involved in controlling cell morphology and septation in fission yeast

Background: Rho family small GTPases have been shown to be involved in various cellular activities, including the organization of actin cytoskeleton in eukaryotic cells. There are six rho genes in the fission yeast Schizosaccharomyces pombe. Cdc42 is known to control the polarity of the cell. Rho1, Rho2 and Rho3 play important roles in controlling cell shape and septation. On the other hand, Rho4 and Rho5 have not yet been characterized. Here we report the function of rho4+ in fission yeast.

Localization of Rho GTPase in sea urchin eggs

We isolated the urho1 ( rchin in English or ni in Japanese) gene from the sea urchin cDNA library which encodes a Rho GTPase. Anti‐URho1 antibodies specifically recognized a 22 kDa protein in the extracts of echinoderm eggs. URho1 was concentrated in the cortices from both unfertilized and fertilized eggs as judged by immunoblot analysis. URho1 may bind directly to the cell membrane but not be a component of the cortical layer. Immunofluorescence microscopy revealed that URho1 is localized to the cleavage furrow and the midbody during cytokinesis.

Fission yeast IQGAP arranges actin filaments into the cytokinetic contractile ring.

The contractile ring (CR) consists of bundled actin filaments and myosin II; however, the actin‐bundling factor remains elusive. We show that the fission yeast Schizosaccharomyces pombe IQGAP Rng2 is involved in the generation of CR F‐actin and required for its arrangement into a ring. An N‐terminal fragment of Rng2 is necessary for the function of Rng2 and is localized to CR F‐actin. In vitro the fragment promotes actin polymerization and forms linear arrays of F‐actin, which are resistant to the depolymerization induced by the actin‐depolymerizing factor Adf1. Our findings indicate that Rng2 is involved in the generation of CR F‐actin and simultaneously bundles the filaments and regulates its dynamics by counteracting the effects of Adf1, thus enabling the reconstruction of CR F‐actin bundles, which provides an insight into the physical properties of the building blocks that comprise the CR.

Rho1‐GEFs Rgf1 and Rgf2 are involved in formation of cell wall and septum, while Rgf3 is involved in cytokinesis in fission yeast

The Rho GTPase acts as a binary molecular switch by converting between a GDP‐bound inactive and a GTP‐bound active conformational state. The guanine nucleotide exchange factors (GEFs) are critical activators of Rho. Rho1 has been shown to regulate actin cytoskeleton and cell wall synthesis in the fission yeast Schizosaccharomyces pombe. Here we studied function of fission yeast RhoGEFs, Rgf1, Rgf2, and Rgf3. It was shown that these proteins have similar molecular structures, and function as GEFs for Rho1. Disruption of either rgf1 or rgf2 did not show a serious effect on the cell. On the other hand, disruption of rgf3 caused severe defects in contractile ring formation, F‐actin patch localization, and septation during cytokinesis. Rgf1 and Rgf2 were localized to the cell ends during interphase and the septum. Rgf3 formed a ring at the division site, which was located outside the contractile ring and inside the septum where Rho1 was accumulated. In summary, Rgf1 and Rgf2 show functional redundancy, and roles of these RhoGEFs are likely to be different from that of Rgf3. Rho1 is likely to be activated by Rgf3 at the division site, and involved in contractile ring formation and/or maintenance and septation.