Ritsuko Arai

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.

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.

Directionality of F-actin cables changes during the fission yeast cell cycle

Longitudinal F-actin cables are thought to be important for transporting materials for polarized cell growth in fission yeast. We show that most F-actin in the cables is oriented such that the barbed end faces the nearest cell tip during interphase; however, this directionality is reversed during mitosis. These orientations of F-actin ensure proper transport of materials to growing sites during these cell-cycle stages.

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.

Overproduction of elongation factor 1α, an essential translational component, causes aberrant cell morphology by affecting the control of growth polarity in fission yeast

Elongation factor 1α (EF1α), an essential component of the eukaryotic translational machinery, has been shown to possess various biochemical and biological activities, including F‐actin‐binding and ‐bundling, microtubule‐ severing, and the activity of making fibroblasts highly susceptible to transformation. However, our understanding of the biological significance of EF1α with respect to these various biochemical or biological activities remains limited. Here we report the identification of EF1α‐encoding genes as genes whose over‐expression causes aberrant cell morphology in fission yeast.

Small GTPase Rho5 is a functional homologue of Rho1, which controls cell shape and septation in fission yeast

The small GTPase Rho1 plays an essential role in controlling the organization of the actin cytoskeleton and synthesis of the cell wall in the fission yeast Schizosaccharomyces pombe. Here we studied the role of Rho5 whose primary structure is very similar to that of Rho1. It was found that elevated expression of Rho5 was able to compensate for the lethality of cells lacking Rho1. Rho5 was localized to the ends of interphase cells and the mid‐region of mitotic cells. Overexpression of Rho5 caused depolarization of F‐actin patches and abnormal formation of the cell wall, as did Rho1. Although rho5 + was not essential for maintaining the cell shape, rho1 rho5‐double null cells showed more severe defects in cell viability than rho1‐null cells. Thus, it is likely that Rho5 has an overlapping function with Rho1 in controlling cell growth and division in S. pombe.