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 bni1⌬ 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 spa2⌬ cla4-75-td, bud6⌬ cla4-75-td, and pea2⌬ 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 cla4⌬ 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. INTRODUCTIONThe development of both unicellular and multicellular organisms requires cells to respond to intracellular and extracellular cues that direct growth and division. These signals regulate polarized cell growth, maintenance of cell shape, cell motility, and cytokinesis (Drubin and Nelson, 1996). Polarized cell growth is a complex process that requires the directed organization of the actin cytoskeleton and the coordinated function of many polarity proteins and signal transduction cascades. Cells of the yeast Saccharomyces cerevisiae grow by budding, a process in which a rigid cell wall is locally expanded as a result of polarized secretion (Pruyne and Bretscher, 2000a). Before bud emergence, cells polarize the actin cytoskeleton toward the future bud site and assemble a septin ring at that site.The septins are a conserved family of filament-forming proteins that play important roles in a variety of cell functions in fungal and animal cells (Longtine et al., 1996;Trimble, 1999;Gladfelter et al., 2001;Longtine and Bi, 2003).Typical septins have a variable N-terminal region, a conserved core that includes the element of a GTP-binding site, and a variable C-terminal region. Septins were first identified as temperature-sensitive cdc mutants in S. cerevisiae (Hartwell, 1971). Septins are assembled into a ring before bud formation and remain as a collar subjacent to the plasma membrane at the mother-bud neck for most of the cell cycle. The septin ring is assembled by the copolymerization of the septins Cdc3p, Cdc10p, Cdc11p, Cdc12p, and Shs1p/Sep7p into filaments (Frazier et al., 1998;Field and Kellogg, 1999). Purified septins from yeast form filaments of 7-9-nm diameter and of v...

Budding yeast mutants in assembly of actin cables, which are thought to be the only actin structures essential for budding, still could form a small bud. Mutations in actin patch endocytic machineries/endocytic recycling factors inhibited this budding, suggesting a mechanism that promotes polarized growth by local recycling of endocytic vesicles.

A formin Bni1p nucleates actin to assemble actin cables, which guide the polarized transport of secretory vesicles in budding yeast. We identified mutations that suppressed both the lethality and the excessive actin cable formation caused by overexpression of a truncated Bni1p (BNI1DN ). Two recessive mutations, act1-301 in the actin gene and sla2-82 in a gene involved in cortical actin patch assembly, were identified. The isolation of sla2-82 was unexpected, because cortical actin patches are required for the internalization step of endocytosis. Both act1-301 and sla2-82 exhibited synthetic growth defects with bni1D. act1-301, which resulted in an E117K substitution, interacted genetically with mutations in profilin (PFY1) and BUD6, suggesting that Act1-301p was not fully functional in formin-mediated polymerization. sla2-82 also interacted genetically with genes involved in actin cable assembly. Some experiments, however, suggested that the effects of sla2-82 were caused by depletion of actin monomers, because the temperature-sensitive growth phenotype of the bni1D sla2-82 mutant was suppressed by increased expression of ACT1. The isolation of suppressors of the BNI1DN phenotype may provide a useful system for identification of actin amino-acid residues that are important for formin-mediated actin polymerization and mutations that affect the availability of actin monomers.

Arsenic trioxide (ATO), which is known to be an effective carcinostatic against acute promyelocytic leukemia (APL), has drawn attention throughout the world. Studies on the effectiveness of ATO against solid tumors have started, and progress is expected. In this study, we evaluate the differences in the suppressive effects on tumor growth between different combinations of ATO administration and hyperthermia. Mice in which a squamous cell carcinoma (SCC-VII) had grown to 5-7 mm in the right femoral region were subjected to experiments in 5 groups. The groups consisted of the control group, hyperthermia alone group, ATO alone group, A-H group (ATO administration followed by 1-hour hyperthermia at an interval of 2 hours), and H-A group (1-hour hyperthermia followed by ATO administration at an interval of 2 hours). In each group, 10 mice were used for the measurement of the tumor volume, and 1 mouse for the pathological examination. The suppressive effects on tumor growth were examined in the A-H and H-A groups using changes in the tumor volume and pathological findings of the tumor. The changes in the tumor volume demonstrated synergistic effects of combined hyperthermia and ATO administration, but there was no significant difference in the suppressive effects between the A-H and H-A groups. The histopathological examination demonstrated more histological changes in the