Hiromi Kobori

Relationship of actin organization to growth in the two forms of the dimorphic yeastCandida tropicalis

SummaryCandida tropicalis is a dimorphic yeast capable of growing both as a budding yeast and as filamentous hyphae depending upon the source of the carbon used in the culture medium. The organization of F-actin during growth of the yeast form (Y-form) and the hyphal form (H-form) was visualized by rhodamine-conjugated phalloidin by using a conventional fluorescence microscope as well as a laser scanning confocal fluorescence microscope. In single cells without a bud or non-growing hyphae, actin dots were evenly distributed throughout the cytoplasm. Before the growth of the bud or hypha, the actin dots were concentrated at one site. During bud growth, actin dots were located solely in the bud. They filled the small bud and then filled the apical two-thirds of the cytoplasm of the middlesized bud. During growth of the large bud, actin dots which had filled the apical half of the cytoplasm gradually moved to the tip of the bud. In the formation of the septum, actin dots were arranged in two lines at the conjunction of the bud and the mother cell. During hyphal growth, the majority of actin dots were concentrated at the hyphal apex. A line of clustered spots or a band of actin was observed only at the site where the formation of a new septum was imminent. This spatial and temporal organization of actin in both categories of cells was demonstrated to be closely related to the growth and local deposition of new cell wall material by monitoring the mode of growth with Calcofluor staining. Treatment of both forms of cells with cytochalasin A (CA) confirmed the close relationship between actin and new cell wall deposition. CA treatment revealed lightly stained unlocalized actin which was associated with abnormal cell wall deposition as well as changes in morphology. These results suggest that actin is required for proper growth and proper deposition of cell wall material and also for maintaining the morphology of both forms of cells.

Interspecific protoplast fusion between Candida tropicalis and Candida boidinii: Characterization of the fusants

Abstract The polyethylene glycol (PEG) and electroporation protoplast fusion methods were applied to imperfect yeasts of the genus Candida to induce interspecific fusion. Auxotrophic strains of Candida tropicalis (an n -alkane-utilizing yeast) and Candida boidinii (a methanol-utilizing yeast) were used as parental strains and fusion products were selected by complementation on minimal medium. The frequency of fusion induced by filter-sterilized PEG was high, ranging from 10 −4 to 10 −5 . Scanning electron microscopy showed that the fusants varied considerably in shape, surface structure, and cell volume. Various electrophoretic karyotypes of fusants were observed, although their karyotypes were basically the same as that of C. boidinii . Morphological, biochemical, and physiological studies indicated that the characteristics of the fusants were mainly those of C. boidinii , but with some of those of C. tropicalis . The fusants could utilize methanol and n -alkane as sole carbon sources when they were first isolated, but their ability to utilize n -alkane was lost during their prolonged storage.

Changes in microfilaments and microtubules of yeasts induced by pressure stress

Abstract Changes in cytoskeletal elements induced by pressure stress in a budding yeast Saccharomyces cerevisiae , a dimorphic yeast Candida tropicalis and a fission yeast Schizosaccharomyces pombe were investigated by fluorescence microscopy. The cell cycle-specific organization of microfilaments and microtubules in the three yeasts was altered by exposure to hydrostatic pressure of 50–150 MPa for 10 min and their complete disassembly was observed at 150–300 MPa. Similar morphological changes in the cytoskeleton were caused in the three yeasts by acceleration of pressure stress, although their sensitivity differed from that of yeasts; hyphal-form cells of C. tropicalis and S. pombe cells were sensitive to pressure stress.

Morphological effects of pressure stress on yeasts

Abstract To investigate the induction of polyploidy by pressure stress, the ultra- structure and microtubules of Saccharomyces cerevisiae and Schizosaccharomyces pombe were studied by conventional and immunoelectron microscopy. The nuclear membrane was disrupted even at 100 MPa and with increasing pressure mitochondria had electron-dense areas, the cytoplasmic substances changed dramatically and the cellular organelles could hardly be detected. S. pombe cells were more sensitive to low pressure stress than were S. cerevisiae cells. Immunoelectron microscopy confirmed that the microtubules were damaged by pressure stress. The damage to spindle pole bodies, microtubules and the nuclear membrane was thought to be followed by breakdown of the nuclear division apparatus and inhibition of nuclear division.

Ultrastructural effects of pressure stress to Saccharomyces cerevisiae cells revealed by immunoelectron microscopy using frozen thin sectioning

Abstract Effects of hydrostatic pressure on ultrastructure of Saccharomyces cerevisiae were studied by immunoelectron microscopy using frozen thin sections. At 100 MPa bundles of the microtubules (MTs) extended in the nucleus, but spindle pole bodies were not visible. At 150 MPa the deposition of gold particles for anti α-tubulin was recognized in the nucleus, although the filamentous structure of the MTs was not seen. At 200 MPa fewer gold particles were scattered in the nucleus. These results show that elements of the nuclear division apparatus are susceptible to pressure stress. These events were reversible at below 200 MPa.