Effects of extracellular pH on internal pH in cell cycle defective Schizosaccharomyces pombe
Haworth, R. S.; Jia, Z.-P.; Young, P. G.; Sykes, Brian L.; Fliegel, Larry
Spon. by D. Pellman.) Nitrogen starvation of budding yeast in the presence of a carbon source stimulates a dimorphic transition from round cell (RND) to pseudohyphal (PH) growth. When grown on agar, RND cells form hemispherical mounds fed by nutrient diffusion. In contrast, PH cells form branching chains of spindleshaped cells which invade the agar, greatly increasing colony boundaries. We performed time-lapse video contrast-enhanced Nomarski-DIC microscopy to compare the growth of RND and PH
... lonies at high resolution. We found that the growth pattern of PH cells is strikingly different from the classical description of the cell cycle of budding yeast. In RND cells, buds are initiated in GI after a cell-size checkpoint, START, is passed. Mitosis, septation and separation immediately follow completion of S phase; RND cells spend little or no time in G2. When nutrient starvation limits growth, RND daughters may stall in GO, having been released from their mothers at a size well below the START threshold. By contrast, mother-daughter pairs of PH cells do not septate before doubling size. Upon separation, both cells form new buds in synchrony. In our study, RND cells in low nitrogen/high carbon media required 152+/-6 min (mean+/-SEM, n=18) to progress from nascent buds to first bud initiation; second buds required only 131+/-5 min. For PH cells these times were 142+/4 and 147+/-5 min (n=1l). In contrast to the RND cell cycle, the PH growth pattern favors the daughter, the cell closer to a potential nutrient source. The results of flow cytometry of DNA content and fluorescence imaging of nuclear and spindle morphology suggest that growth of PH daughter cells to the size of the mother follows DNA synthesis but precedes nuclear division. These data provide evidence for a second cell-size checkpoint present in G2. DIFFERENTIAL EFFECTS OF MIMOSINE ON DNA REPLICATION IN The plant amino acid mimosine reversibly arrests the cell cycle of cultured mammalian cells near the Gl/S boundary by blocking initiation of DNA replication (Dikwel and Hamlin, Mol. Cell. Biol. 12, 1992). In our study, mimosine did not inhibit DNA replication in cell-free extracts of Xenopus laevis eggs, nor did it block M to S phase transition in these extracts. Incubation of Xenopus embryos in 2 mM mimosine had no effect on development for at least 3 days after fertilization. Moreover, microinjection of mimosine (2 mM final concentration) into 4-cell embryos had no detectable effect on embryogenesis through hatching. Neither [3H]-thymidine incorporation nor replication of microinjected DNA during early embryogenesis were inhibited by the microinjected mimosine. Microinjection of mimosine, even after the midblastula transition when Xenopus embryonic cells first acquire the Gl and G2 phases of the cell cycle, did not inhibit DNA replication or cell cycle progression. In contrast, the growth of Xenopus kidney epithelial A6 cells in culture was blocked at the Gl/S boundary by mimosine as were mammalian NRK-49F cells, protozoan (Tetrahymena) cells, and plant (Black Mexican sweet maize) cells. We conclude that mimosine affects cell cycle progression and DNA replication differently in somatic cells than in embryonic X. laevis cells. AN UNUSUAL BUDDING PATTERN IN SACCHAROMYCES CEREVISIAE. We are reporting here the first definitive example of a haploid strain (GS 1731) of the budding yeast S. cerevisiae that forms both multiple buds and short filaments during the early phases of growth in a rich medium. In all strains examined previously each mother cell produces only one bud at a time. Certain cell cycle mutants and osmoregulationdefective mutants have been shown to arrest as multiple budded cells under non-permissive conditions. Recently diploid, but not haploid, S. cerevisiae cells have been shown to produce short filaments. By the end of lag phase of the GS 1731 growth curve multiple budded cells constituted 50-60% of the budded cells, with slightly more of the cells possessing three or more buds. During log phase the % cells showing multiple buds decreased. We have shown by electron microscopy that the multiple buds are attached to a common mother cell. Comparison of the data from the lag phase of a normal growth curve and those after removal of a hydroxyurea block suggests that during the S-phase after a growth arrest GS 1731 can initiate several buds. We have observed that cells of GS 1731 have multiple bundles of cytoplasmic microtubules. This would explain why the multiple buds on a mother cell all tend to be at the same pole.