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ABSTRACTCandida albicansis an opportunistic fungal pathogen whose virulence is related to its ability to switch between yeast, pseudohyphal, and true-hyphal morphologies. To ask how long-distance nuclear migration occurs in C. albicanshyphae, we identified the fundamental properties of nuclear movements and microtubule dynamics using time-lapse microscopy. In hyphae, nuclei migrate to, and divide across, the presumptive site of septation, which forms 10 to 15 µm distal to the basal cell. The mother nucleus returns to the basal cell, while the daughter nucleus reiterates the process. We used time-lapse microscopy to identify the mechanisms by which C. albicansnuclei move over long distances and are coordinated with hyphal morphology. We followed nuclear migration and spindle dynamics, as well as the time and position of septum specification, defined it as the presumptum, and established a chronology of nuclear, spindle, and morphological events. Analysis of microtubule dynamics revealed that premitotic forward nuclear migration is due to the repetitive sliding of astral microtubules along the cell cortex but that postmitotic forward and reverse nuclear migrations are due primarily to spindle elongation. Free microtubules exhibit cell cycle regulation; they are present during interphase and disappear at the time of spindle assembly. Finally, a growth defect in strains expressing Tub2-green fluorescent protein revealed a connection between hyphal elongation and the nuclear cell cycle that is coordinated by hyphal length and/or volume.

The ability of Candida albicansto switch cellular morphologies is crucial for its ability to cause infection. Because the cell cycle machinery participates in Saccharomyces cerevisiaefilamentous growth, we characterized in detail the two C. albicansB-type cyclins, CLB2and CLB4, to better understand the molecular mechanisms that underlie the C. albicansmorphogenic switch. Both Clb2p and Clb4p levels are cell cycle regulated, peaking at G2/M and declining before mitotic exit. On hyphal induction, the accumulation of the G1 cyclin Cln1p was prolonged, whereas the accumulation of both Clb proteins was delayed when compared with yeast form cells, indicating that CLB2and CLB4are differentially regulated in the two morphologies and that the dynamics of cyclin appearance differs between yeast and hyphal forms of growth. Clb2p-depleted cells were inviable and arrested with hyper-elongated projections containing two nuclei, suggesting that Clb2p is not required for entry into mitosis. Unlike Clb2p-depleted cells, Clb4p-depleted cells were viable and formed constitutive pseudohyphae. Clb proteins lacking destruction box domains blocked cell cycle progression resulting in the formation of long projections, indicating that both Clb2p and Clb4p must be degraded before mitotic exit. In addition, overexpression of either B-type cyclin reduced the extent of filamentous growth. Taken together, these data indicate that Clb2p and Clb4p regulate C. albicansmorphogenesis by negatively regulating polarized growth.