The G 1 /S transition is a critical control point for cell proliferation and involves essential transcription complexes termed SBF and MBF in Saccharomyces cerevisiae or MBF in Schizosaccharomyces pombe. In the fungal pathogen Candida albicans, G 1 /S regulation is not clear. To gain more insight into the G 1 /S circuitry, we characterized Swi6p, Swi4p and Mbp1p, the closest orthologues of SBF (Swi6p and Swi4p) and MBF (Swi6p and Mbp1p) components in S. cerevisiae. The mbp1⌬/⌬ cells showed minor growth defects, whereas swi4⌬/⌬ and swi6⌬/⌬ yeast cells dramatically increased in size, suggesting a G 1 phase delay. Gene set enrichment analysis (GSEA) of transcription profiles revealed that genes associated with G 1 /S phase were significantly enriched in cells lacking Swi4p and Swi6p. These expression patterns suggested that Swi4p and Swi6p have repressing as well as activating activity. Intriguingly, swi4⌬/⌬ swi6⌬/⌬ and swi4⌬/⌬ mbp1⌬/⌬ strains were viable, in contrast to the situation in S. cerevisiae, and showed pleiotropic phenotypes that included multibudded yeast, pseudohyphae, and intriguingly, true hyphae. Consistently, GSEA identified strong enrichment of genes that are normally modulated during C. albicans-host cell interactions. Since Swi4p and Swi6p influence G 1 phase progression and SBF binding sites are lacking in the C. albicans genome, these factors may contribute to MBF activity. Overall, the data suggest that the putative G 1 /S regulatory machinery of C. albicans contains novel features and underscore the existence of a relationship between G 1 phase and morphogenetic switching, including hyphal development, in the pathogen.

The conserved anaphase-promoting complex/cyclosome (APC/C) system mediates protein degradation during mitotic progression. Conserved coactivators Cdc20p and Cdh1p regulate the APC/C during early to late mitosis and G 1 phase. Candida albicans is an important fungal pathogen of humans, and it forms highly polarized cells when mitosis is blocked through depletion of the polo-like kinase Cdc5p or other treatments. However, the mechanisms governing mitotic progression and associated polarized growth in the pathogen are poorly understood. In order to gain insights into these processes, we characterized C. albicans orthologues of Cdc20p and Cdh1p. Cdc20p-depleted cells were blocked in early or late mitosis with elevated levels of Cdc5p and the mitotic cyclin Clb2p, suggesting that Cdc20p is essential and has some conserved functions during mitosis. However, the yeast cells formed highly polarized buds in contrast to the large doublets of S. cerevisiae cdc20 mutants, implying a distinct role in morphogenesis. In comparison, cdh1⌬/cdh1⌬ cells were viable but showed enrichment of Clb2p and Cdc5p, suggesting that Cdh1p may influence mitotic exit. The cdh1⌬/cdh1⌬ phenotype was pleiotropic, consisting of normal or enlarged yeast, pseudohyphae, and some elongated buds, whereas S. cerevisiae cdh1⌬ yeast cells were reduced in size. Thus, C. albicans Cdh1p may have some distinct functions. Finally, absence of Cdh1p or Cdc20p had a minor or no effect on hyphal development, respectively. Overall, the results suggest that Cdc20p and Cdh1p may be APC/C activators that are important for mitosis but also morphogenesis in C. albicans. Their novel features imply additional variations in function and underscore rewiring in the emerging mitotic regulatory networks of the pathogen.

The Polo-like kinases (Plks) are conserved, multifunctional cell cycle regulators that are induced in many forms of cancer and play additional roles in metazoan development. We previously identified plkA in Aspergillus nidulans, the only Plk investigated in filamentous fungi to date, and partially characterized its function through overexpression. Here, we report the plkA null phenotype. Surprisingly, plkA was not essential, unlike Plks in other organisms that contain a single homologue. A subset of cells lacking PLKA contained defects in spindle formation and chromosome organization, supporting some conservation in cell cycle function. However, septa were present, suggesting that PLKA, unlike other Plks, is not a central regulator of septation. Colonies lacking PLKA were compact with multibranched hyphae, implying a role for this factor in aspects of hyphal morphogenesis. These defects were suppressed by high temperature or low concentrations of benomyl, suggesting that PLKA may function during vegetative growth by influencing microtubule dynamics. However, the colonies also showed reduced conidiation and precocious formation of sexual Hülle cells in a benomyl-and temperature-insensitive manner. This result suggests that PLKA may influence reproduction through distinct mechanisms and represents the first example of a link between Plk function and development in fungi. Finally, filamentous fungal Plks have distinct features, and phylogenetic analyses reveal that they may group more closely with metazoan PLK4. In contrast, yeast Plks are more similar to metazoan proteins PLK1 to PLK3. Thus, A. nidulans PLKA shows some conservation in cell cycle function but may also play novel roles during hyphal morphogenesis and development.

The fungal pathogen Candida albicans differentiates between yeast, hyphae and pseudohyphae in order to enhance survival in the human host. Environmental cues induce hyphal development and expression of hyphal-specific genes. Filaments also result from yeast cell cycle arrest, but the nature of these cells and their mechanisms of formation are less clear. We previously demonstrated that depletion of the mitotic polo-like kinase Cdc5p resulted in the production of filaments under yeast growth conditions that were distinct from hyphae with respect to several criteria, yet expressed hyphal-specific genes at later stages of development. In order to clarify the identity of these growth forms and their relationship to true hyphae, we conducted time course-based investigations of aspects of the polar growth machinery, which can distinguish cell types. During later stages of Cdc5p depletion, the myosin light chain Mlc1p demonstrated a Spitzenkörper-like localization in the tips of some filaments, and the Cdc42p GAP Rga2p became hyper-phosphorylated, as in true hyphae. Hyphal-specific genes HWP1, UME6 and HGC1 were strongly expressed at approximately the same time. HWP1 expression was dependent on Ume6p, and absence of Ume6p or Hgc1p influenced late-stage filament morphology and integrity. Finally, polarized growth and UME6 expression in Cdc5p-depleted cells were independent of the transcription factor Hms1p. Thus, depleting Cdc5p generates elongated buds that switch to a hyphal fate over time through a mechanism that involves UME6 and HGC1 induction, possibly in response to maintenance of polarized growth. The results expand on the multiple strategies with which C. albicans can modulate growth mode and expression of virulence determinants.