Motor protein Myo5p is required to maintain the regulatory circuit controlling WOR1 expression in Candida albicans.

Motor Protein Myo5p Is Required To Maintain the Regulatory Circuit Controlling WOR1 Expression in Candida albicans Nadezda Kachurina,a Bernard Turcotte,a and Malcolm Whitewayb,c Department of Medicine, Division of Experimental Medicine, McGill University, Royal Victoria Hospital, Montreal, Quebec, Canadaa; Biotechnology Research Institute, National Research Council, Montreal, Quebec, Canadab; and Department of Biology, McGill University, Montreal, Quebec, Canadac The Candida albicans MYO5 gene encodes myosin I, a protein required for the formation of germ tubes and true hyphae. Because the polarized growth of opaque-phase cells in response to pheromone results in mating projections that can resemble germ tubes, we examined the role of Myo5p in this process. We localized green fluorescent protein (GFP)-tagged Myo5p in opaquephase cells of C. albicans during both bud and shmoo formation. In vegetatively growing opaque cells, Myo5p is found at sites of bud emergence and bud growth, while in pheromone-stimulated cells, Myo5p localizes at the growing tips of shmoos. Intriguingly, cells homozygous for MTLa in which the MYO5 gene was deleted failed to switch efficiently from the white phase to the opaque phase, although ectopic expression of WOR1 from the MET3 promoter can convert myo5 mutants into mating-competent opaque cells. However, when WOR1 expression was shut off, the myo5-defective cells rapidly lost both their opaque phenotype and mating competence, suggesting that Myo5p is involved in the maintenance of the opaque state. When MYO5 is expressed conditionally in opaque cells, the opaque phenotype, as well as the mating ability of the cells, becomes unstable under repressive conditions, and quantitative real-time PCR demonstrated that the shutoff of MYO5 expression correlates with a dramatic reduction in WOR1 expression. It appears that while myosin I is not directly required for mating in C. albicans, it is involved in WOR1 expression and the white-opaque transition and thus is indirectly implicated in mating. U nder different environmental conditions, the human pathogen Candida albicans is able to grow in different morphological forms. In response to changes in nutrient status, temperature, and pH, for example, Candida cells can switch from the yeast growth form to pseudohyphae or true hyphae, and this well-studied switch has led to the classification of C. albicans as dimorphic. However, C. albicans can exist in other morphological forms. A second well-studied morphological transition is the white-opaque switch. White and opaque cells are easily distinguished under the microscope; white-form cells are of the classic ovoid yeast shape, and opaque-form cells are much more elongated (2). On agar plates, white cells form domed colonies of a whitish color, while opaque cells form colonies that are flattened and greyish (2). Multiple environmental stimuli that slow cell growth lead to an increase in white-to-opaque switching in C. albicans (1). The opaque phase is stable at 25°C; however, upon shifting of the temperature to 30 to 37°C, cells switch en masse to the white phase (47). More than 450 genes are modulated in expression during the white-opaque transition (26), and recently a master regulator of white-opaque switching, the WOR1 gene (22, 46, 54), has been identified. The data suggest that Wor1p is autoregulated. That means that once Wor1p expression is established, it tends to remain on, with a positive-feedback loop keeping the cells in the opaque state (54). In addition there is chromatin level regulation; several chromatin-modifying enzymes, such as Hst2p, Set3, and Hos2, are involved in the modulation of white-opaque switching (20). The white-opaque transition plays a key role in the mating of C. albicans. The mating type locus of C. albicans controls the white-opaque transition. Only cells of the a or ␣ mating type are permissive for switching, and only opaque-form cells are able to mate with high efficiency. C. albicans cells are diploid and have never been observed to undergo meiosis; however, the morpho- 626 ec.asm.org logical changes that opaque cells undergo in response to pheromone are similar to those seen in Saccharomyces cerevisiae haploid cells. Mating-competent cells form mating projections in response to pheromone, and although these forms have been termed shmoos in both cell types, C. albicans can generate very long hypha-like conjugation tubes, while S. cerevisiae forms only short projections (29, 49, 53). Although the variety of cellular forms exhibited by C. albicans can be triggered by distinct cellular processes, in each instance polarized growth events must take place. Polarized growth is directed by the actin cytoskeleton, which consists of actin patches and actin cables (31, 42, 43). The myosin family proteins appear to interact with actin filaments in different ways to promote polarized growth in yeasts (8). In particular, the myosin type I protein encoded by MYO5 in C. albicans is required for the organization and polarized distribution of cortical actin patches. Myosin I is found to be colocalized with actin patches at the bud and hyphal tips, and a myosin I-null mutant shows impaired polarized growth and is incapable of hyphal formation (38). Genetic studies have revealed that myosin I functions in many actin-based processes, in particular endocytosis and exocytosis (17, 33, 37, 42, 43). In C. albicans, it has been observed that the ability to form hyphae is strictly correlated with MYO5 function in endocytosis (37). Received 13 January 2012 Accepted 29 February 2012 Published ahead of print 9 March 2012 Address correspondence to Malcolm Whiteway, malcolm.whiteway@cnrc -nrc.gc.ca. This article is publication 53122 of the National Research Council of Canada. Supplemental material for this article may be found at http://ec.asm.org/. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/EC.00021-12 1535-9778/12/$12.00 Eukaryotic Cell p. 626 – 637 Myo5p and WOR1 Expression in Candida albicans TABLE 1 C. albicans strains used in this study Strain Genotype TABLE 2 Plasmids used in this study Source MTLa ⌬ura3::imm34/⌬ura3::imm434 his1/his1 32 arg5,6/arg5 3315 MTL␣ trp1/trp1 lys2/lys2 32 CaNK01 CaI-4 MTL␣ MYO5/myo5::pVEC MYO5-GFP-URA3 This study CaNK02 3294 MYO5/myo5::pVEC MYO5-GFP-URA3 This study CaNK03 CaI-4 MTL␣ CDC12/cdc12::pVEC CDC12-GFP-URA3 This study CaNK06 BWP17 MTLa NOP1/NOP1::YFP-HIS1 MYO5/myo5::pVEC MYO5-GFP-URA3 This study CaNK07 3294 MYO5/myo5::hisG URA3 hisG This study CaNK08 3294 MYO5/myo5::hisG This study CaNK09 3294 myo5::hisG/myo5::hisG This study CaNK10 3294 myo5⌬/myo5⌬ Rp10/rp10::pRZ25 MET3-WOR1 This study CaNK11 3294 myo5::hisG/URA3-MET3-MYO5 This study CaNK12 3294 pVEC This study CaNK17 3294 sla2⌬871-1789::URA3/sla2⌬871-1789::HIS1 This study 3294 Because shmoo formation in C. albicans involves long polarized mating projections, we were interested in the potential role (...truncated)