Cell Biology of Mating inCandida albicans

Abstract

It was recently demonstrated that strains homozygous for either of the mating type-like loci MTLa and MTL␣ of Candida albicans undergo white-opaque switching and that expression of the opaque-phase phenotype greatly enhances mating between strains. Exploiting the latter property to obtain high-frequency mating, we have characterized the cell biology of the mating process of C. albicans. Employing continuous videomicroscopy, computer-assisted three-dimensional reconstruction of living cells, and fluorescence microscopy, we have monitored the mating-associated processes of conjugation, tube formation, fusion, budding, septum formation, and daughter cell development and the spatial and temporal dynamics of nuclear migration and division. From these observations, a model for the stages in C. albicans mating is formulated. The stages include shmooing, chemotropism of conjugation tubes, fusion of tubes and nuclear association, vacuole expansion and nuclear separation in the conjugation bridge, asynchronous nuclear division in the zygote, bud growth, nuclear migration into the daughter cell, septation, and daughter cell budding. Since there was no cytological indication of karyogamy, genetic experiments were performed to assess marker segregation. Recombination was not observed, suggesting that mating takes place in the absence of karyogamy between naturally occurring, homozygous a and ␣ strains. This study provides the first description of the cell biology of the mating process of C. albicans.Candida albicans is a diploid organism that, until quite recently, was believed to have no sexual cycle. However, in 1999 Hull and Johnson (13) demonstrated that C. albicans strain CAI4 contained MTLa1 on one chromosome and MTL␣1 and MTL␣2 on the homolog. Soon after, fusion was genetically demonstrated to occur between MTLa and MTL␣ strains (a/Ϫ and ␣/Ϫ, respectively) in vivo (14) and in vitro (20). Recently, Miller and Johnson (21) demonstrated that the homozygous MTLa and MTL␣ strains engineered from strain CAI4 underwent white-opaque switching and that when both strains were in the opaque phase, mating occurred 10 6 times more efficiently than when one or both strains were in the white phase. Lockhart et al. (17) subsequently demonstrated that approximately 3% of 220 clinical isolates of C. albicans representing the major clades worldwide were homozygous for the mating type locus (i.e., either MTLa or MTL␣), that all tested clinical strains that underwent white-opaque switching were homozygous for MTL, that most clinical strains that were homozygous for MTL underwent the white-opaque transition, and that heterozygotes did not undergo white-opaque switching. Together, these recent results demonstrate for the first time that the mating type locus regulates switching and that switching facilitates mating.To visualize the mating process of C. albicans, we took advantage of the recent discovery that mating is facilitated by the opaque-phase phenotype (21). Using information obtained from videomicroscopy, computer-assisted dynami...

The Same Receptor, G Protein, and Mitogen-activated Protein Kinase Pathway Activate Different Downstream Regulators in the Alternative White and Opaque Pheromone Responses ofCandida albicans

“…Opaque cells of the mutants ste2⌬ and ste4⌬ were also compared with opaque cells of parent strain P37005 for their ability to mate with opaque cells of the ␣/␣ strain WO-1, by using microscopically identified fusion as an assay (Lockhart et al, 2003a) Together with the pheromone response data, these results indicated that the ␣-pheromone receptor,Ste2p and the ␤-subunit of the heterotrimeric G protein Ste4p were essential for ␣-pheromone-induced shmooing and fusion of opaque a/a cells, and that the a-pheromone receptor Ste3p was essential for a-pheromone-induced shmooing and fusion of opaque ␣/␣ cells.…”

“…Opaque cells of the mutants ste2⌬ and ste4⌬ were also compared with opaque cells of parent strain P37005 for their ability to mate with opaque cells of the ␣/␣ strain WO-1, by using microscopically identified fusion as an assay (Lockhart et al, 2003a) observed between opaque ste2⌬ or ste4⌬ cells, and opaque WO-1 cells ( Figure 3, B and C, respectively; and K). Opaque cells of the complemented strains ste2⌬/STE2 and ste4⌬/ STE4 regained the capacity to mate with opaque WO-1 cells (data not shown).…”

“…But C. albicans has one additional and unique response to mating pheromones that so far has not been identified in other yeast . To mate, MTL-heterozygous strains of C. albicans must undergo homozygosis to a/a or ␣/␣ (Hull et al, 2000;Magee and Magee, 2000), then switch from the mating-incompetent white phenotype to the mating-competent opaque phenotype Lockhart et al, 2003a). Although pheromones induce mating responses in opaque a/a and ␣/␣ cells, including G1 arrest, polarization, and shmooing, as in S. cerevisiae, they do not induce these responses in white cells (Bennett et al, 2003; Lockhart et al, 2003a,b;Zhao et al, 2005a;Daniels et al, 2006).…”

“…Cell concentration was also monitored over time. To test for a-pheromone-induced shmoo formation, a transwell assay was performed according to Daniels et al (2006).To test for mating (Lockhart et al, 2003a), opaque cells of an a/a or ␣/␣ mutant were grown to early saturation phase and mixed with an equal concentration of WO-1 (␣/␣) or P37005 (a/a) opaque cells, respectively, in liquid culture. The mating mixtures were incubated at 25°C in a rotary shaker (250 rpm), and they were monitored for fusants microscopically over a 48-h period.…”

“…Whereas opaque cells of parent strain P57072 cells were induced to form shmoos ( Figure 2B), opaque cells of ste3⌬ were not ( Figure 2C). Opaque cells of the complemented strain ste3⌬/STE3 regained shmoo formation in response to a-pheromone (data not shown).Opaque cells of the mutants ste2⌬ and ste4⌬ were also compared with opaque cells of parent strain P37005 for their ability to mate with opaque cells of the ␣/␣ strain WO-1, by using microscopically identified fusion as an assay (Lockhart et al, 2003a) Together with the pheromone response data, these results indicated that the ␣-pheromone receptor,Ste2p and the ␤-subunit of the heterotrimeric G protein Ste4p were essential for ␣-pheromone-induced shmooing and fusion of opaque a/a cells, and that the a-pheromone receptor Ste3p was essential for a-pheromone-induced shmooing and fusion of opaque ␣/␣ cells.White Cell Pheromone Response of ste2⌬, ste3⌬, and ste4⌬ White cells of the mutants ste2⌬ and ste4⌬ were compared with white cells of the parental strain P37005 for the ␣-pheromone-stimulated white cell response, which included dramatic increases in cohesion, adhesion, and enhanced biofilm development . To assess pheromoneinduced cohesion, ste2⌬ and ste4⌬ cells were incubated in suspension either in the absence or in the presence of ␣-pheromone for 6 h. Cells were then distributed on a slide, and the average number of cells per aggregate was calculated.…”