The Ashbya gossypii fimbrin SAC6 is required for fast polarized hyphal tip growth and endocytosis.

Ashbya gossypii has been an ideal system to study filamentous hyphal growth. Previously, we identified a link between polarized hyphal growth, the organization of the actin cytoskeleton and endocytosis with our analysis of the A. gossypii Wiskott-Aldrich Syndrome Protein (WASP)-homolog encoded by the AgWAL1 gene. Here, we studied the role of AgSAC6, encoding a fimbrin in polarized hyphal growth and endocytosis, and based on our functional analysis identified genetic interactions between AgSAC6 and AgWAL1. SAC6 mutants show severely reduced polarized growth. This growth phenotype is temperature dependent and sac6 spores do not germinate at elevated temperatures. Spores germinated at 30°C generate slow growing mycelia without displaying polarity establishment defects at the hyphal tip. Several phenotypic characteristics of sac6 hyphae resemble those found in wal1 mutants. First, tips of sac6 hyphae shifted to 37°C swell and produce subapical bulges. Second, actin patches are mislocalized subapically. And third, the rate of endocytotic uptake of the vital dye FM4-64 was reduced. This indicates that actin filament bundling, a conserved function of fimbrins, is required for fast polarized hyphal growth, polarity maintenance, and endocytosis in filamentous fungi.

Candida albicans Vrp1 is required for polarized morphogenesis and interacts with Wal1 and Myo5.

Recently, a link between endocytosis and hyphal morphogenesis has been identified in Candida albicans via the Wiskott-Aldrich syndrome gene homologue WAL1. To get a more detailed mechanistic understanding of this link we have investigated a potentially conserved interaction between Wal1 and the C. albicans WASP-interacting protein (WIP) homologue encoded by VRP1. Deletion of both alleles of VRP1 results in strong hyphal growth defects under serum inducing conditions but filamentation can be observed on Spider medium. Mutant vrp1 cells show a delay in endocytosis - measured as the uptake and delivery of the lipophilic dye FM4-64 into small endocytic vesicles - compared to the wild-type. Vacuolar morphology was found to be fragmented in a subset of cells and the cortical actin cytoskeleton was depolarized in vrp1 daughter cells. The morphology of the vrp1 null mutant could be complemented by reintegration of the wild-type VRP1 gene at the BUD3 locus. Using the yeast two-hybrid system we could demonstrate an interaction between the C-terminal part of Vrp1 and the N-terminal part of Wal1, which contains the WH1 domain. Furthermore, we found that Myo5 has several potential interaction sites on Vrp1. This suggests that a Wal1-Vrp1-Myo5 complex plays an important role in endocytosis and the polarized localization of the cortical actin cytoskeleton to promote polarized hyphal growth in C. albicans.

Candida albicans SH3-domain proteins involved in hyphal growth, cytokinesis, and vacuolar morphology.

This report describes the analyses of three Candida albicans genes that encode Src Homology 3 (SH3)-domain proteins. Homologs in Saccharomyces cerevisiae are encoded by the SLA1, NBP2, and CYK3 genes. Deletion of CYK3 in C. albicans was not feasible, suggesting it is essential. Promoter shutdown experiments of CaCYK3 revealed cytokinesis defects, which are in line with the localization of GFP-tagged Cyk3 at septal sites. Deletion of SLA1 resulted in strains with decreased ability to form hyphal filaments. The number of cortical actin patches was strongly reduced in Deltasla1 strains during all growth stages. Sla1-GFP localizes in patches that are found concentrated at the hyphal tip. Deletion of the first two SH3-domains of Sla1 still resulted in cortical localization of the truncated protein. However, the actin cytoskeleton in this strain was aberrant like in the Deltasla1 deletion mutant indicating a function of these SH3 domains to recruit actin nucleation to sites of endocytosis. Deletion of NBP2 resulted in a defect in vacuolar fusion in hyphae. Germ cells of Deltanbp2 strains lacked a large vacuole but initiated several germ tubes. The mutant phenotypes of Deltanbp2 and Deltasla1 could be corrected by reintegration of the wild-type genes.

An Ashbya gossypii cts2 mutant deficient in a sporulation-specific chitinase can be complemented by Candida albicans CHT4.

The Candida albicans genome encodes four chitinases, CHT1, CHT2, CHT3 and CHT4. All four C. albicans chitinase-encoding genes are non-essential. The corresponding proteins belong to two groups in which Cht1, Cht2 and Cht3 are more similar to Saccharomyces cerevisiae Cts1, while Cht4 is more similar to ScCts2. In the filamentous fungus Ashbya gossypii, a CTS2 homolog (ACL166w) was identified as the sole chitinase gene. The AgCts2 is 490 aa in Length and shows 42.3% overall identity to ScCts2 (511 aa) and 33.2% identity to CaCht4 (388 aa). The A. gossypii cts2 deletion mutant showed no growth retardation or vegetative morphogenetic defects. However, upon sporulation Agcts2 mutants revealed a defect in spore formation. Expression of AgCts2 using a lacZ reporter gene was only found in the centre of a mycelium corresponding to the sporogenous part of a colony. The mutant spore phenotype of Agcts2 could be complemented by either AgCTS2, the S. cerevisiae CTS2, or the C. albicans CHT4 gene when expressed by either the AgCTS2 or the AgTEF1 promoter.