Sigyn Jorde:
Functional analysis of proteins involved in actin cytoskeleton organization in Ashbya gossypii and Candida albicans

Date: 04-11-2010    Supervisor: Steen Holmberg

This thesis deals with some of the aspects of endocytosis in fungi. The human pathogen Candida albicans and the filamentous Ashbya gossypii were used as models when investigating some of the core mechanisms in this process. The virulence of C. albicans is dependent on its ability to switch between yeast and hyphal growth, which is why these dynamic processes are of special interest. A. gossypii has been used in comparison to study the extended polarized growth in hyphae. First, a set of SH3-domain containing proteins in C. albicans were tagged with GFP to allow for visualization and localization. Three proteins were successfully visualized with a C-terminal GFP, namely Bbc1, Sla1 and Cyk3. Cyk3 localized at septal sites and Bbc1 and Sla1, proteins involved in endocytosis, were seen in cortical patches. Second, this PhD thesis addressed interactions formed in C. albicans among the core proteins in actin filament nucleation. For this we used the yeast two-hybrid assay, which indicates physical interactions between Vrp1 (WIP homolog) and the Arp2/3 nucleators Wal1 and Myo5 (WASP and myosin I homologs, respectively); a similar complex is formed in S. cerevisiae. Also, functional analysis of the A. gossypii fimbrin mutant sac6 was performed, a gene that is synthetic lethal with SLA2 in S. cerevisiae. SLA2 is essential for endocytosis in other studied fungi but the gene is missing in A. gossypii. Deletion of SAC6 leads to impaired endocytosis, greatly affected organization of the actin cytoskeleton and swelling hyphae when grown at elevated temperature. These phenotypic characteristics are similar to the wal1 (WASP) mutant implying that the two proteins take part in the same pathway. Finally, proteins of the eisosomes, Pil1, Lsp1 and Sur7 in A. gossypii were investigated. As in S. cerevisiae deletion of PIL1 produces the more severe phenotype. Agpil1 is barely viable under the growth conditions tested in this assay but investigation of a few germinating mutants showed no obvious defects in endocytosis or actin organization. A GFP tagged AgPil1 localized in a punctate pattern beneath the plasma membrane but in separate structures from cortical actin patches. The same localization was observed in all eisosome mutants as well as in sac6 and wal1. This implies that eisosomes are not directly involved in the endocytosis process but might have a regulatory function.