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Title: Characterisation of the domain in Sla1p required for regulation of the yeast actin cytoskeleton
Author: Dewar, Hilary
ISNI:       0000 0001 3423 0873
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2002
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Slalp is a protein required for cortical actin cytoskeleton structure and organisation in the budding yeast Saccharomyces cerevisiae. Slal null cells display aberrant cortical actin patches which are fewer in number but significantly enlarged. Movement of these patches is also significantly reduced in slal null cells. Expression of mutant forms of Slalp in which distinct domains within its primary sequence are deleted has shown that phenotypes associated with the full deletion are functionally separable. In particular, the C-terminal repeat region is required if cells are to grow in the absence of cortical patch protein Abplp and a region encompassing the third of its three SH3 domains is important for the highly polarised and punctate appearance of cortical actin patches in wildtype cells. To investigate the role of Slalp in actin dynamics further an slal mutant lacking the third SH3 domain has been integrated into the yeast genome (slal-Delta118-511) and expressed as the sole form of Slalp in cells. Rhodamine-phalloidin staining of this mutant showed aberrant cortical actin patches similar to the slal null phenotype. Both myc and GFP tagged mutant proteins localise to the cell cortex but do not show any co-localisation with the aberrant actin patches, implicating this domain in actin cytoskeleton interaction but showing it to be unnecessary for cortical localisation. A role for Slalp in enhancing actin turnover is also suggested by the observation that yeast expressing the mutant Slalp exhibit a significantly reduced sensitivity to Latrunculin compared to wild-type cells (indicating increased F-actin stability). A two-hybrid screen using the slal-118-511 domain as bait identified two actin- associated proteins as binding partners: Sla2p and Ysc84p. Sla2p was identified in the same synthetic-lethal screen as Slalp and is required for a normal actin cytoskeleton and endocytosis. Ysc84p is a novel protein that requires Abp1p (but not Slalp) for localisation to cortical actin patches. Ysc84p is not essential for viability, actin organisation or uptake of vital dyes. However, cells that lack Ysc84p and a novel protein encoded by the ORP YCL034W were inviable at 37°C. YCL034W encodes a protein that has previously been identified in a two-hybrid screen as interacting with the S. cerevisiae Arp2/3 complex activator and WASp homologue Lasl7p. Thus the slalp-118-511 domain, was found to play an essential role in regulating cortical patch structure and organisation as it couples actin cytoskeleton associated proteins and endocytic complex associated proteins.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Eukaryotic cytoskeleton