An investigation into the influence of nucleation factors on yeast F-actin structure and organisation

The actin cytoskeleton is a ubiquitous feature of eukaryotic cells capable of performing diverse cellular functions in response to the needs and specialisms of the cell. The core component of the actin cytoskeleton is the globular protein actin, which reversibly polymerises into filaments (F-actin). The cytoskeleton undergoes continuous reorganisation, and its regulation is performed on many levels, including isoform variation, post-translational modifications, interaction with actin binding proteins, local ionic conditions, and ATP hydrolysis. Actin nucleation – the formation of new filaments – undergoes tight spatiotemporal regulation, directed by a set of actin binding proteins called nucleators. There is evidence from cell-based studies that actin nucleators define not only the spatiotemporal nature of a new actin network, but also the properties of the actin network they establish. The collective action of discrete nucleators allows the generation of distinct actin networks in a common cytoplasm. This project used cryogenic electron microscopy (cryo-EM) to investigate the hypothesis that the influence of actin nucleators on actin filaments has a structural basis, with nucleators influencing the structure of the actin filaments themselves. Our chosen actin nucleator for this work was the endocytic protein Las17, which functions at actin patches at the cortex of the budding yeast Saccharomyces cerevisiae. Our use of actin from the same species allowed structural characterisation of actin filaments from S. cerevisiae, which has been studied only at low resolution until now. Our investigation re-assesses previous reports that F-actin from S. cerevisiae has greatly reduced inter-strand connectivity and a more open nucleotide-binding cleft than F-actin from vertebrate skeletal muscle. An additional, unexpected outcome of this project was the discovery of actin bundling as a novel in vitro functionality of the polyproline rich region of Las17.