The role of tropomyosin in regulating actin filament dynamics in fission yeast

Actin is an essential cytoskeletal protein which is conserved in all eukaryotic organisms examined to date. It is actin's ability to polymerise into dynamic filaments which allows a cell's growth and even movement to be rapidly affected by both intra- and extra-cellular demands. Upon cellular signalling the actin polymerises to form filamentous or F-actin which form both cables and lattice structures, known as patches in yeasts. These actin cables have been shown to have a role in a large number of cellular processes which include cell polarity; cytokinesis; cell growth and movement; providing cortical tension; endocytosis; and acting as 'pathways' along which molecular motors (myosins) can travel. Actin's functions are conserved within yeasts, and forms cables and patches which localise predominantly to regions of cell growth, facilitating the cells increase in size during interphase, or division during cytokinesis. The fission yeast, Schizosaccharomyces pombe, is cylindrical in shape, with growth occurring in a polarised manner at the cell pole. Actin is seen to localised predominantly to patch structures at these growing cell poles and to actin cables throughout the cytoplasm during interphase. During mitosis actin cables exist as a major component of the cytokinetic ring, which contracts in order for a cell to divide. This project make use of the cross-discipline approaches allowed by this experimentally tractable organism to determine the role actin filaments play in regulating and maintaining polarised cell growth. Using the versatile fission yeast model system allows us to not only examine actin filaments in both an in vivo and in vitro context, but allows us to make use of mutants to modulate filaments' dynamics as well as their ability to interact with motor proteins. Thus it will be possible to elucidate their function(s) within the cell.

This project will address two main questions. The first is to determine the role actin filaments play in regulating and maintaining polarised cell growth in the fission yeast cell. The second is to determine whether tropomyosin plays a role in regulating myosin's interaction with actin and how it does this in a non-muscle cell. We will use real time live cell imaging to study actin filament dynamics in vivo, and examine the affect mutations within their component proteins (e.g. tropomyosin) has upon their dynamic properties, as well as establishing the affect the mutations have upon myosin function. We will go on to use a cross discipline approach to screen for novel tropomyosin mutants both in vivo (by identifying cells with abnormal actin filament function in the fission yeast cell) and in vitro (by identifying mutations which affect the protein's physical properties and interactions with actin). We will also explore the significance of why only a proportion of the tropomyosin in the fission yeast cell is constantly acetylated (80%) during the cell division cycle. This will be examined in detail using in vitro motility assays, as well as other biochemical techniques, and will examine how this post-translational modification affects tropomyosin's assocation with actin in a structural context using EM reconstruction techniques.