Characterisation and functional analysis of actin cytoskeleton components and development of a novel hydrostatic pressure live cell imaging system

The main aims of this project were to use a variety of techniques to study various components of the actin cytoskeleton using Caenorhabditis elegans and Schizosaccharomyces pombe, in addition to developing a novel hydrostatic pressure imaging system.

The function and localisation of the C. elegans class I myosins: HUM-1 and HUM-5, were first explored using fluorescent protein fusions and phenotype analysis. The impact of a conserved phosphorylation event on myosin Ie function and localisation was then examined in C. elegans and S. pombe HUM-1 and Myo1 respectively. The results show HUM-1 and HUM-5 are non-essential, with HUM-1 expressed in a variety of tissues whereas HUM- 5 was expressed exclusively in the nervous system. Loss of HUM-1 reduces maximal brood size, inducing delayed embryo release. The conserved serine residue is required for the function and localisation of both HUM-1 and Myo1. Mutations of the residue altered HUM-1 localisation in a tissue specific manner and abolished Myo1 membrane association in addition to inducing spore formation.

In vitro and in vivo studies were next undertaken to explore the impact of fluorescent protein fusions and temperature sensitive mutations upon the the stability and function of S. pombe tropomyosin. While both Ts mutants, Cdc8-27 and Cdc8-110, had a two-step thermal unfolding transition lower than that of wild-type Cdc8, fluorescent fusions did not impact stability. Amino- terminal fusions however, mimic acetylation whereas carboxyl terminal fusions abolished polymerisation, localisation and functionality. The final part of this project used a methodical approach to develop a hydrostatic imaging system that could be used to follow fluorescently labelled protein dynamics in a live cell context. Preliminary data revealed the application of 100 bar of pressure induced a cell cycle delay in both S. pombe and Candida albicans, however the current limitations of the system make it incompatible with fluorescence microscopy.