Wheatley, Sally Paula; (1993) The actin cytoskeleton of Schizosaccharomyces pombe. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In this study a number of approaches were taken to investigate actin and the contribution of its associated proteins, particularly myosin, during the cell cycle of S. pombe. Methods used included actin purification, drug treatment of whole cells, and attempted cloning of genes defective in the actin-dependent event, cytokinesis. S. pombe is a particularly amenable organism for cell cycle studies as it grows only by extension at the tips, hence measurement of cell length provides a convenient and accurate marker for cell cycle progression. Actin also undergoes predictable rearrangements during the course of the cell cycle anticipating areas of cell wall deposition thus providing an alternative cell cycle gauge which is particularly useful when cell polarity has been disturbed eg. by pharmacological agents and mutational alterations. The main emphasis of this study assesses the effects of three reagents on cell cycle events hence this latter strategy for monitoring cell cycle position is employed. The drugs used are the actin inhibitors cytochalasins B and D, and a myosin inhibitor, 2,3-butanedione-2-monoxime (BDM). S. pombe was sensitive to both cytochalasin B and cytochalasin D in the range of 50-100μgml-1. The effects of both drugs were reversible at 50ugml-1. Whilst the population numbers and septation indices were similarly affected by each drug, the actin configurations were markedly distinct. Cytochalasin B treated cells exhibited an increased number of actin rings in the population but, most strikingly, tip staining was not lost in these cells, whereas in untreated wild type controls these patterns were mutually exclusive. The ability to construct an equatorial actin ring without loss of polar staining suggests that, in contrast to mammalian cells, S. pombe is able to assemble a cytokinetic ring from a population of actin distinct from that seen at the cell tips, thus, in this case, global redistribution of actin during the cell cycle may not occur. Additionally, the concentration of actin at both localizations was enhanced in the presence of cytochalasin B and cells were correspondingly swollen at these regions. By contrast, polarity in cytochalasin D treated cells appeared to be diminished, the cells swelling uniformly to create a more rounded phenotype than controls. Background staining of actin was high although there was a bias towards staining at the cell tips. Where present, actin rings appeared to be contracted. The role of actin-associated motors, or myosins, has not previously been addressed in the fission yeast. In this study, a myosin cross-bridge inhibitor, BDM, was used to assess the possible roles of myosin(s) in S. pombe. BDM was found to reversibly inhibit S. pombe growth at 20mM. BDM treated cells were uniformly short and less polarized than untreated controls. Calcofluor staining, actin localization and FACS analysis suggested that BDM arrested the majority of cells (90%) in G1 with a 1C DNA content. The remainder of the population appeared to be arrested in cytokinesis with a 2C content. The rate of cell elongation was estimated to be inhibited by up to 47% by the addition of 20mM BDM, hence although growth was markedly reduced, it was not abolished at this concentration. Electron microscopy revealed a complete absence of large vacuoles which are normally abundant in S. pombe, and an accumulation of abnormally large vesicles close to the nucleus which contained electron dense material. Although the origin and nature of these structures remains unresolved, the presence and location of these vesicles indicates that BDM may be interfering with intracellular transport. Using synchronized cultures of the cell cycle mutant, cdc25.22, it was found that BDM increased the length of time required to complete cytokinesis. Thus treatment with BDM suggested the involvement of myosin(s) in normal cell growth and cytokinesis. Screening of cell division cycle mutants defective in cytokinesis revealed two hypersensitive strains, cdc4.8 and cdc7.2A. As cdc4 is known to encode myosin light chain, this exemplifies the potential use of BDM as a tool for identifying myosin interacting proteins or myosin dependent events. In summary, BDM points to the potential role of myosin(s) in cell growth and cytokinesis in S. pombe.

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