May, Karen Marie; (1997) Identification and characterisation of a type II myosin in the fission yeast <italic>Schizosaccharomyces pombe</italic>. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The fission yeast Schizosaccharomyces pombe divides by medial fission and, like many higher eukaryotic cells, requires the function of a F-actin ring for cytokinesis. At the onset of mitosis actin forms an equatorial ring, analogous to the contractile ring of higher eukaryotes. This precedes the formation of the septum and subsequent cytokinesis. During cytokinesis in higher eukaryotes, F-actin, interacting with cytoplasmic myosin, forms the contractile ring structure that generates the force required for cytokinesis. It has yet to be determined conclusively if the actin ring of S. pombe is a contractile structure or if it is essential only for the deposition of septal material. Exhaustive screens have identified number of mutants defective in cytokinesis, including the actin binding proteins tropomyosin (Cdc8p), profilin (Cdc3p) and a putative myosin light chain (Cdc4p). To date all of the screens have failed to identify a myosin. This thesis describes the isolation and characterisation of the first myosin to be identified in S. pombe . The complete gene encoding a type II cytoplasmic myosin in the fission yeast Schizosaccharomyces pombe was cloned from a cosmid library. Sequencing of the gene, designated myo2+, revealed a single open reading frame, encoding a protein of 1526 amino acids, with significant homology to the 'conventional' type II class of myosin heavy chain. Genetic mapping revealed linkage to the ade6+ gene on chromosome III. To determine if myo2+ is an essential gene, a diploid strain was created with one copy of the gene deleted. When sporulated the myo2 null spores germinated but were unable to undergo cytokinesis and died, but occasionally a microcolony of aberrantly shaped cells with a hyphal morphology was formed. Further analysis indicated the presence of a suppressor mutation, designated smy2-, which partially rescues the lethality of the myo2 deletion. Cells overexpressing Myo2p became long and multinucleate with diffuse regions of septal material. Actin accumulated in patches throughout the cell. Localisation of actin to the cell tips and the site of septum formation did occur but no actin ring structure was formed and the cells failed to divide. These results suggest that myosin II is essential for completion of cytokinesis in fission yeast and for the reestablishment of growth polarity in the next cell cycle.

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