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Thesis Abstract

Title: Purification and Characterization of a 270 kD actin-binding protein from Dictyostelium that localizes to the centrosome

Author: Niebling, Kenneth Robert, Ph.D.

Date: Stanford University, 1993

Using a null mutant of Dictyostelium discoideum (Manstein et. al., 1989) as a biochemical background in which to identify novel cytoskeletal proteins, I have purified a 270 kD protein from and actin-rich cytoskeletal fraction derived from extracts of these cells. The cytoskeletal precipitate derived from extracts of wild type cells contains actin, myosin, and a number of other unidentified proteins (Clarke and Spudich, 1974). The profile of the precipitate derived from the myosin null cells is remarkably similar except for the enrichment of the 270 kD protein and the absence of myosin. This proteinís association with the cytoskeletal dialysis precipitate is Mg2+ sensitive. In Chapter 1, I show that this protein has a submicromolar affinity for filamentous actin in cosedimentation assays. Electron microscopy reveals an oval shaped structure with dimensions 100 x 165 angstroms. Using polyclonal antibodies for indirect fluorescence immunomicroscopy, I demonstrate that in cells fixed in the presence of 0.04% Triton X-100 that this protein localizes to the centrosome, to the nucleus, and to a reticular meshwork throughout the cell. Also within chapter 1, three examples of subcellular localization of the cytoskeletal proteins actin, and tubulin are shown. Particular emphasis is focused on the methods and treatment of the cells used in these experiments, and the results of these localizations and that of the 270 kD protein are discussed in this context. We conclude that this subcellular localization makes this protein a candidate to participate in centrosomal function, and that its actin binding activity suggests that it may be part of a class of proteins that link important cellular structures to the actin cytoskeleton.

Molecular genetic methods have been used within Dictyostelium to support the biochemical work that has been done with purified proteins. In order to define the role of the 270 kD protein in vivo, I have begun to isolate and sequence this 8 kb gene. This work, along with experiments designed to disrupt the function of this protein in vivo using the recovered sequences in gene targeting experiments are described in the second chapter. Strategies aimed at isolating the unrecovered portions of this gene, and for disruption and genetic analysis of mutants within Dictyostelium are also discussed within this chapter.

Equipped with approaches which use both biochemistry and molecular genetics within Dictyostelium, proteins of unknown function can be characterized both in vitro and in vivo. In the final chapter, I describe the results of a related but distinct project. My work in collaboration with my colleagues (Nature 328 (6130):536-539.) using in vitro motility assays led to the identification of a domain restricted to the myosin head which is sufficient to produce actin sliding motility. The production and purification of single-headed myosins and the examination of their properties within this defined in vitro system is the subject of the final chapter.