MOLECULAR GENETIC ANALYSIS OF THE CYTOSKELETON

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The cytoskeleton is a complex and dynamic structure that plays a crucial role in the determination of cell shape, motility and morphogenesis. These processes are critical for normal development, wound healing and the immune response. In addition, an understanding of the regulation of motility can be applied to the prevention of tumor cell metastasis. We are using molecular genetic techniques to determine the in vivo function and interactions of two cytoskeletal proteins. Dictyostelium amoebae are remarkably like mammalian motile cells both in behavior and in the complement of proteins that organize the cytoskeleton. Dictyostelium ABP-120 and ABP-240 both cross-link actin filaments into orthogonal arrays and are both related to human non-muscle filamin. Human filamin has recently been shown to be important for cancer cell motility. Structural, functional and immunological evidence indicates that ABP-240 is the Dictyostelium equivalent of filamin. Dictyostelium ABP-120 has an actin binding site that is related to filamin but the protein has a different structural conformation. It is presumed that Dictyostelium ABP-120 and ABP-240 would perform related but distinct functions in cytoskeletal organization. This project will accomplish the cloning and sequencing of the Dictyostelium ABP-240 gene in order to define its structural and primary sequence similarity to both Dictyostelium ABP-120 and filamin. In order to assess the in vivo function of ABP-240, we will create mutant cell lines either lacking the ABP-240 gene possessing mutations in predicted functional sites. The possibility that ABP-120 and ABP-240 have overlapping functions will be addressed by making mutants that lack both genes. In order to minimize the cancer of compensatory mutations caused by the lack of either or both of these proteins, a new technique will be employed which will allow either protein to be conditionally expressed in cells. The mutant cell lines will be extensively analyzed to determine the in vivo function of these two proteins. The rate of growth and behavior during development will assess the general viability of the cells. Monoclonal antibodies specific for ABP-120 and ABP-240, in combination with fluorescent phalloidin will be used to determine the relative 3- dimensional localization of actin filaments and the two cross-linking proteins in wild-type and mutant cells. Computerized analysis of cell motility will be used on vegetative and chemotactically oriented cells to determine what behavior aspects of pseudopod extension and movement are altered by the mutations. Most models of cell movement and shape control focus on the organization of the actin cytoskeleton. The actin filaments in cells are found primarily in the form of orthogonal networks. The described experiments will directly test these models by altering the proteins responsible for F-actin cross-linking and investigating how these perturbations affect cytoskeletal organization and motility.