Molecular Genetic Analysis of Actin-interacting Protein 1 During Myofibril Assembly

Emory University, Atlanta GA

Investigators

Abstract

Actin is a ubiquitous and conserved protein among eukaryotes, but the structure and function of the actin cytoskeleton is remarkably diverse among cell types. This differentiation occurs by specific regulation of dynamic assembly and maintenance of actin filaments, a process mediated by a number of actin-regulatory proteins. Myofibrils in striated muscles are highly differentiated forms of actin cytoskeleton that are specialized for muscle contraction. The mechanisms by which the actin cytoskeleton is reorganized and assembled into sarcomeric structures during muscle development are largely unknown. The objective of this project is to understand the functional significance of regulators of actin polymerization and polymerization during myofibril assembly in body wall muscle of the nematode Caenorhabditis elegans. This project will focus on the function of the unc-78 gene that encodes a homolog of actin-interacting protein 1 (AIP1). AIP1 has been identified in a variety of eukaryotes and shown to be involved in dynamic reorganization of actin cytoskeleton. However, its role in multicellular organisms has not been clear due to lack of an AIP1 mutation in genetic model systems. Mutations recently isolated in unc-78 now make possible analysis of the role of AIP1 in muscle differentiation. In C. elegans, mutations in unc-78 cause slow-moving nematodes that have large accumulations of actin filament in body wall muscle cells. This phenotype suggests that the unc-78 gene is important for the regulation of actin filament dynamics in muscle cells. Therefore, this system provides a unique opportunity to study the in vivo function of AIP1 during myofibril assembly. In this project, the function of the unc-78 gene will be analyzed by the following five approaches: (a) Phenotypic consequences of unc-78 mutations will be characterized with respect to motility and the organization of myofibrillar components. (b) The expression pattern and subcellular localization of UNC-78 protein will be determined. (c) Biochemical interactions of wild-type or mutant UNC-78 proteins with actin and UNC-78 with unc-60 (the gene coding for ADF/cofilin) and other muscle genes will be tested. (e) The second AIP1 isoform in C. elegans will be examined for functional redundancy and specificity. These molecular genetic and biochemical analyses of the unc-78 gene and its related gene will potentially reveal an important aspect of actin filament dynamics in muscle cells and provide insight into the function of the evolutionarily conserved AIP1 family of proteins.