The Cysteine Rich Protein (CRP) family of cytoskeletal LIM domain proteins has recently emerged as a class of molecules important for maintaining muscle cytoskeleton integrity. The molecular architecture of CRPs suggests that they act as cytoskeletal scaffolds, bring together multi-protein complexes. A mouse knock out of one family member, CRP3/MLP, results in cardiomyofibrillar disorganization, implicating CRPs in some aspect of muscle sarcomere stabilization. Two Drosophila CRP family members, designated M1p60A and M1p84B, have been identified that share many of the same characteristics of their vertebrate counterparts. The goal of this proposal is to investigate the molecular mechanisms of M1p84B function in Drosophila myogenesis. Mutations in mlp84B will be generated and studied in detail, in order to determine, on a cell biological level, how this protein functions in regulation of the muscle cytoskeleton. A second approach to defining M1p84B function is to identify the proteins with which it physically interacts. I will use the yeast two- hybrid method to screen for potential binding partners of Mlp84B. These interactions will be characterized using a variety of biochemical methods, such as immunoprecipitation and blot overlay. Similar biochemical methods will be employed to identify the docking sites on M1p84B for its binding partners, thus defining functional domains in the protein. The significance of these domains will be assessed by an in vivo structure/function analysis of M1p84B. Overall, these proposed experiments will add to our current understanding of muscle development, and have broader applications to the study of cytoskeletal regulation and assembly of macromolecular complexes.
