Fellowship for Steven Smith: The Role of Talin in Cell Adhesion and Migration, and How a C-terminal Modular element Affects its Function

Cardimyopathies are caused by structural defects that occur in cardiomyocytes. These defects arise from changes in cell-cell or cell-matrix contacts such as focal adhesions (FAs), costameres, and intercalated discs. These contacts are formed through interactions involving the extracellular matrix (ECM), integrins, cytoskeletal proteins, and the actin cytoskeleton. Talins are large, modular proteins that link the actin cytoskeleton with the ECM by interactions with integrins. Talin1 is a key member of FAs, while Talin2 is found in costameres and intercalated discs in skeletal and cardiac muscle. Both vertebrate Talin1 and Talin2 are part of the I/LWEQ module superfamily, which is a class of actin-binding proteins that contains an ancient, conserved C-terminal actin binding element named the I/LWEQ module. Our goal is to understand the molecular interactions involved in the function and regulation of the Talin1 IILWEQ module. We hypothesize that the I/LWEQ module is an essential element in Talin1 and Talin2 that connects the actin cytoskeleton with the ECM, thereby promoting the integrity of FAs, costameres, and intercalated discs. We will test our hypothesis by addressing the following specific aims (1) determining if actin binding and dimerzation of the IILWEQ module are coupled and linked to block 4 and (2) by identifying the intrasteric regulatory mechanism of the I/LWEQ module. Talin1 will be used as our exemplar of I/LWEQ module-containing proteins; however, we will extend our work to Talin2 in order to improve upon our knowledge on the structure and function of costameres and intercalated discs in striated muscle. We will construct double and triple alanine mutations on the inner conserved residues in block 4 of the I/LWEQ module and test the F-actin binding capacity, dimerization, subcellular localization of these mutants to determine if these properties are linked. In addition, these same mutations will be made into fulllength mouse Talin1 to test the effects on cell adhesion and migration. To determine the regulation of the II LWEQ module, truncations will be made to biochemically identify the structural determinants of the USH-II LWEQ module interaction as well as GST-pull down assays to identify the protein partner(s) that relieve the USH intrasteric inhibition. These studies will improve the understanding of the functions of the I/LWEQ module and how these functions are regulated in Talin1 and other IILWEQ module-containing proteins.