Fellowship/Singiser: Talin2 and Intercalated Disc Assembly in Cardiac Cells

Our goal is to understand how Talin2 interacts with various muscle specific proteins. Vertabrate talins are large, modular proteins that link the extracellular matrix (ECM) with the actin cytoskeleton in plasma membrane-associated multicomponent adhesion complexes. We have recently found that Talin2 is a component of intercalated disks in cardiac muscle. Talin1 is essential for mouse embryonic development, which is not compensated for by Talin2, indicating that the two isoforms of talin are not redundant. Talin1 contains a calpain2 proteolytic site that contributes to the dynamic nature of focal adhesions. This cleavage site is not found in Talin2, which we hypothesize contributes to the stablitiy of intercalated discs and costameres. We hypothesize that Talin2 functions to link the actin cytoskeleton to the plasma membrane at intercalated discs in cardiac myocytes, and dysregulation of Talin2 may lead to cardiac diseases such as cardiomyopathy. While previous work has studied Talin1 function in focal adhesions, this proposal seeks to understand the isoform-specific functions of Talin2 in intercalated discs. Based on substantial preliminary studies, we hypothesize that Talin2 is required for the assembly of intercalated discs and provides stability to intercalated discs that is not found in Talin1-containing adhesion complexes. To address this hypothesis we will (1) determine the molecular interactions that drive N-RAP and metavinculin binding with Talin2 at intercalated discs and (2) determine the stabilizing features that allow Talin2 to function in intercalated discs. Various techniques will be used to determine Talin2 interacting partners and Talin2 stability. While determining properties of Talin2 binding partners we will do coimmunoprecipitations using our isoform specific antibodies, and express recombinant GST fusion protein constructs to determine Talin2 interacting regions. To determine stabilizing effects of Talin2 we will produce GFP-Talin2 fusion proteins that will be introduced into chicken cardiomyocytes. We will monitor localization with confocal microscopy. This research will provide insight into the structure and assembly of intercalated discs, which has been relatively unstudied. Ultimately, this work may lead to a better understanding of the fundamental mechanisms in cardiac muscle development which will lead to better understanding and treatment of cardiomyopathies.