Muscle is essential for the function of the circulatory, urogenital, respiratory, and digestive systems. Although much is understood about the force-generating capacity of acto-myosin, little is known about how the contractile machinery is organized within muscle cells and about how it is stabilized during contraction cycles. Sites of actin filament anchorage represent key sites for organization, integration, and tethering of the contractile apparatus. The actin crosslinking protein, a-actinin, is concentrated at actin anchorage sites in muscle. We have identified members of two families of LIM domain proteins, the Cysteine-Rich Protein (CRP) family and the ALP-Enigma family as a-actinin binding partners. We have demonstrated that these proteins are enriched in muscle where they co-localize with a-actinin at actin filament anchorage sites. Here we propose to define the mechanisms by which these proteins contribute to muscle cytoarchitecture and function. By analysis of lossof-function mutations that we have generated, we will defme the contributions of these proteins to normal muscle structure and function. We will examine the consequences of these genetic alterations for the ultrastructural organization and molecular architecture of actin anchorage sites in muscle, including Z-discs, dense bodies, and regions of cell adhesion. We will identify the proteins that complex with CRP and ALPEnigma in order to gain a more comprehensive view of their molecular mechanisms of action. Regions of actin filament anchorage in muscle are proposed to play a critical role in muscle stability, and these areas may serve as architectural sensors that monitor the integrity and function of the contractile machinery. In humans, deficiencies in components of actin-anchorage sites are associated with cardiomyopathies. Therefore, our results will contribute to an understanding of both normal muscle function as well as aspects of muscle pathology.
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