Molecular and phylogenetic analysis of the cytoskeletal linker proteins Talin-1 and Talin-2 and their role in cell adhesion during myogenesis.

摘要

The function of the cytoskeleton and its associated proteins is absolutely essential. Defects in cell adhesion due to mutation induced disruption of the cytoskeleton can lead to a variety of disorders including muscular dystrophies, neurodegenerative diseases including Alzheimer's and Parkinson's, and skin fragility diseases including epidermolysis bullosa. My dissertation focused on the mechanisms by which cells transduce environmental cues to the actin cytoskeleton. I provide substantial evidence that the focal adhesion protein Talin1 and its C-terminal actin-binding I/LWEQ module are vital to this process. I also provide substantial data that Talin1 and Talin2 have distinct functions during skeletal muscle myogenesis.;I show that the I/LWEQ module is found in a unique subset of cytoskeletal linker proteins and that the I/LWEQ module promotes actin-binding, dimerization, and F-actin stabilization among I/LWEQ module proteins. I also show that the Talinl I/LWEQ module is necessary and sufficient for targeting Talinl to focal adhesions. I demonstrate that actin-binding and subcellular localization, but not dimerization, is regulated by an intrasteric interaction with an upstream alpha-helix in Talinl. These data define a novel role for the I/LWEQ module as the primary adhesion-complex targeting determinant of Talinl and suggest that pathways that relieve inhibition of I/LWEQ module function will be important for regulating structural and signaling properties of adhesion complexes.;Using a bioinformatics approach, I show that TLN1 and TLN2 are found in all available vertebrate genomes. By Western blot and Q-RT-PCR I show that Talin2 expression is upregulated during skeletal muscle differentiation in parallel to myogenic markers. In contrast Talinl expression remains constant. I also identified putative regulatory sequences of human TLN1 and TLN2 and examined their differences by electrophoretic mobility shift assay. By immunofluorescence, I show that GFP-Talin1 localizes to focal adhesions at the ends of actin stress fibers in myoblasts, whereas GFP-Talin2 targets to large, fibrillar adhesions that decorate actin stress fibers and the cortical actin cytoskeleton. These data suggest that Talin1 is involved in the formation of dynamic focal adhesions required for cell migration and that Talin2 is involved in the formation of stable adhesions required for muscle fiber contractility and stability.