Molecular pathways maintaining homeostasis in the vertebrate muscle sarcomere

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Our lab studies the molecular pathways that control and effect cell determination and differentiation. Of particular interest are the mechanisms of quality control in the metazoan muscle sarcomere, overlapping myosin and actin filaments that are joined by other proteins that give strength and elasticity to the complex in three dimensions while allowing the dramatic contraction of the fibers. Even minor defects in the assembly or stability of these protein complexes can compromise their function. There are highly regulated pathways that control the abundance of nascent structural components, and a strict order in which components are added to the sarcomere. The most abundant protein components of muscle are well known, but little is understood about the factors that regulate assembly, maintain the functional contractile state, and repair or replace the contractile components as they denature or are damaged. These ‘assembly' factors have been the recent focus of our lab towards two goals. (i) use genetics to uncover novel components of this pathway, and (ii) determine how these components regulate sarcomere patterning and assembly in skeletal and cardiac muscle, and test predictions of these models in vivo. The process of metazoan myogenesis is remarkably conserved, allowing us to extend our initial work in C. elegans to vertebrate muscle. The zebrafish is our experimental system of choice for the experiments described here, and recent developments in genome editing technology (CRISPR/Cas9) has made this system even more powerful. ***The first aim is to determine what role the non-structural quality control components (such as unc45b and smyd1b) play in patterning of sarcomeres, and to determine whether they work via ubiquitin-mediated protein degradation. This will monitor spatial and temporal expression of the components during early sarcomere assembly, and examine sarcomere structure in animals mutant for the major components. In the second aim, we will search for novel factors that work in this process, using existing muscle mutants (where the cognate gene is not identified) and analysis of transcript datasets derived from muscle mutants affecting different sarcomere assembly steps. In the last aim, we will use CRISPR/Cas9 to generate knockouts of suspected sarcomere assembly components for which mutants are not available (e.g. myomesin, and some E3 ligases). These mutants will be tested for abnormal sarcomere structure due to defects in assembly. ******