The mechanisms and functions of nuclear spacing in muscle development

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Summary Nuclear movement is evolutionarily conserved throughout eukaryotes, a testament to the fundamentally important nature of this process. The importance of this process is most evident in syncytial muscle cells, which move their nuclei to maximize the distance between each nucleus and its neighbors, a process thought to establish a series of independent myodomains. Furthermore, mispositioned nuclei in muscle cells are prevalent in muscle disease. In recent years, dozens of proteins have been implicated in the movement and spacing of nuclei, but their mechanisms of action, and the consequences of nuclear spacing, remain elusive. The goal of the proposed research is to fill this critical gap in knowledge. Specifically, we will define the features of the microtubule cytoskeleton that are critical for both moving nuclei to establish the spacing and anchor nuclei to maintain the spacing. Only recently have the molecular, physical, and imaging tools emerged to make it possible to address these mechanistic questions in vivo. In the proposed research we will generate and test mutant proteins that target the microtubule specific functions of pleitropic proteins to isolate the specific contribution of the microtubule interactions. We will then apply our recently developed analyses of nuclear movement and microtubule organization combined with classical genetic approaches to determine the genetic and molecular mechanisms by which these factors move and anchor nuclei during muscle development. Within this work we will test whether two emergent properties of the microtubule cytoskeleton, branching and sliding contribute to nuclear spacing in muscle. Finally, we will directly tie these data to disease pathogenesis by determining how dystrophin, the gene mutated in the most common and debilitating muscle disease contributes to these processes. Successful completion of these aims will provide the first mechanistic understanding of nuclear movement in an in-tact developing muscle cell making it possible to manipulate nuclear spacing to understand how the position of the nucleus impacts its function.