DESCRIPTION (provided by applicant): Skeletal muscle is critical for survival and quality of life, as it is required for respiration, ingestion, and locomotion. Aberrant muscle differentiation impairs these activities and can lead to disease and death. During myogenesis, myoblasts differentiate into myocytes, which fuse to form mature myotubes containing thousands of nuclei. Gene expression must be correctly regulated spatially and temporally to ensure proper myogenesis and maintenance of normal skeletal muscle function. Mature myotubes in vivo display regional specialization in function and protein expression and myonuclei exhibit nonequivalence of protein localization and transcript production. An open question is how transcription factors and other nuclear proteins can be targeted to one nucleus and not to its neighbors in a multinucleated myotube. Proteins that regulate gene expression are synthesized in the cytoplasm and must traverse the nuclear pore to access their targets in the nucleus. To enter the nucleus, proteins must translocate through nuclear pore complexes (NPC) composed of multiple nucleoporins (Nups). The NPC allows free movement of small molecules between the nucleus and cytoplasm but excludes proteins larger than 40 kDa. Larger proteins must be bound to a nuclear transport receptor, which recognizes a nuclear localization signal (NLS) within the cargo protein, in order to pass through the NPC. We examined nuclear import in cultured primary murine myotubes using an in vitro nuclear import assay and found that a subset of nuclei were incompetent to import a protein reporter containing the most common NLS, the classical NLS (cNLS). When cNLS import-competence was investigated across myogenesis, we found that, while almost all myoblast nuclei are cNLS import-competent, as myoblasts differentiate into myocytes, only a low percentage of myonuclei are cNLS import-competent. However, as myocytes develop into mature myotubes, cNLS import is restored. We hypothesize that modulation of nuclear import competence in differentiating muscle cells is a mechanism by which transcriptional activity of a nucleus is regulated. In Specific Aim 1, we will determine if cNLS import-incompetent nuclei are competent for other import pathways by analyzing the ability of myonuclei to import reporter proteins containing a cNLS compared to other NLS motifs. In Specific Aim 2, we will identify differences in Nup stoichiometry and post- translational modifications among myoblast, myocyte, and myotube nuclei and analyze their functional significance by mass spectrometry analysis of NPCs from three stages of myogenesis and subsequent inhibitor and siRNA experiments. In Specific Aim 3, we will compare transcriptional activity of cNLS import- competent and -incompetent nuclei by examining markers of active transcription by immunocytochemistry and the expression of specific transcripts by in situ hybridization in relation to the cNLS import-competent nuclei. Understanding muscle differentiation and coordination of transcriptional activity between many nuclei in a common cytoplasm could provide novel targets for preventing loss of muscle mass in disease and aging.

PUBLIC HEALTH RELEVANCE: Changes in gene expression are critical to proper development of skeletal muscle. Proteins that regulate gene expression are synthesized in the cytoplasm but act in the nucleus; consequently nuclear transport is a key regulatory step in gene expression. These experiments will increase our understanding of muscle differentiation and coordination of transcriptional activity among many nuclei in a common cytoplasm, which could yield novel targets for preventing loss of muscle mass in disease and aging.