PROJECT SUMMARY/ABSTRACT Skeletal muscle allows for controlled movement and fine-tuned coordination throughout the entire life of an individual. In all cells, the response to physical forces is critical; however, this is particularly important in skeletal muscles to facilitate movement, contraction, and force generation. Mechanotransduction allows cells to sense and respond to the external environment. In muscle cells, forces transmit through the Z-disc which are interspersed between sarcomeres and anchor actin filaments. In numerous muscular diseases and myopathies, mechanotransduction is altered and leads to loss of force, muscle wasting, and increased stiffness. At the molecular level, these pathological alterations are accompanied by extensive transcriptional changes and mis- regulation of alternative splicing, an RNA processing mechanism that allows single genes to code for multiple protein isoforms. A unique feature of skeletal muscle is that it exhibits one of the highest levels of tissue-specific and evolutionarily conserved alternative splicing. During muscle development, extensive alternative splicing changes occur to facilitate maturation of the tissue. Interestingly, numerous genes developmentally regulated by splicing encode proteins that are involved in membrane trafficking and localize to the sarcomere. One of these genes encodes the Capping Actin Protein of Muscle Z-Line Subunit Beta (CAPZB) protein. Besides its critical function in capping actin, CAPZB plays unconventional roles in sarcomere organization. Surprisingly, how mechanotransduction and alternative splicing are interconnected in muscles has not been deeply investigated and can reveal new insights about muscle diseases. In my proposal, I hypothesize that alternative splicing regulation contributes to the development of the mechanical properties of skeletal muscle. I will test this hypothesis in two specific aims. In aim 1, I will identify the role of two splicing regulators, the poly-pyrimidine tract binding protein 1 (PTBP1) and quaking protein (QK), in controlling the mechanical properties of muscle cells by stretching cells and investigating effects on mechanosensitive pathways. In aim 2, I will determine how CAPZB and its splice forms contribute to the mechanosensitivity of muscle cells by using force microscopy and functional studies. My long-term goal is to be an independent scientific leader who can lead a team. Therefore, the training I will receive through this fellowship will facilitate my growth in becoming a muscle biologist with expertise in RNA processing, and solid skills in mentorship, writing and teaching. My sponsor and co-sponsor are experts in muscle physiology, cell biology, and, alternative splicing and I have recruited collaborators and mentors with expertise in physics and mechanotransduction to facilitate a multidisciplinary dissertation. All of them are strongly committed to mentoring and education and will support me during my Ph.D. and as I...