T-tubule membrane remodeling in Drosophila myofiber function and models of myopathy

University Of California, San Diego, La Jolla CA

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Abstract

Project Summary Muscles are highly specialized cells that provide contraction essential for animal life. A disruption of the elaborate muscle cell organization underlies human myopathy diseases. In particular, differentiation of the muscle cell membrane into an extensive tubular membrane network called Transverse (T)-tubules is needed to enable signaling that coordinates power of muscle contraction. T-tubule disorganization or loss is observed in certain human skeletal myopathies and cardiovascular diseases, and interestingly, associated mutant genes encode for membrane regulators with known roles outside of muscle in endocytosis or endosomal trafficking. In flies, we discovered a regulated T-tubule remodeling program in development, along with the identity of conserved genes involved at distinct steps: disassembly, remodeling, or reassembly. Importantly, two fly genes function as key switches for T-tubule disassembly also have human homologs associated with centronuclear myopathy (CNM). This signifies that regulated T-tubule remodeling and the use of fly models are both likely important contexts to study, understand and treat human CNM disease. The fly system affords the unique and key advantages of in vivo imaging of T-tubule membrane dynamics in live, intact muscle cells within a stereotypical developmental timeframe in combination with a wealth of genetic tools. We established a new framework for understanding T- tubule dynamics needed both in development and adult muscle remodeling. We identified that class II PI3-kinase (PI3KC2) and dynamin large GTPase act together as a molecular switch that controls T-tubule membrane scission and disassembly with muscle cell remodeling. In these studies, we identified that there is concurrent muscle membrane remodeling not just of T-tubule membranes, but also of the adjacent encircling striations of costamere integrin adhesion complexes (IACs) that link the underlying contractile sarcomeres to the cell membrane. We hypothesize a shared PI3KC2-Dynamin switch activity coordinates muscle cell membrane disassembly of both T-tubules and IACs, and that this switch must be tightly regulated to ensure appropriate muscle dynamics and prevent myopathy disease. Using our innovative genetic, cellular and molecular approaches in intact fly muscles at different stages, we will build on our novel findings to determine the functional relationship between IAC dynamics and T-tubule disassembly. We will, (1) establish a timeline of normal IAC disassembly and protein components that depend on PI3KC2-dynamin switch activity in normal and induced muscle remodeling, (2) determine the signaling and molecular mechanisms that promote and mediate a switch in PI3KC2 and dynamin physical interactions, localization and enzymatic activities for disassembly, and (3) define how PI3KC2 functions with Rab21 GTPase, another molecular switch, collaborate to integrate T-tubule and IAC disassembly with integrin endocytosis and endosomal pathways. Importantly, we will establish conservation and consequences of pathway activity for T-tubule membrane reorganization in ongoing adult muscle function, with relevance to understanding human muscle disease.