Formation and remodeling of the muscle cell T-tubule membrane network. Amy Kiger, Jen Nguyen, Ines Ribeiro, Naonobu Fujita. Cell & Dev Biol, Univ California, San Diego, La Jolla, CA.

   Muscle cells rely on the extensive membrane network of the excitation-contraction coupling system for synchronous contraction. Transverse (T)-tubule membranes extend continuously from the plasma membrane into the muscle interior to couple surface signals with sarcomere function. Whereas the physiological significance of T-tubules is established in fly and human mobility and myopathy, little is understood about T-tubule formation or remodeling with muscle use. We discovered that the T-tubule network undergoes a dramatic, regulated remodeling during wildtype metamorphosis, providing an ideal developmentally programmed system in flies to study mechanisms of T-tubule remodeling. In pupal abdominal myofibers, we show that T-tubules disassemble at the larval-pupal transition and then rapidly reform in pharates. The extent of T-tubule disassembly correlates with an accumulation of membrane inclusions marked with T-tubule proteins, suggesting a membrane reservoir that serves in T-tubule reassembly. Using this system, we found that shibire (shi) dynamin GTPase and myotubularin (mtm) phosphoinositide 3-phosphatase play opposite roles in T-tubule remodeling. Muscle-targeted depletion of shi blocked regulated T-tubule disassembly, while in contrast, mtm depletion blocked T-tubule reassembly. Moreover, GTPase-dependent shi overexpression was sufficient to drive persistent T-tubule disassembly and Shi localization at T-tubule remnants. Double mutant analyses indicated that shi and mtm genetically interact in a common pathway dependent on the Class II PI3-kinase, Pi3K68D. Altogether, our results indicate that dynamin acts directly in T-tubule membrane scission under phosphoinositide regulation. Importantly, these and our additional findings with disease mutations provide insight into dominant and recessive forms of human centronuclear myopathy associated independently with Dynamin-2 and MTM1, respectively. Our work here and from ongoing screens reveals the dynamic nature and specific mechanisms of T-tubule membrane formation.