Stress-induced myosin folding and assembly mechanisms

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Muscle function and maintenance is governed by the spatiotemporal organization of structural and motor proteins into contractile sarcomeres. Given the constant use and mechanical stress exposure of the muscle tissue, the sarcomeric integrity is continuously protected by a complex network of protein folding and degradation pathways. An important regulator of sarcomere formation and muscle function is the HSP90 co-chaperone UNC-45. According to its central role in myosin assembly, UNC-45 protein amount and localization are tightly coordinated with mechanical stress conditions or induced damage to the myofiber. We are particularly interested in the myosin-directed UNC-45 chaperone network, orchestrating myofibrillogenesis from Caenorhabditis elegans to man. Besides elucidating the disease relevance of the human orthologue UNC-45B, we successfully developed powerful experimental approaches during the first funding period for the in-depth investigation of muscle-specific quality control pathways. In order to define myosin-directed stress response mechanisms, we have established optogenetic methods, transgenic reporter assays, and electrical pulse stimulation both in worms and murine myotubes in combination with optimized pull-down and mass spectrometry protocols. Moreover, we identified a so far undiscovered muscle-specific waste management pathway that is regulated by UNC-45. The central objective of the proposed research is to understand how protein folding and degradation networks are coordinated with the dynamics of muscle organization and repair in the context of mechanical stress conditions. The suggested project will address myosin-directed stress response programs and systematically analyze: the role of UNC-45 in protein synthesis, protein degradation, and exopher formation (Aim 1), mechanical stress-induced UNC-45 regulation and myosin-directed quality control (Aim 2), and the conserved regulation of UNC-45 function (Aim 3). To this end, optogenetic induction of mechanical stress, proximity labeling technologies, CRISPR/Cas9 gene editing, automated locomotor activity measurement, and large-scale genetic screenings will be performed. Given our recent discoveries of myopathy-related UNC-45 mutations and a novel muscle-specific waste management system, combined with state-of-the-art technologies established during the first funding period, we are ideally positioned to define mechanical stress response mechanisms that protect muscle function during acute physical exercise or continued use.