Regulation of bipolar myosin filaments by the giant scaffold protein obscurin

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The overall objective of my NSERC research program is to understand the modulation and regulation of the actomyosin cytoskeleton during development. Here we propose to investigate the giant scaffold protein obscurin, a myosin-binding protein. While there are three obscurin family members in vertebrates, there is only a single obscurin gene in Drosophila. We have previously shown that obscurin localizes to the M-line of sarcomeres, the smallest functional contractile unit of muscles, and plays a crucial role in myofibril assembly, especially required for the proper anchoring of myosin thick filaments at the M-line. Here we plan to analyze genetic obscurin mutants for their role in epithelial development, to identify and characterize proteins, in particular nonmuscle myosin, interacting with obscurin, and to investigate bipolar filament architecture in nonmuscle cells. This will help us understand the cell biological function of obscurin and its various protein domains. Aim 1: Investigate cellular defects in obscurin mutants. We have so far analyzed the function of hypomorphic obscurin mutants in myofibrils. We will now analyze these mutants in epithelial tissues and propose to generate null mutants by targeted mutagenesis using the CRISPR-Cas9 technique. We hypothesize that obscurin in addition to its muscle functions plays a role in epithelial development, because obscurin localizes to the cell cortex of epithelial cells and shows defects in morphogenesis and cell shape when depleted. We will analyze larval salivary glands, imaginal discs, and embryonic epithelia. This will identify novel nonmuscle functions common to all obscurin family members. Aim 2: Investigate bipolar filament architecture. We hypothesize that obscurin interacts directly with nonmuscle myosin in epithelial cells. We will purify tagged nonmuscle myosin rod domains and tagged obscurin domains to test interaction in vitro. We raised an antibody against the DH domain of obscurin, which will be used together with other antibodies to employ superresolution microscopy. This will help determine the relative localization and orientation of obscurin within actin fibers. We have also generated a transgene covering the N-terminus of obscurin. Affinity purification (AP) from adults and mass spectrometry (MS) identified two strong interactors: titin, which is known to interact with vertebrate obscurin, and tropomyosin-1, demonstrating the feasibility of this approach. We will repeat AP-MS with embryonic extracts to identify nonmuscle obscurin interactors and analyze them biochemically and genetically. This will uncover how obscurin regulates nonmuscle myosin filaments. Conclusion: This proposal offers the unique chance to better understand the function of a crucial actomyosin-regulating protein in a model organism, providing insights into the basic cell biological and developmental functions of obscurin.