Myosin motor proteins play fundamental roles in a multitude of cellular processes. Myosin generates force on cytoskeletal actin filaments to control cell shape, most dramatically during cytokinesis, and has a conserved role in defining cell polarity. Myosin contracts the actin cytoskeleton, ensuring prompt turnover of cellular adhesion sites, retracting the cell body during migration and development, and contracting muscle among diverse other functions. How myosins work, and why force generation is essential for their function, is in many cases an open question. Chapter 2 presents a structure-function analysis of the amoebozoan myosin 7 (DdMyo7) in live Dictyostelium discoideum cells. DdMyo7 bears structural resemblance to human Myosin 7 (a protein involved in maintenance of the retina, stereocilia of the ear, and gut microvilli) but has functional similarity to human Myosin 10, a regulator of cell adhesion that is also essential in formation of actin-based structures called filopodia. Phylogenetic analysis of these related proteins shows that DdMyo7 is not directly related to any human myosin but rather represents a molecular ancestor of several vertebrate myosins (Myo7, Myo10 and Myo15). Functional analysis focused on rescue of myo7– cells. The two MyTH4-FERM domains were fully redundant in rescuing formation of filopodia. A conserved Myo7 regulatory motif in the C-terminal FERM domain was found to stimulate filopodia formation when mutated, establishing DdMyo7 as a filopodial motor with features of Myo7 and Myo10. A molecular chimera of DdMyo7 motor/lever arm region fused to the MF domain of human Myo10 partially rescued filopodia formation, suggesting the MF domain plays a similar role in filopodia in divergent organisms. Structural information must be combined with physiological data to understand the mechanism of myosin motor function. Structural studies have long focused on conventional myosin 2 as a model due to ease of protein expression and purification. This approach has yielded considerable data regarding the static structures and in vitro kinetics of the myosin mechanochemical cycle; however, high-resolution methods to observe the dynamics of myosin activation in cells have been lacking. Chapter 4 introduces methods and instrumentation for rapid, precise measurement of fluorescence lifetime. This is a necessary step toward Myo2-based live cell FRET sensors described in Chapter 5. Implications of this work for future studies of myosin physiological function are discussed in Chapter 6.
University of Minnesota Ph.D. dissertation.December 2016. Major: Biochemistry, Molecular Bio, and Biophysics. Advisors: Margaret Titus, David Thomas. 1 computer file (PDF); xv, 171 pages.
Structure-Function Analysis of Motor Proteins: Insights from Conventional and Unconventional Myosins.
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