Browsing by Subject "myosin"

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    3D Orientation of Alpha Helix in Muscle Myosin Regulatory Light Chain Using Bifunctional Electron Paramagnetic Resonance
    (2021-06) Savich, Yahor
    Muscle contraction is a coordinated work of nanometer-sized force generators, myosin molecules. These molecules are out of equilibrium: they use the energy stored in the form of ATP to move collectively along the track protein actin. The myosin molecules transfer their work via lever arms that connect force generators to their cargo. Orientation of these lever arms has been studied thoroughly since 1) their structural dynamics is fundamental for understanding the muscle contraction and 2) their particular orientations are associated with disease states of cardiac and skeletal muscle. Electron microscopy, fluorescence polarization, and X-ray diffraction have provided insight into the structure of muscle, but there is still no high-resolution data of the vertebrate lever arm orientation available at ambient (not vitrified or crystallized) conditions. The present work establishes a method of measuring the orientation of the alpha helices in three dimensions using electron paramagnetic resonance (EPR). Chapter 3 introduces the use of EPR with bifunctional spin labels attached to different helices of the myosin regulatory light chain (RLC) protein with and without ATP. Demembranated skeletal muscle fibers were aligned with the slowly-varying magnetic field; RLC was chemically substituted by labeled RLC; axial orientational dynamics of the probe with respect to the muscle axis was determined. Chapter 4 utilizes 1) directional statistics that replaces the previous use of a Gaussian distribution and provides new insights into the degree of disorder and 2) a new bifunctional probe that adds an azimuthal dimension to the orientational data. Together, these techniques allow determination of the tilt and roll angles of the alpha helix without relying on the myosin structure.
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    MyTH4-FERM myosin based filopodia initiation
    (2020-07) Arthur, Ashley
    Filopodia are thin actin-based structures that cells use to interact with their environments. Filopodia initiation requires a suite of conserved proteins but the mechanism remains poorly understood. The actin polymerase VASP and a MyTH-FERM (MF) myosin, DdMyo7 in amoeba and Myo10 in animals, are essential for initiation. DdMyo7 is localized to dynamic regions of the ac-tin-rich cortex. Analysis of VASP with altered activity reveals that localized actin polymerization is required for myosin recruitment and activation in Dic-tyostelium. Targeting of DdMyo7 to the cortex is not sufficient for filopodia ini-tiation; VASP activity is required as well. The actin regulator locally produces new actin filaments which activates a MF myosin. Myosin then shapes or crosslinks the actin network so parallel bundles of actin can extend during filo-podia formation. This work reveals cooperativity of an actin binding protein and the actin cytoskeleton on mediating myosin activity during filopodia initia-tion.
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    Structure-Function Analysis of Motor Proteins: Insights from Conventional and Unconventional Myosins
    (2016-12) Petersen, Karl
    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.