title: Using fluorescent myosin to directly visualize cooperative activation of thin filaments.
abstract: Contraction of striated muscle is tightly regulated by the release and sequestration of calcium within myocytes. At the molecular level, calcium modulates myosin's access to the thin filament. Once bound, myosin is hypothesized to potentiate the binding of further myosins. Here, we directly image single molecules of myosin binding to and activating thin filaments. Using this approach, the cooperative binding of myosin along thin filaments has been quantified. We have found that two myosin heads are required to laterally activate a regulatory unit of thin filament. The regulatory unit is found to be capable of accommodating 11 additional myosins. Three thin filament activation states possessing differential myosin binding capacities are also visible. To describe this system, we have formulated a simple chemical kinetic model of cooperative activation that holds across a wide range of solution conditions. The stochastic nature of activation is strongly highlighted by data obtained in sub-optimal activation conditions where the generation of activation waves and their catastrophic collapse can be observed. This suggests that the thin filament has the potential to be turned fully on or off in a binary fashion.
authors.affiliation: From the School of Biosciences, University of Kent, Canterbury CT2 7NH, United Kingdom.
authors.forename: Rama
authors.initials: R
authors.lastname: Desai
authors.affiliation: From the School of Biosciences, University of Kent, Canterbury CT2 7NH, United Kingdom.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Geeves
authors.affiliation: From the School of Biosciences, University of Kent, Canterbury CT2 7NH, United Kingdom n.kad@kent.ac.uk.
authors.forename: Neil M
authors.initials: NM
authors.lastname: Kad
mesh_terms: D008841:Actin Cytoskeleton / Q000737:chemistry*; D000199:Actins / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D002118:Calcium / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D006868:Hydrolysis; D009119:Muscle Contraction / Q000502:physiology; D018482:Muscle, Skeletal / Q000378:metabolism; D024462:Myosin Type II / Q000737:chemistry; D009218:Myosins / Q000737:chemistry*; D016011:Normal Distribution; D011485:Protein Binding; D012518:Sarcomeres / Q000378:metabolism; D013269:Stochastic Processes; D014335:Tropomyosin / Q000737:chemistry; D014336:Troponin / Q000737:chemistry
chemical_list: D000199:Actins; D005456:Fluorescent Dyes; D014335:Tropomyosin; D014336:Troponin; D000255:Adenosine Triphosphate; D024462:Myosin Type II; D009218:Myosins; D002118:Calcium
keywords: Actin; Cooperativity; Muscle Regulation; Myosin; Single Molecule Biophysics; Tropomyosin; Troponin