If you have any problems related to the accessibility of any content (or if you want to request that a specific publication be accessible), please contact (email@example.com)
The Interactions of Smooth, Skeletal, and Cardiac Myosin Filaments with Actin Provide Evidence for a Novel Mixed-Kinetic Model of Actomyosin Mechanochemistry
AuthorBrizendine, Richard K.
AdvisorCremo, Christine R.
AltmetricsView Usage Statistics
There are numerous diseases known to affect contractility of the three major types of muscles in humans: smooth, skeletal, and cardiac. A current problem in the field of muscle biophysics that this dissertation will address is that there is a lack of an integrated approach to understanding the underlying mechanisms (kinetic and structural) of factors that perturb the normal function of the contractile apparatus. Due to the complexity of muscle, the most often used approach to probe the kinetics and mechanics of the system is the in vitro motility assay. These assays are useful because experimental conditions can be tightly controlled and specific effects of a single perturbation can be discovered. We have recently developed a novel in vitro motility assay (Mf/A assay) that allows us to measure unloaded sliding velocities (V) of myosin filaments (Mf) moving over surface-attached actin (A). This assay is more ‘muscle-like’ and avoids some of critical disadvantages of the standard in vitro motility assay (A/Mm assay) where actin moves over surface-attached monomeric myosin (Mm). In this dissertation, we further develop this assay and others, and show that when myosin is in its physiologically functional filamentous form, classical models of actomyosin mechanochemistry cannot explain the observed results.Instead we use these novel assays to develop a novel model of actomyosin mechanochemistry called the mixed-kinetic model. This model incorporates structural, kinetic, and mechanical components that were previously unrecognized as important affecters of muscle contraction and provides a powerful tool to analyze data from all types of motility assays. In this model, velocity is influenced by kinetic constants defining myosin attachment to actin as well as myosin detachment from actin. The relative contributions of each vary with p, the probability that a myosin head will remain attached to actin long enough to reach the end of its S2 tether (distance dictating the length parameter, L). This model has two key features: 1) at low N, V is primarily influenced by attachment kinetics (V = Nvd), and 2) as N increases, V becomes influenced increasingly by detachment kinetics, limited by L and ton (the time myosin remains bound to actin). We show that this model can account for the velocity of smooth (SMM), skeletal SKM), and cardiac (CMM) myosin filaments.This dissertation also presents a strategy for specifically labeling myosin filaments with quantum dots (QDs) after the filament has already been formed. We use a novel approach for myosin by implementing the SpyTag002 SpyCatcher002 and SnoopTag SnoopCatcher systems to prepare SMM filaments labeled with QDs at the regulatory light chain (RLC) and the filament backbone. We show that filament assembly, actin-activated steady-state ATPase activities, ability to be phosphorylated, and selected enzymatic and mechanical properties were essentially unaffected by the presence of the Spy or Snoop systems. Using these SMM filaments incorporating these systems, this thesis presents groundwork to simultaneously observe the behavior of a single SMM head and filament backbone.