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Interactions of Smooth Muscle Myosin Filaments with Actin and Myosin Light Chain Kinase: Insights into the Mechanics of Smooth Muscle Contraction
AuthorHaldeman, Brian D.
AdvisorCremo, Christine R.
Biochemistry and Molecular Biology
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The functional form of smooth muscle myosin (SMM) within cells is the filament. Smooth muscle contraction is thick (myosin) filament-regulated by phosphorylation of the myosin regulatory light chain (RLC). Upon phosphorylation of Serine 19 of the RLC of SMM by myosin light chain kinase (MLCK), SMM filaments and actin (thin) filaments slide over one another and the cell contracts. The effect of the filamentous conformation of SMM on the kinetics underlying the interactions with MLCK and actin is unknown. The filamentous structure of unphosphorylated smooth muscle myosin is not stable under physiological conditions, so there is a lack of research studying filaments of smooth muscle myosin. Our lab is interested in studying the kinetics and the interactions between smooth muscle myosin filaments with actin filaments and MLCK to further understand the mechanics behind smooth muscle contraction. In this dissertation we present a preparation of smooth muscle myosin filaments that have been stabilized to ATP-induced depolymerization. This preparation has allowed us to develop a more physiological in vitro model system for studying the interactions of SMM filaments with actin filaments and MLCK.The classical approach for studying acto-myosin interactions is using the in vitro motility assay. Previous research investigating the kinetics underlying the interaction of soluble sub fragments of smooth muscle myosin with actin filaments showed that this is a detachment-limited model, with ADP-release from myosin being the rate limiting step. In this dissertation we present a novel in vitro motility assay to investigate interactions of SMM filaments with actin filaments and show that the underlying kinetics are fundamentally different than the classic in vitro motility assay. We propose that the kinetics observed in this in vitro model system are more representative of unloaded muscle shortening seen in the cell.Solution kinetic studies have also been used to study the interactions of SMM with actin and nucleotide to gain a further understanding of the mechanics of muscle contraction. The vast majority of these experiments have been done using soluble subfragments of SMM because the filaments are not stable in the presence of ATP and the subfragments remain soluble at low ionic strength. Here we present a kinetic characterization of stabilized SMM filaments and compare the kinetics to soluble subfragments under the same conditions. This dissertation also presents groundwork in developing an assay to watch real-time activation of SMM filaments by MLCK using single molecule fluorescence microscopy. Our lab has previously investigated the kinetics and the mechanics of MLCK interactions with monomeric SMM (Hong et al (2013) Biochemistry 52, 8489-8500) as well as with purified F-actin and skinned smooth muscle cells (Hong et al (2015) manuscript in preparation). Applying the stabilized SMM filaments, the novel in vitro model system described previously, and the development of an expression system for MLCK we can gain insights into the mechanics of how MLCK is able to activate smooth muscle contraction.