From single molecules to single cells : mechanistic studies of myosin VI and cardiac myosin - Chuan, Peiying; Spudich, James A; Dunn, Alexander Robert; Harbury, Pehr; Herschlag, Daniel; Stanford University, Department of Biochemistry (2011)
Myosins are a superfamily of molecular motors that couple the chemical energy derived form adenosine triphosphate (ATP) hydrolysis to precise mechanical movements along the filamentous cytoskeletal protein actin. There exist more than 20 distinct classes of myosins within this superfamily, and their specific biophysical and biochemical properties likely allow them to function accordingly in vivo. My research has focused on understanding the how the structural and kinetic characteristics of a particular myosin, myosin VI, relate to its proposed trafficking and tethering functions. In the first section of this dissertation, I perform single molecule studies on the contribution of an unexpected structural domain in the myosin VI tail to the efficiency of processive stepping, especially against external forces. I additionally use optical trapping to examine the load-dependent kinetics of the motor in the second section, and propose that external load in vivo likely regulates myosin VI's dual functions as cellular transporter and cytoskeletal anchor. The specific cellular cargoes as well as oligomerization states of different myosins are defined by their tail domain, which is the most divergent structural region among myosin classes. Myosin VI, shown to be monomeric when purified in its native form, likely dimerizes upon cargo binding. As most in vitro work on myosin VI so far have been performed on a truncated, artificially forced myosin VI dimer, I discuss in the third section steps taken toward achieving a more native, biochemically reconstituted full-length myosin VI molecule bound to its bona fide cargo. Having gained a better understanding of the biophysics, kinetics and biochemistry of myosin at the single molecule level, I moved on to studying myosin function at the cellular level. In the forth section of this dissertation, I examine the effects of a familial hypertrophic cardiomyopathy (FHC) mutation, R403Q in cardiac myosin, on the contractile function of single mouse cardiomyocytes. I find that the functional effects of the R403Q mutation in the diseased FHC heart are manifest at the level of cardiomyocytes as well, suggesting that these effects are cell autonomous. From these studies, we can better appreciate the structural and kinetic properties of myosin VI that make it uniquely suited for its functions. This knowledge is also beneficial in aiding the understanding of other molecular motors that may employ similar mechanisms. We are now also aware of the cell-autonomy of particular dysfunctions exhibited by diseased R403Q FHC hearts, and are better poised to understand the complex disorder as a whole.
Submitted to the Department of Biochemistry.
Thesis (Ph.D.)--Stanford University, 2011.
Rights: (c) Copyright 2011 by Peiying Chuan
Rights: This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
Keywords: theses