title: Motor and tail homology 1 (Th1) domains antagonistically control myosin-1 dynamics.
abstract: Class 1 myosins are monomeric motor proteins that fulfill diverse functions at the membrane/cytoskeletal interface. All myosins-1 contain a motor domain, which binds actin, hydrolyzes ATP, and generates forces, and a TH1 domain, which interacts directly with membrane lipids. In most cases, TH1 is needed for proper subcellular localization and presumably function, although little is known about how this domain regulates the behavior of class 1 myosins in live cells. To address this, we used single molecule total internal reflection fluorescence microscopy to examine the dynamics of the well-characterized myosin-1a isoform during interactions with the cortex of living cells. Our studies revealed that full-length myosin-1a exhibits restricted mobility relative to TH1 alone. Motor domain mutations that disrupt actin binding increased the mobility of full-length myosin-1a, whereas mutations to the TH1 domain that are known to reduce steady-state targeting to the plasma membrane unexpectedly reduced mobility. Deletion of the calmodulin-binding lever arm in Myo1a mimicked the impact of actin-binding mutations. Finally, myosin-1b, which demonstrates exquisite sensitivity to mechanical load, exhibited dynamic behavior nearly identical to myosin-1a. These studies are the first, to our knowledge, to explore class 1 myosin dynamics at the single-molecule level in living cells; our results suggest a model where the motor domain restricts dynamics via a mechanism that requires the lever arm, whereas the TH1 domain allows persistent diffusion in close proximity to the plasma membrane.
authors: Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, Tennessee.
authors: Jessica N
authors: JN
authors: Mazerik
authors: Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee.
authors: Lewis J
authors: LJ
authors: Kraft
authors: Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee.
authors: Anne K
authors: AK
authors: Kenworthy
authors: Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, Tennessee. Electronic address: matthew.tyska@vanderbilt.edu.
authors: Matthew J
authors: MJ
authors: Tyska
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D009154:Mutation; D018995:Myosin Heavy Chains / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024461:Myosin Type I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D013552:Swine
chemical_list: D000199:Actins; D020033:Protein Isoforms; D024461:Myosin Type I; D018995:Myosin Heavy Chains