title: Myosin VI altered at threonine 406 stabilizes actin filaments in vivo.
abstract: Myosin VI is a minus-end directed actin-based molecular motor implicated in uncoated endocytic vesicle transport. Recent kinetic studies have shown that myosin VI displays altered ADP release kinetics under different load conditions allowing myosin VI to serve alternately as a transporter or as an actin tether. We theorized that one potential regulatory event to modulate between these kinetic choices is phosphorylation at a conserved site, threonine 406 (T406) in the myosin VI motor domain. Alterations mimicking the phosphorylated (T406E) and dephosphorylated state (T406A) were introduced into a GFP-myosin VI fusion (GFP-M6). Live cell imaging revealed that GFP-M6(T406E) expression changed the path myosin VI took in its transport of uncoated endocytic vesicles. Rather than routing vesicles inwards as seen in GFP-M6 and GFP-M6(T406A) expressing cells, GFP-M6(T406E) moved vesicles into clusters at distinct peripheral sites. GFP-M6(T406E) expression also increased the density of the actin cytoskeleton. Filaments were enriched at the vesicle cluster sites. This was not due to a gross redistribution of the actin polymerization machinery. Instead the filament density correlated to the fixed positioning of GFP-M6(T406E)-associated vesicles on F-actin, leading to inhibition of actin depolymerization. Our study suggests that phosphorylation at T406 changes the nature of myosin VI's interaction with actin in vivo.
authors.affiliation: Section of Cell and Developmental Biology, Division of Biological Sciences, University of California at San Diego, La Jolla, California 92093, USA. snaccache@gmail.com
authors.forename: Samia N
authors.initials: SN
authors.lastname: Naccache
authors.forename: Tama
authors.initials: T
authors.lastname: Hasson
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D018938:Caco-2 Cells / Q000378:metabolism; D002478:Cells, Cultured; D015638:Cytochalasin D / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D018995:Myosin Heavy Chains / Q000502:physiology*; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010766:Phosphorylation; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism; D013552:Swine; D013912:Threonine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D022161:Transport Vesicles / Q000378:metabolism
chemical_list: D019384:Nucleic Acid Synthesis Inhibitors; C089785:myosin VI; D049452:Green Fluorescent Proteins; D015638:Cytochalasin D; D013912:Threonine; D018995:Myosin Heavy Chains