title: The bacterial virulence factors VopL and VopF nucleate actin from the pointed end.
abstract: VopL and VopF (VopL/F) are tandem WH2-domain actin assembly factors used by infectiousVibriospecies to induce actin assembly in host cells. There is disagreement about the filament assembly mechanism of VopL/F, including whether they associate with the filament barbed or pointed end. Here, we used multicolor total internal reflection fluorescence microscopy to directly observe actin assembly with fluorescently labeled VopL/F. In actin monomer assembly reactions, VopL/F exclusively nucleate actin filament assemblies, remaining only briefly associated with the pointed end. VopL/F do not associate with the ends of preassembled filaments. In assembly reactions with saturating profilin, ∼85% of VopL/F molecules also promote nucleation from the pointed end, whereas a smaller fraction (<15%) associate for ∼25 s with the barbed end of preassembled filaments, inhibiting their elongation. We conclude that VopL/F function primarily as actin nucleation factors that remain briefly (∼100 s) associated with the pointed end.
authors.affiliation: Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL 60637.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Burke
authors.affiliation: Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL 60637.
authors.forename: Alyssa J
authors.initials: AJ
authors.lastname: Harker
authors.affiliation: Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104.
authors.forename: Roberto
authors.initials: R
authors.lastname: Dominguez
authors.affiliation: Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL 60637 drkovar@uchicago.edu.|Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637.
authors.forename: David R
authors.identifier: 0000-0002-5747-0949
authors.initials: DR
authors.lastname: Kovar
mesh_terms: D000199:Actins / Q000737:chemistry* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D058105:Polymerization; D014733:Vibrio / Q000378:metabolism*; D037521:Virulence Factors / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D000199:Actins; D001426:Bacterial Proteins; D037521:Virulence Factors