title: Molecular mechanism of Ena/VASP-mediated actin-filament elongation.
abstract: Ena/VASP proteins are implicated in a variety of fundamental cellular processes including axon guidance and cell migration. In vitro, they enhance elongation of actin filaments, but at rates differing in nearly an order of magnitude according to species, raising questions about the molecular determinants of rate control. Chimeras from fast and slow elongating VASP proteins were generated and their ability to promote actin polymerization and to bind G-actin was assessed. By in vitro TIRF microscopy as well as thermodynamic and kinetic analyses, we show that the velocity of VASP-mediated filament elongation depends on G-actin recruitment by the WASP homology 2 motif. Comparison of the experimentally observed elongation rates with a quantitative mathematical model moreover revealed that Ena/VASP-mediated filament elongation displays a saturation dependence on the actin monomer concentration, implying that Ena/VASP proteins, independent of species, are fully saturated with actin in vivo and generally act as potent filament elongators. Moreover, our data showed that spontaneous addition of monomers does not occur during processive VASP-mediated filament elongation on surfaces, suggesting that most filament formation in cells is actively controlled.
authors.affiliation: Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany.
authors.forename: Dennis
authors.initials: D
authors.lastname: Breitsprecher
authors.forename: Antje K
authors.initials: AK
authors.lastname: Kiesewetter
authors.forename: Joern
authors.initials: J
authors.lastname: Linkner
authors.forename: Marlene
authors.initials: M
authors.lastname: Vinzenz
authors.forename: Theresia E B
authors.initials: TE
authors.lastname: Stradal
authors.forename: John Victor
authors.initials: JV
authors.lastname: Small
authors.forename: Ute
authors.initials: U
authors.lastname: Curth
authors.forename: Richard B
authors.initials: RB
authors.lastname: Dickinson
authors.forename: Jan
authors.initials: J
authors.lastname: Faix
mesh_terms: D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D002465:Cell Movement / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics; D058105:Polymerization; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D059008:Time-Lapse Imaging
chemical_list: D000199:Actins; D004268:DNA-Binding Proteins; C092744:ENA-VASP proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins