Actin-based motility demands the spatial and temporal coordination of numerous regulatory actin-binding proteins (ABPs) , many of which bind with affinities that depend on the nucleotide state of actin filament. Cofilin, one of three ABPs that precisely choreograph actin assembly and organization into comet tails that drive motility in vitro , binds and stochastically severs aged ADP actin filament segments of de novo growing actin filaments . Deficiencies in methodologies to track in real time the nucleotide state of actin filaments, as well as cofilin severing, limit the molecular understanding of coupling between actin filament chemical and mechanical states and severing. We engineered a fluorescently labeled cofilin that retains actin filament binding and severing activities. Because cofilin binding depends strongly on the actin-bound nucleotide, direct visualization of fluorescent cofilin binding serves as a marker of the actin filament nucleotide state during assembly. Bound cofilin allosterically accelerates Pi release from unoccupied filament subunits, which shortens the filament ATP/ADP-Pi cap length by nearly an order of magnitude. Real-time visualization of filament severing indicates that fragmentation scales with and occurs preferentially at boundaries between bare and cofilin-decorated filament segments, thereby controlling the overall filament length, depending on cofilin binding density.
Bibliographical noteFunding Information:
This work was supported by grants from Agence Nationale de la Recherche to J.-L.M. and L.B. (ANR-08-BLANC-0022 and ANR-08-SYSC-013), the American Heart Association (0940075N awarded to E.M.D.L.C.), the National Institutes of Health (GM071688 and GM071688-03S1 awarded to E.M.D.L.C.), and the Institute of Complex Systems IXXI, Rhône-Alpes (awarded to J.-L.M.). E.M.D.L.C. is an American Heart Association Established Investigator, an NSF-CAREER Award recipient (MCB-0546353), and a Hellman Family Fellow. We thank Pekka Lappalainen and Bruce Goode for the ADF/cofilin D34C, C62A construct.