title: Assembly and turnover of short actin filaments by the formin INF2 and profilin.
abstract: INF2 (inverted formin 2) is a formin protein with unique biochemical effects on actin. In addition to the common formin ability to accelerate actin nucleation and elongation, INF2 can also sever filaments and accelerate their depolymerization. Although we understand key attributes of INF2-mediated severing, we do not understand the mechanism by which INF2 accelerates depolymerization subsequent to severing. Here, we show that INF2 can create short filaments (<60 nm) that continuously turn over actin subunits through a combination of barbed end elongation, severing, and WH2 motif-mediated depolymerization. This pseudo-steady state condition occurs whether starting from actin filaments or monomers. The rate-limiting step of the cycle is nucleotide exchange of ADP for ATP on actin monomers after release from the INF2/actin complex. Profilin addition has two effects: 1) to accelerate filament turnover 6-fold by accelerating nucleotide exchange and 2) to shift the equilibrium toward polymerization, resulting in longer filaments. In sum, our findings show that the combination of multiple interactions of INF2 with actin can work in concert to increase the ATP turnover rate of actin. Depending on the ratio of INF2:actin, this increased flux can result in rapid filament depolymerization or maintenance of short filaments. We also show that high concentrations of cytochalasin D accelerate ATP turnover by actin but through a different mechanism from that of INF2.
authors.affiliation: From the Department of Biochemistry, Geisel School of Medicine and.
authors.forename: Pinar S
authors.initials: PS
authors.lastname: Gurel
authors.affiliation: From the Department of Biochemistry, Geisel School of Medicine and.
authors.forename: Mu
authors.initials: M
authors.lastname: A
authors.affiliation: the Department of Chemistry, Dartmouth College, Hanover, New Hampshire 03755.
authors.forename: Bingqian
authors.initials: B
authors.lastname: Guo
authors.affiliation: From the Department of Biochemistry, Geisel School of Medicine and.
authors.forename: Rui
authors.initials: R
authors.lastname: Shu
authors.affiliation: the Department of Chemistry, Dartmouth College, Hanover, New Hampshire 03755.
authors.forename: Dale F
authors.initials: DF
authors.lastname: Mierke
authors.affiliation: From the Department of Biochemistry, Geisel School of Medicine and henry.higgs@dartmouth.edu.
authors.forename: Henry N
authors.initials: HN
authors.lastname: Higgs
mesh_terms: D008841:Actin Cytoskeleton / Q000737:chemistry* / Q000235:genetics; D020816:Amino Acid Motifs; D000080865:Formins; D006801:Humans; D008840:Microfilament Proteins / Q000737:chemistry* / Q000235:genetics; D051304:Profilins / Q000737:chemistry* / Q000235:genetics; D017510:Protein Folding
chemical_list: D000080865:Formins; C546959:INF2 protein, human; D008840:Microfilament Proteins; D051304:Profilins
keywords: ATP; actin; fluorescence; formin; profilin