ABSTRACT
Formins are a conserved group of proteins that nucleate and processively elongate actin filaments. Among them, the formin homology domain-containing protein (FHOD) family of formins contributes to contractility of striated muscle and cell motility in several contexts. However, the mechanisms by which they carry out these functions remain poorly understood. Unlike other formins, mammalian FHOD1 and FHOD3 do not accelerate actin assembly in vitro, and have instead been suggested to act as barbed end cappers or bundlers. Here, we show that purified Drosophila Fhod, in contrast with the mammalian homologues, potently accelerates actin assembly by nucleation. We found that Fhod binds tightly to barbed ends, where it slows elongation in the absence of profilin and allows elongation in the presence of profilin. Fhod protects barbed ends from capping protein, but dissociates from barbed ends relatively quickly. Finally, we used cosedimentation assays to determine that Fhod binds the sides of actin filaments and bundles filaments. This work establishes that Fhod shares the capacity of other formins to nucleate and bundle actin filaments, but is notably less effective at processively elongating barbed ends.
Footnotes
This work was supported by the following grants from the National Institutes of Health: NRSA F30 HL137263 (A.A.P.), UCLA Medical Scientist Training Program T32 GM008042 (A.A.P.), and R01 GM096133 (M.E.Q.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
The abbreviations used are: FHOD, formin homology domain-containing protein; TIRF, total internal reflection fluorescence; FH, formin homology domain; IPTG, isopropyl β-D-thiogalactoside; DTT, dithiothreitol; PMSF, phenylmethanesulfonyl fluoride; EGTA, ethylene glycol tetraacetic acid; GOPTS, (3-glycidyloxypropyl)trimethoxysilane