Formin tails act as a switch, inhibiting or enhancing processive actin elongation.

Formins are large, multidomain proteins that nucleate new actin filaments and accelerate elongation through a processive interaction with the barbed ends of filaments. Their actin assembly activity is generally attributed to their eponymous formin homology (FH) 1 and 2 domains; however, evidence is mounting that regions outside of the FH1FH2 stretch also tune actin assembly. Here, we explore the underlying contributions of the tail domain, which spans the sequence between the FH2 domain and the C terminus of formins. Tails vary in length from ∼0 to >200 residues and contain a number of recognizable motifs. The most common and well-studied motif is the ∼15-residue-long diaphanous autoregulatory domain. This domain mediates all or nothing regulation of actin assembly through an intramolecular interaction with the diaphanous inhibitory domain in the N-terminal half of the protein. Multiple reports demonstrate that the tail can enhance both nucleation and processivity. In this study, we provide a high-resolution view of the alternative splicing encompassing the tail in the formin homology domain (Fhod) family of formins during development. While four distinct tails are predicted, we found significant levels of only two of these. We characterized the biochemical effects of the different tails. Surprisingly, the two highly expressed Fhod-tails inhibit processive elongation and diminish nucleation, while a third supports activity. These findings demonstrate a new mechanism of modulating actin assembly by formins and support a model in which splice variants are specialized to build distinct actin structures during development.

Drosophila and human FHOD family formin proteins nucleate actin filaments.

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. Mammalian FHOD proteins were reported not to accelerate actin assembly in vitro; instead, they were proposed to act as barbed end cappers or filament bundlers. Here, we show that purified Drosophila Fhod and human FHOD1 both accelerate actin assembly by nucleation. The nucleation activity of FHOD1 is restricted to cytoplasmic actin, whereas Drosophila Fhod potently nucleates both cytoplasmic and sarcomeric actin isoforms. Drosophila Fhod binds tightly to barbed ends, where it slows elongation in the absence of profilin and allows, but does not accelerate, elongation in the presence of profilin. Fhod antagonizes capping protein but dissociates from barbed ends relatively quickly. Finally, we determined that Fhod binds the sides of and bundles actin 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 than most well studied formins.