Biochemical and Microscopic Characterization of INFT-1: an Inverted Formin in C. elegans
Formins are potent regulators of actin dynamics that can remodel the actin cytoskeleton to control cell shape, cell cytokinesis, and cell morphogenesis. The defining feature of formins is the formin homology 2 (FH2) domain (Paul and Pollard, 2008), which promotes actin filament assembly while processively moving along the polymerizing filament barbed end. INFT-1 is one of six formin family members present in Caenorhabditis elegans (Hunt-Newbury et al., 2007) and is most closely related to vertebrate INF2, an inverted formin with regulatory domains in the C- rather than N-terminus. Nematode INFT-1 contains both formin homology 1 (FH1) and formin homology 2 (FH2) domains. However, it does not share the regulatory N-terminal Diaphanous Inhibitory Domain (DID) domain and C-terminal Diaphanous Autoregulatory Domain (DAD) domain found in mammalian INF2. In contrast to mammalian INF2, the sequence of INFT-1 starts immediately at FH1 domain and C-terminal region of INFT-1 shares little homology with INF2, suggesting that elegans INFT-1 is regulated by other mechanisms. We used fluorescence spectroscopy to determine the effect of INFT-1 FH1FH2 on actin assembly and total internal reflection fluorescence microscopy to investigate how INFT-1 formin homology 1 and formin homology 2 domains (FH1FH2) mediate filament nucleation and elongation. INFT-1 FH1FH2 nucleates actin filament and promote actin assembly. However, INFT-1 FH1FH2 reduces filament barbed-end elongation rates in the absence or presence of profilin. Evidences demonstrated that INFT-1 is non-processive, indicating a unique mechanism of nucleation. INFT-1 nucleation efficiency is similar to the efficiency of Arabidopsis FORMIN1 (AFH1), another non-processive formin. High phosphate affected the assembly activity of INFT-1 FH1FH2 in the absence or presence of profilin. INFT is thus the second example of a non-processive formin member and will allow a more detailed understanding of the mechanistic difference between processive and non-processive formins.