DEV02709 Supplementary Material

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• Supplemental Figure 1 -

  Fig. S1. The FH2 domain of FOZI-1 is divergent from other eukaryotic formins. (A) An alignment report from Megalign (DNAStar, Inc.) of the FH2 domain of FOZI-1 with the FH2 domains of the S. cerevisiae formins Bni1p (ScBni1p; Jansen et al., 1996; Kohno et al., 1996) and Bnr1p (ScBnr1p; Imamura et al., 1997); the M. muscularis formin mDia1 (Watanabe et al., 1997); C. elegans CYK-1 (Swan et al., 1998); and C. briggsae (CbFOZI-1) and C. remanei FOZI-1 (CrFOZI-1) homologs (Wormbase). The FH2 domain was defined as the minimal portion of the S. cerevisiae formin Bni1p (residues 1348 to 1750) able to nucleate actin filaments (Moseley et al., 2004), plus nine additional residues that were resolved in the Bni1p FH2-domain crystal structure (Xu et al., 2004). Numbering of residues begins with the first residue of each FH2 domain. Color scheme is as follows: yellow, lasso residues; red, actin-interacting residues; blue, post residues; purple, dual post/actin-interacting residues. Criteria for conserved residues are as previously described (Otomo et al., 2005; Shimada et al., 2004; Xu et al., 2004). Asterisks denote conserved residues in the lasso domain that are found at the hydrophobic pocket of the post domain in Bni1p FH2 dimers (Xu et al., 2004). Arrowheads denote actin-binding residues from Bni1p crystal structure (Xu et al., 2004). (B) The FH2 domain of FOZI-1 does not nucleate actin filaments in vitro. Actin polymerization is represented as a measure of pyrene fluorescence over time. CYK-1^FH1FH2COOH and GST-Bni1p^FH1FH2COOH (used as positive controls) show a robust amount of actin polymerization over 10 minutes. Varying concentrations of FOZI-1 (not shown) and FOZI-1^FH2 are unable to promote actin polymerization to levels significantly higher than buffer alone or GST controls.

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