Fig. S1. Recombinant FRG-1 and FRG1 proteins bundle F-actin. (A) Bacterially generated recombinant human FRG1 (Hs) and C. elegans FRG-1 (Ce) used in F-actin binding and bundling assays. (B) Low-speed actin-bundling cosedimentation assay showing FRG-1-dependent F-actin bundling. FRG-1 was treated as a dimer with a molecular mass of 60 kDa. The bundling of F-actin by FRG-1 dimers was saturated at a molar ration of 7:1 (actin:FRG-1 dimer).

Fig. S2. Characterization of the FRG-1 antibody. Transgenic animals expressing FRG-1::GFP preferentially localize FRG-1 in the nucleus. P_frg-1::frg-1::gfp transgenic adults were (A,D) immunostained for FRG-1 and (B,E) visualized for FRG-1::GFP fluorescence. Transgenic and non-transgenic embryos within the uterus showing (A) the endogenous FRG-1 and (B) the FRG-1::GFP transgene product signals (C) overlap in the transgenic animal and appear nuclear localized. (D-F) Immunostaining of adult P_frg-1::frg-1::gfp animals for FRG-1 detects both nuclear and cytoplasmic FRG-1 in body-wall muscle. Scale bar: 50 m. (G) Western blot analysis of whole C. elegans protein extracts from P_frg-1::frg-1::GFP worms probed with antibodies against GFP (left) or αFRG-1 (right).

Fig. S3. FRG-1 is the only fascin-like domain protein in C. elegans. A search of the C. elegans database revealed that FRG-1 is the only fascin domain protein known or predicted. A Pfam database search for human proteins identified four fascin domains in both Fascin1 and Fascin2, in addition to one in FRG1. Phylogeny performed with Clustal W. Rooted Phylogenetic Tree from alignment shows that the C. elegans FRG-1 fascin-like domain is more closely related to its counterpart in human FRG1 than human Fascin1 and Fascin2. Each one of the fascin domains in Fascin1 is more closely related to its counterpart in Fascin2.

Table S1. PCR primers.