Fig. 1.

Filamin A (FlnA) interacts and overlaps with formin 2 (Fmn2) in developing mouse brain and neural cells. (A) FlnA directly interacts with Fmn2, as assessed by yeast two-hybrid assay. Filter assay demonstrates activation of lacZ reporter gene following binding of VP16-Fmn2 to LexA FLNA/B. DNA sequencing of the prey bound to FLNA/B identified the Fmn2 FH1 domain (amino acids 788-870) as the FLNA/B-interacting region. ABD, actin-binding domain; RBD, receptor-binding domain. (B) Co-immunoprecipitation shows that full-length Fmn2-GFP precipitates FLNA/B C-terminal fragments when a Fmn2-GFP construct is overexpressed with FLNA-C and FLNB-C constructs in HEK 293 cells. SUP, input supernatant; IgG, sample precipitated by normal IgG; GFP, sample precipitated by anti-GFP. (C) Endogenous FLNA is co-immunoprecipitated by full-length Fmn2-GFP using anti-GFP antibody in HEK 293 cells. (D) Endogenous FlnA is strongly co-immunoprecipitated from neonatal WT mouse lysates by anti-Fmn2 antibody. (E) Brightfield light microscopy of coronal E16 mouse brains shows overlapping mRNA expression of Flna, Flnb and Fmn2 by in situ hybridization. Flna and Fmn2 show strong and nearly identical expression patterns along the ventricular zone and within the cortical plate. (F) Fluorescent photomicrographs along the lateral ventricles of E16 Fmn2-GFP transgenic mouse embryos demonstrate that FlnA and Fmn2 share overlapping expression along the ventricular zone at the protein level. (G) Cultured neural progenitor cells expressing Fmn2-GFP, showing that Fmn2 overlaps with FlnA along the cytoplasmic membrane (arrow). Scale bars: 500 μm in E; 50 μm in F; 10 μm in G.