Figure 3. The dimerization mutant W630A FH1FH2mDia2 induces stable Glu MTs. (A) Elution proﬁles of the indicated His-FH1FH2mDia2 proteins from analytical Superdex 200 gel ﬁltration. An aliquot of each fraction was analyzed by Western blotting using an anti-His antibody. The arrows indicate elution volumes of standards run under the same conditions. (B) Diagram of W630A His-FH1FH2mDia predicted to be a dimerization mutant (red star indicates the position of the mutation) and comparison of elution proﬁles of W630A and WT His-FH1FH2mDia2 proteins from analytical Superdex gel ﬁltration analyzed by Western blotting as in A. (C) Pyrene-actin assembly assays using His-tagged wild type (WT) and increasing concentrations of W630A FH1FH2mDia2 (W630A). Fluorescence is expressed in arbitrary units (A.U.) and all reactions were performed under identical conditions. (D) Actin immunostaining of serum- starved NIH3T3 cells expressing microinjected EGFP-tagged W630A FH1FH2mDia2. (E) Glu and Tyr tubulin immunostaining of serum-starved NIH3T3 cells expressing microinjected EGFP-tagged wild-type or W630A FH1FH2mDia2. (F) Quantiﬁcation of cells that exhibited stable Glu MTs after expression of indi- cated EGFP-tagged constructs. Data are mean ± SD from three independent experiments (n > 70 cells). Asterisk indicates P < 0.001, calculated by 2 test (one degree of freedom). (G) Glu tubulin immunostaining of nocodazole-resistant MTs in cells expressing EGFP-tagged W630A FH1FH2mDia2. Bars, 10 μm. W630A FH1FH2 mDia2 dimerization mutant induces stable Glu MTs A dimer appears to be the functional state of formin FH2 do- mains and is required to promote actin nucleation and elonga- tion (Moseley et al., 2004; Shimada et al., 2004; Xu et al., 2004). We questioned whether a functional dimer is also necessary for the stabilization of the MT cytoskeleton and analyzed the di- merization properties of purified wild-type and mutant His- FH1FH2mDia2 proteins by analytical gel filtration (Fig. 3 A). The elution profile for all of these proteins was identical with a single peak at a volume consistent with the elution properties of analogous dimeric fragments of both mDia1 and mDia3 (Li and Higgs, 2003; Shimada et al., 2004). We next tested whether a dimerization mutant, based on the crystal structure of the Bni1p FH2 domain, was capable of inducing stable Glu MTs in cells. We substituted Trp 630 with an Ala, as this residue localizes at the dimerization interface of Bni1 (Xu et al., 2004). Trp 630 is also among the most conserved amino acids in the FH2 domain (Moseley et al., 2004; Xu et al., 2004). Gel filtration analyses of the W630A FH1FH2mDia2 MDIA2 STABILIZES MICROTUBULES • Bartolini et al. 527