Figure 4. FH1FH2mDia2 binds directly to MTs. (A) SDS gel analysis of the cosedimentation of His- (His-mDia2), GST- (GST-mDia2), or untagged (mDia2) FH1FH2mDia2 or GST alone (GST) with taxol-assembled MTs or BSA. Matching input and pellet fractions were resuspended in SDS loading buffer and run on 10% SDS-PAGE followed by Coomassie staining. tub, tubulin. (B) Concentration dependence of FH1FH2mDia2 binding to taxol stabilized MTs (1.5 μM tubulin). Error bars show SEM of three independent experiments. (C) SDS gel analysis of MT cosedimentation assay, as described in A, with His-tagged versions of wild-type (WT) and mutant (K853A, I704A, and W630A) FH1FH2mDia2. (D) Concentration dependence of wild-type and mutant His-FH1FH2mDia2 bind- ing to MTs. Input material (I) and MT (or BSA) pellets material are shown. (E) MT cosedimentation analyses of wild-type and mutant His-FH1FH2mDia2 with intact and sheared MTs. (F) Immunostaining of His-FH1FH2mDia2 on immobilized MTs as described in Materials and methods. Bar, 10 μm. protein showed that it eluted as a major peak at a volume sug- gesting that it behaves as a monomer rather than a dimer (Fig. 3 B). We conclude that the W630A mutation generates a protein that is impaired in forming a stable dimer. Because dimerization is a necessary requirement for pro- cessive actin filament association, we tested the ability of W630A FH1FH2mDia2 to promote F-actin assembly in vitro and in vivo. Pyrene-labeled actin polymerization assays showed that the W630A mutant was >250 times less active than the wild type in nucleating actin (Fig. 3 C). Actin staining of serum-starved cells microinjected with W630A EGFP-FH1FH2mDia2 showed no induction of actin fibers as was observed with the wild type (Fig. 3 D and Fig. 1 C). Moreover, virtually no directional movement of bright GFP particles was visualized in cells expressing this mutant mDia2 and no accumulation in filopodia protrusions was observed (Video 4, available at http://www.jcb.org/cgi/content/ full/jcb.200709029/DC1). We conclude that the W630A mutant lost its ability to polymerize actin in vitro and in vivo. When we tested the same construct for induction of stable Glu MTs, we observed that cells expressing W630A EGFP- FH1FH2mDia2 displayed more stable MTs than uninjected neigh- boring cells, although at a frequency 20% lower than that of cells expressing the wild-type protein (Fig. 3, E and F). Most of the Glu MTs ( 90%) induced by the W630A mutant displayed the “knotted” phenotype that was observed with the most severe actin binding mutant. The stability of these Glu MTs was con- firmed by testing their resistance to nocodazole treatment (Fig. 3 G). These data show that a stable dimer is not necessary to generate stable Glu MTs by mDia. Combined with the data from the actin- deficient mutants, these results reinforce the conclusion that promotion of actin polymerization and induction of MT stability by mDia2 can be functionally segregated. 528 JCB • VOLUME 181 • NUMBER 3 • 2008