Figure 2. K853A and I704A FH1FH2mDia2 induce stable Glu MTs. (A) Glu and Tyr tubulin immunostaining of serum-starved NIH3T3 cells expressing indicated wild-type (WT) or actin mutant (I704A and K853A) EGFP-FH1FH2mDia2 constructs. (B) Quantiﬁcation of cells injected with indicated constructs that exhibited stable Glu MTs (Glu). Data are mean ± SD from three independent experiments (n > 50 cells). Asterisks indicate P < 0.001, calculated by 2 test (one degree of freedom). (C) Glu and Tyr tubulin immunostaining of serum-starved NIH3T3 cells expressing indicated constructs and then treated with 2 μM nocodazole for 30 min. (D) Quantiﬁcation of cells that exhibited stable Glu MTs after injection of indicated constructs and nocodazole treatment as in C. Data are mean ± SD from three independent experiments (n > 50 cells). Asterisks indicate P < 0.001, calculated by 2 test (one degree of freedom). (E) Western blot analysis of pulldown experiments using GST-EB1, GST-APC-C, or GST alone incubated with the indicated His-FH1FH2mDia2 proteins. Bound proteins were eluted and resolved by SDS-PAGE and mDia2 proteins were detected by mouse RGS-His antibody. Bars, 10 μm. EGFP-tagged cDNAs (unpublished data). The Glu MTs induced by all the mDia2 constructs were resistant to 30-min incubation with 2 μM nocodazole, a treatment that depolymerizes dynamic MTs (Fig. 2, C and D). With the I704A mutant, the induced Glu MTs were frequently ( 50% of cells) concentrated in a peri- nuclear area rather than extended to the cell periphery as observed in cells expressing the wild-type or K853A mutant. The I704A mutant was the most impaired in nucleating actin and in proces- sive movements (Fig. 1, D and E), suggesting that these activities may still be required for producing or maintaining stable Glu MTs in the cell periphery. Alternatively, this defect could be caused by the inability of the I704A mutant to bind to either EB1 or APC, two necessary partners for mDia in stabilizing MTs downstream of Rho signaling. To test whether the mutants still bound to EB1 and APC, in vitro pulldown assays were performed with purified proteins (Fig. 2 E). Both mutants bound to EB1 or APC to a simi- lar extent as with wild-type mDia2, suggesting a role for the retention of these interactions in the induction of stable Glu MTs and that the lack of extended Glu MTs in cells expressing the I704A mutant is presumably caused by another activity. 526 JCB • VOLUME 181 • NUMBER 3 • 2008