Figure 1. K853A and I704A FH1FH2mDia2 point mutants are defective in actin polymerization. (A) K853A and I704A point mutations (top, red stars) were introduced into a constitutively active fragment of mDia2 that includes the FH1 and 2 domains but lacks the GBD and DAD domains. HIS-tagged versions of the mutant (K853A and I704A) and wild-type (WT) proteins were expressed in Escherichia coli, afﬁnity puriﬁed, and run on 10% SDS-PAGE followed by Coomassie staining. M, protein markers. (B) Pyrene-labeled monomeric actin was assembled in the presence of the indicated concentrations of wild-type and mutant His-FH1FH2mDia2. The panels below the graph show the rates of actin assembly at 50% polymerization in the presence of different concentrations of wild-type and mutant mDia2. Fluorescence is expressed in arbitrary units (A.U.), and all reactions were performed under identical conditions. (C) Rhodamine- conjugated phalloidin staining of serum-starved NIH3T3 cells microinjected with 8 μM of wild-type, I704A, or K853A His-FH1FH2mDia2 and human IgG as a marker. (D) Quantiﬁcation of the percentage of cells injected with the indicated constructs of His-FH1FH2mDia2 that exhibit actin ﬁbers (n > 40 cells). (E) TIRF microscopy of EGFP-FH1FH2mDia2 puncta in NIH3T3 cells. Shown is a single frame from movies taken of cells expressing wild-type or actin mutant EGFP- FH1FH2mDia2. Each color line indicates the track of a single EGFP punctum that could be followed for more than three frames. A kymograph of a selected region (white line) from the movie taken with a wild-type–expressing cell is shown below. Bars: (C) 10 μm; (E) 5μm. wild-type EGFP-FH1FH2mDia2 puncta was 0.31 ± 0.16 μm/s, an order of magnitude faster than the flow rate of the actin network yet slower than the velocity estimated for GFP-FH1FH2mDia1 puncta (Higashida et al., 2004). Conversely, the K853A mutant generally traveled over shorter distances with very limited lo- calization to filopodia tips, whereas the I704A mutant exhibited no clear directional movement with no accumulation at the cell periphery (Videos 2 and 3). We conclude that both mDia2 mutants do not associate processively with the barbed ends of growing actin filaments in vivo. I704A and K853A FH1FH2mDia2 mutants induce stable Glu MTs and bind to APC and EB1 To test whether the actin-deficient FH1FH2mDia2 mutants were able to induce stable Glu MTs, we microinjected them into serum- starved NIH3T3 cells, which contain low levels of stable Glu MTs. Both EGFP-tagged mutants generated stable MTs, although the I704A mutant was reduced 20% compared with the wild type (Fig. 2, A and B). Similar results were observed if His- or GST-tagged proteins were microinjected instead of expressing MDIA2 STABILIZES MICROTUBULES • Bartolini et al. 525