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.