Figure 3. The dimerization mutant W630A... | Rockefeller University Press

Figure 3.

$Figure 3. The dimerization mutant W630A FH1FH2mDia2 induces stable Glu MTs. (A) Elution profiles of the indicated His-FH1FH2mDia2 proteins from analytical Superdex 200 gel filtration. An aliquot of each fraction was analyzed by Western blotting using an anti-His antibody. The arrows indicate elution volumes of standards run under the same conditions. (B) Diagram of W630A His-FH1FH2mDia predicted to be a dimerization mutant (red star indicates the position of the mutation) and comparison of elution profiles of W630A and WT His-FH1FH2mDia2 proteins from analytical Superdex gel filtration analyzed by Western blotting as in A. (C) Pyrene-actin assembly assays using His-tagged wild type (WT) and increasing concentrations of W630A FH1FH2mDia2 (W630A). Fluorescence is expressed in arbitrary units (A.U.) and all reactions were performed under identical conditions. (D) Actin immunostaining of serum-starved NIH3T3 cells expressing microinjected EGFP-tagged W630A FH1FH2mDia2. (E) Glu and Tyr tubulin immunostaining of serum-starved NIH3T3 cells expressing microinjected EGFP-tagged wild-type or W630A FH1FH2mDia2. (F) Quantification of cells that exhibited stable Glu MTs after expression of indicated EGFP-tagged constructs. Data are mean ± SD from three independent experiments (n > 70 cells). Asterisk indicates P < 0.001, calculated by χ2 test (one degree of freedom). (G) Glu tubulin immunostaining of nocodazole-resistant MTs in cells expressing EGFP-tagged W630A FH1FH2mDia2. Bars, 10 μm.$

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 indicated EGFP-tagged constructs. Data are mean ± SD from three independent experiments (n > 70 cells). Asterisk indicates P < 0.001, calculated by χ² test (one degree of freedom). (G) Glu tubulin immunostaining of nocodazole-resistant MTs in cells expressing EGFP-tagged W630A FH1FH2mDia2. Bars, 10 μm.