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Fig. 2. Latrunculin B at low dose induces processive movement of mDia1. (A) Processive movement of EGFP-mDia1Full was induced after 100 nM LatB perfusion. Arrowheads indicate mDia1Full speckles moving processively at indicated time points. Marked speckles moved in one direction for at least five consecutive frames. Scale bar: 5 µm. (B) Number of speckles moving processively after perfusion with 100 nM LatB. Each colored line indicates data from an individual cell. Dotted black line indicates the average speed of processive speckles in five cells. (C) Normalized frequency of processive mDia1 speckles in cells treated with various concentrations of LatB for 100 seconds. Normalized frequency was calculated by dividing the number of speckles by total intensity of EGFP fluorescence in the imaged region. (D) F-actin structures were well preserved 90 seconds after 100 nM LatB treatment except for a gradual loss in the EGFP-actin signal (12%) in the lamellipodial actin meshwork. Scale bar: 5 µm. (E) The density of capping protein speckles (EGFP-CPβ1) did not change before and 80 seconds after 100 nM LatB treatment. Scale bar, 2 µm. (F) Treatment of EGFP-mDia1Full expressing cells with 500 nM SwinA. Arrowheads indicate mDia1Full speckles moving processively. Scale bar: 5 µm. (G) The number of processive mDia1Full speckles before and 11 minutes after 500 nM SwinA perfusion. Data were analyzed using a two-tailed Student's t-test. *P<0.02. (H) Flag-tagged actins, WT (wild-type), R62D or G13R, were coexpressed with EGFP-mDia1Full. Normalized frequency of processive mDia1 speckles in cells expressing actin WT, R62D or G13R is shown. Data were normalized by dividing the number of processive speckles by total intensity of EGFP fluorescence in the imaged region (mean ± s.d., n=5 cells for WT; n=12 cells for G13R; n=16 cells for R62D) and analyzed using a two-tailed Student's t-test. *P<0.02; **P<0.01.
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