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Fig. 3. Rho is required, but the FH2 region is sufficient for processive movement induced by LatB. (A) Cells were electroporated in the presence (right) or absence (left) of C3-exoenzyme and allowed to spread on the PLL-coated glass coverslips for 30 minutes. Phalloidin staining shows the loss of actin stress fibers in C3-treated cells indicating the efficient incorporation of C3-exoenzyme. Scale bar: 20 µm. (B) Cells expressing EGFP-mDia1Full were electroporated in the presence (lower panels) or absence (upper panels) of C3. Then time-lapse images were taken before and 80 seconds after treatment with 100 nM LatB. Arrowheads indicate processively moving mDia1Full speckles. Scale bar: 5 µm. (C) The number of processive mDia1Full speckles in the electroporated cells was counted before and 100 seconds after 100 nM LatB perfusion. The same area within each cell was used for measurement before and after the LatB treatment. C3 inhibited the induction of processive speckles by LatB. (n=5 cells; ^**P<0.01, two-tailed paired t-test.) (D) A low dose of LatB increases the number of processive speckles of the FH2 region mutant (mDia1F2) and the FH1-FH2 region mutant (mDia1N3) in a C3-insensitive manner. Cells expressing EGFP-mDia1F2 or EGFP-mDia1N3 were electroporated in the presence or absence of C3. For mDia1F2, speckles were counted before and 50 seconds after 50 nM LatB perfusion. For EGFP-mDia1N3, speckles were counted before and 80 seconds after 100 nM LatB perfusion. The same area within each cell was used for the measurement. (^*P<0.03 and ^**P<0.005, two-tailed paired t-test.) Since the measured cell areas are different, the number of processive speckles is not comparable between different constructs.

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