Figure 10. Multipoint FDAP analysis... | Rockefeller University Press

Figure 10.

$Figure 10. Multipoint FDAP analysis showing spatial distribution of G-actin concentration in lamellipodium and cell body. (A) Fluorescence images of Dp-actin (left) and mCherry (right) in Rac(V12)-expressing N1E-115 cells. Positions of ROIs (ROI-1 and -2 in the middle and rear of the lamellipodium and ROI-3 to -5 in the cell body) are indicated as white circles. Red circles indicate the areas of photoactivation. Relative fluorescence intensities of Dp-actin and mCherry in each ROI are shown on the bottom. Arrows and asterisks indicate the tip of the lamellipodium and the nucleus, respectively. Bar, 20 µm. (B) Total Dp-actin concentration, calculated from the fluorescence intensities of Dp-actin and mCherry. *, P < 0.05. (C) Multipoint single FDAP analysis. Time courses of Dp-actin fluorescence decay after single photoactivation were measured in each ROI. Fluorescence intensity was normalized to total Dp-actin fluorescence intensity in each ROI and fitted using the diffusion coefficient of Dp-actin (13.7 µm2/s). Data are means ± SEM of 28 cells from at least three independent experiments. (D) Mobile fraction of Dp-actin in each ROI, calculated from the time curve of the mean fluorescence intensity of Dp-actin in C. Error bars are standard errors of mobile fraction. *, P < 0.05. (E) The population of G-actin in each ROI, calculated by the total Dp-actin fluorescence intensity and the mobile fraction. Error bars were calculated by the law of propagation of errors, using the error bars in A and D. *, P < 0.05. (F) The concentration of G-actin in each ROI, calculated by the total Dp-actin concentration and the mobile fraction. Error bars were calculated by the law of propagation of errors, using the error bars in B and D.$

Multipoint FDAP analysis showing spatial distribution of G-actin concentration in lamellipodium and cell body. (A) Fluorescence images of Dp-actin (left) and mCherry (right) in Rac(V12)-expressing N1E-115 cells. Positions of ROIs (ROI-1 and -2 in the middle and rear of the lamellipodium and ROI-3 to -5 in the cell body) are indicated as white circles. Red circles indicate the areas of photoactivation. Relative fluorescence intensities of Dp-actin and mCherry in each ROI are shown on the bottom. Arrows and asterisks indicate the tip of the lamellipodium and the nucleus, respectively. Bar, 20 µm. (B) Total Dp-actin concentration, calculated from the fluorescence intensities of Dp-actin and mCherry. *, P < 0.05. (C) Multipoint single FDAP analysis. Time courses of Dp-actin fluorescence decay after single photoactivation were measured in each ROI. Fluorescence intensity was normalized to total Dp-actin fluorescence intensity in each ROI and fitted using the diffusion coefficient of Dp-actin (13.7 µm²/s). Data are means ± SEM of 28 cells from at least three independent experiments. (D) Mobile fraction of Dp-actin in each ROI, calculated from the time curve of the mean fluorescence intensity of Dp-actin in C. Error bars are standard errors of mobile fraction. *, P < 0.05. (E) The population of G-actin in each ROI, calculated by the total Dp-actin fluorescence intensity and the mobile fraction. Error bars were calculated by the law of propagation of errors, using the error bars in A and D. *, P < 0.05. (F) The concentration of G-actin in each ROI, calculated by the total Dp-actin concentration and the mobile fraction. Error bars were calculated by the law of propagation of errors, using the error bars in B and D.