MAAP of PA-GFP–actin fluorescence decay curves allows for accurate, simultaneous calculation of the G-actin:F-actin ratio and F-actin disassembly rates. (A) Simulated data for PA-GFP–actin showing the effect that changing the local G-actin:F-actin (G:F) ratio has on the fluorescence decay curves. All data was simulated with an F-actin depolymerization rate (Depol. rate) of 0.06/s. (B) Simulated data for PA-GFP–actin showing the effect that changing the depolymerization rate has on the fluorescence decay curves. All data was simulated with a G:F ratio of 1:1. (C) A data set of 105 PA-GFP–actin fluorescence decay curves and their mean (red). (D) Examples of individual curves (blue) from E matched with the simulated data with the combination of G:F ratio and depolymerization rate that gave the best fit (red). (E) Representative photoactivation images from live cells that were pre-treated with DMSO or 100 nM Jasplakinolide for 15 min. (F) Mean decay curves for DMSO (n=40) and Jasplakinolide-treated (n=20) data sets. Error bars are 95% confidence intervals. (G) Box-and-whisker plot showing the distribution of calculated depolymerization rates from individual experiments. (H) Box-and-whisker plot showing the distribution of calculated G:F ratios from individual experiments. Box-and-whisker plots denote the 95th (top whisker), 75th (top edge of box), 25th (bottom edge of box) and 5th (bottom whisker) percentiles, and the median (bold line in box). P-values are from a two-tailed Student's t-test.

Accuracy of MAAP fitting of experimental data. Line graphs showing the nine closest MAAP fits to a single experiment. The fit in the middle, outlined in red, is the one chosen by MAAP because it had the lowest root-mean-square deviation (RMSD).

The significance of cellular geometry in analyzing photoactivation data. (A) A round cell geometry similar to the one used for MAAP of CAD cells (shown in Fig. 1) except that it is 7 μm tall and has a 15 μm radius. (B) A line graph showing the results of simulations from a diffusion-only version of MAAP in two different round cell geometries (shown in Fig. 1 and Fig. 3A) using two different diffusion rates (annotated D). (C) An amoeboid cell geometry with several long, narrow projections extending from the cell body. The region that was photoactivated in the simulations is shown in red. (D) A line graph showing the results of simulations from a diffusion-only version of MAAP using either the round cell geometry (round) or the geometry containing long thin projections (projection) (shown in Fig. 1 and Fig. 3C) using three different diffusion rates (annotated D). (E) Simulated actin photoactivation in the rounded cell geometry. (F) Simulated actin photoactivation within the geometry of a long narrow projection. A close up of the region outlined in red in the middle panel is shown in the images on the left. For E and F, the concentration of photoactivated actin (PA-actin) is color coded according the scale bars underneath the image. (G) Results of simulations from round and projection geometries (shown in Fig. 1 and Fig. 3C) using the parameters listed above the graph. G-actin:F-actin ratio, G:F ratio; depolymerization rate, Depol. rate.

MAAP analyses of β- and γ-actin dynamics. (A) Representative photoactivation images from cells expressing either PA-GFP–β-actin or PA-GFP–γ-actin. (B) Mean fluorescence decay curves for PA-GFP–β-actin (n=92) and PA-GFP–γ-actin (n=105). Error bars are 95% confidence intervals. (C) Box-and-whisker plots showing the distribution of calculated depolymerization rates for PA-GFP–β-actin and PA-GFP–γ-actin from individual experiments. (D) Box-and-whisker plots showing the distribution of the calculated G-actin:F-actin (G:F) ratios of PA-GFP–β-actin and PA-GFP–γ-actin from individual experiments. (E) Scatter plot showing calculated depolymerization rates and G:F ratios from individual experiments. Linear fits of the data and Pearson product-moment correlation coefficients are shown in the same color as the corresponding data set. (F) Representative photoactivation of PA-GFP–β-actin in cells expressing a control scrambled shRNA (Control) or an shRNA knocking down cofilin 1 (Cof1 or CFN1 KD). (G) Box-and-whisker plots showing the distribution of G:F actin ratios and F-actin depolymerization rates of individual decay curves from control (n=92) or Cof1 KD (n=50) cells expressing PA-GFP–β-actin. Cof1 KD cells exhibited a substantial decrease in both the rate of F-actin depolymerization and the G:F ratio. (H) Box-and-whisker plots showing the distribution of G:F actin ratios and F-actin depolymerization rates of individual decay curves from control (n=105) or Cof1 KD (n=50) cells expressing PA-GFP–γ-actin. Cof1 KD cells exhibited a substantial decrease in the rate of F-actin depolymerization but not in the G:F ratio. (I) Scatter plot showing calculated depolymerization rates and G:F ratios of PA-GFP–β-actin and PA-GFP–γ-actin in Cof1 KD cells from individual experiments. Linear fits of the data and Pearson product-moment correlation coefficients are shown in the same color as the corresponding data set. In the absence of cofilin 1, the depolymerization rate and G:F ratio became positively correlated for PA-GFP–β-actin but not PA-GFP–γ-actin. Box-and-whisker plots denote the 95th (top whisker), 75th (top edge of box), 25th (bottom edge of box) and 5th (bottom whisker) percentiles, and the median (bold line in box). P-values are from a two-tailed Student's t-test.