Figure 6: Characterization of actin nodule dynamics by live cell TIRF imaging.

(a) Frames taken from time-lapse TIRF movies of three representative Lifeact-GFP mouse platelets spreading on fibrinogen (See Supplementary Movie 1) showing the dynamic nature of actin nodule formation and turnover. Arrows indicate turnover of three individual actin nodules. Time stamp=min:s. Scale bar, 5 μm. (b) Quantification of the mean number of actin nodules per platelet over time (solid line). Dashed and dotted lines represent±s.d. (c) Frequency histogram of the lifetime of individual actin nodules. (d) Mean surface area of individual actin nodules normalized for lifetime. Bars represent the mean surface area of the nodule at initial appearance (0% of lifetime), disappearance (100% of lifetime) and at 25, 50 and 75% of their lifetime. Error bars indicate the s.d. For (b–d) data are means of 20 platelets from two independent experiments. (e) Representative mean fluorescence intensity plot of a single actin nodule imaged over time in both TIRF (dashed line) and epifluorescence (solid line). Intensity increases are larger in TIRF than in epifluorescence indicating movement of the nodule in the z axis towards the coverslip. This Z movement has been calculated in nm and is shown for this nodule in (f). Appearance of the nodule at T₀ is set at 0 nm and movement towards the coverslip is indicated by negative nm values. (g) Frequency distribution of movement in Z (in nm) of 118 nodules from three independent experiments.