Figure 2.

Correlation of the mean actin fluorescence (actin density) and the decrease in lateral membrane diffusion. (A) Confocal fluorescence images of free-standing membranes containing Atto647N-DOPE (left) and Alexa-488-phalloidin-labeled actin filaments (right) exhibiting a dense filamentous meshwork associated with the membrane. Diffusion coefficients D_i were determined by FCS in the center of numbered free-standing membrane spots i. The corresponding actin density I_i was determined from the average fluorescence intensity in a circular area of the respective spot i. Scale bars: 10 μm. (B) Relative change in diffusion coefficients plotted versus the mean actin intensity (measure of actin density) for the labeled lipid (red circles) and the membrane binding protein (blue squares). Each point represents a pair of measurements of D_i and I_i, the solid line is an empirical fit with an asymptotic function. Changes in diffusion are shown after normalization to D₀, the diffusion coefficient of the respective probe in the membrane after neutravidin addition but before actin coupling. The gray dotted line separates regime I (left) and regime II (right). (C and D) Potential effects on the shape of the FCS autocorrelation curves were investigated by classifying the data according to the actin density (I – low density, II – high density). FCS curves were class-averaged (black) and fitted with a model for 2D diffusion assuming single-component membrane diffusion (Eq. S2 or S3 in the Supporting Material, gray). At the timescales relevant for 2D diffusion, the theoretical models were a reasonable fit to the experimental data for the lipid (C) and the protein (D) independent of the actin density.