Figure 6

FRAP-experiments on polarized protoplasts. FRAP-experiments were performed on polarized areas of 15 h old protoplasts. (A) (Top) 10 μm x 10 μm image of FM4-64 in an FM4-64 enriched region before, right after, 20 s and 40s after the photobleach. (Bottom) Fluorescence recovery graph of FM4-64 from above protoplast region (data points as black squares) and with the recovery fitting (red line) depicting 100% recovery or complete mobility and a diffusion coefficient of 0.124 μm²/s. (B) (Top) 10 μm x 10 μm image of BD-SM in an FM4-64 depleted region before, right after, 20s and 40s after the photobleach. (Bottom) Fluorescence recovery graph of BD-SM from above protoplast region (data points as black squares) with a recovery fitting (blue line) depicting 48% recovery or a 48% mobile fraction and a diffusion coefficient of 3.4 × 10^-3 μm²/s of the mobile fraction. (C &D) Four typical FRAP-measurements are depicted for each dye with the single data point measurements represented as black circles, squares and triangles; Fit of the average of the four typical curves for the manuscript reported values of the mobile fractionand diffusion coefficients for (C) FM4-64 in FM4-64 enriched regions and (D) BD-SM in FM4-64 depleted regions. (C – red curve &D - blue curve) (see manuscript). (See methods section for details of the FRAP acquisition and analysis.)