Fluorescence recovery after photo-bleaching experiments

FRAP experiments were performed at 37 °C and 5% CO₂ using a 1.4 NA × 100 oil-immersion objective on a confocal fluorescence microscope (Zeiss 780, Carl Zeiss AG, Oberkochen, Germany). The fluorescent protein citrine was excited at 488 nm and fluorescence emission was registered at around 525 nm. In the FRAP experiments, a small rectangular ROI (∼5 × 10 μm²) centred on the actin patterns was imaged and a smaller rectangular bleaching ROI (∼2 × 4 μm²) was chosen in its centre. This choice of imaging and bleaching modes minimized the fluorescence loss owing to photo-bleaching during the recovery as well as phototoxic effects on the cell by not exposing the whole cell to light but restricting illumination and thus photo-bleaching to a section of only 0.6 μm in thickness as outlined in ref. ⁵⁴. Fluorescence recovery was monitored over a lnROI parallel to the F-actin strands for actin stress fibres, stars and asters in order to detect the direction of fluorescence regrowth. Bleaching was performed by scanning the 488 nm beam operating at 100% power of the 20 mW laser. In our protocol, bleaching was realized with a single laser pulse of 2 s during which scanning was performed with a pixel dwell time of 8 μs. The recovery of fluorescence was monitored at the same scanning speed with 1–5% of the laser power over 100 frames at 0.05–1-s intervals to minimize photo-bleaching but still allow sampling at a sufficient enough speed⁵⁴. For each recovery, two time-lapse image streams were recorded prior to the initial bleaching, which realized normalization of the fluorescence signal. To assess the loss in fluorescence during observation of the recovery (owing to photo-bleaching), we selected the simultaneously recorded fluorescence signal from a non-bleached region. In all cases, the rate of fluorescence loss due to the observation of recovery was significantly smaller than the rates of fluorescence recovery, with a characteristic time of ∼1,000 s that was one order of magnitude larger than the slowest recovery timescale observed for actin. Hence, imaging-induced fluorescence loss did not significantly affect the measurements. Conventional kymographs for the FRAP experiments were generated by computing a montage of the lnROIs using the standard ImageJ plugin Montage (ImageJ, http://imagej.nih.gov/). Iso-Kymographs were computed in custom-written MATLAB routines (MATLAB Inc.) by using the standard iso-contour functions. The F-actin turnover velocities v_growth were computed by fitting the linear recovery trends in time in the iso-Kymographs using a total pixel size of 29 nm, frame rate of 1 s per frame and a finite actin monomer size of 2.7 nm. FRAP experiments are presented in Fig. 5h–j. For each experimental condition, we acquired FRAP recovery curves and computed the F-actin turnover velocity from at least 20 individual cells over the course of at least 3 independent experiments.