Figure 5.

Analysis of mRNP kinetics in stress granules by FRAP. (A and B) Photobleaching of CPEB1 in CPEB1-induced stress granules. HeLa cells were transfected with CPEB1-GFP. After 20 h, cells harboring stress granules were chosen for analysis. A single stress granule was photobleached, and the fluorescence recovery was recorded over 150 s using a confocal microscope. The procedure was repeated a second time on the same granule to ensure reproducibility. In A, images at indicated times were selected for illustration, with the red spark pointing to the photobleached granule and the green arrow to an unbleached control granule within the same cell. The photobleached granule is enlarged below each frame. In B, the fluorescence associated to the bleached (red) and unbleached (green) granules was quantified and plotted as a function of time. Below, the difference between recovered and initial postbleach fluorescence was plotted as a function of time, for each bleach separately, in order to calculate the time required for half-recovery. (C) Photobleaching of CPEB1 in arsenite-induced stress granules. HeLa/CPEB1 cells were treated with arsenite for 30 min, culture medium was replaced, and single stress granules were bleached and analyzed as described in A. (D) Photobleaching of MS2-tagged RNA in arsenite-induced stress granules. HeLa cells were transfected with MS2-tagged β-Gal reporter and MS2-GFP. Cells were treated with arsenite for 30 min, culture medium was replaced, and single stress granules were bleached as described in A. One bleach experiment is illustrated in the top panel. The average fluorescence of 14 bleached stress granules was plotted as a function of time, after correction for total cell bleaching (middle left panel). Time required for half-recovery was calculated as in A (bottom panel). Control experiments in the cytosol were performed using indicated sizes for the bleached spot, to verify that diffusion was negligible (middle right panel).