Fig. 2.

Quantitative dynamics of Ago2 at PBs and SGs. (a) Time-lapse micrographs of stably expressed EGFP-Ago2 in a single live cell (complete movie is available in Movie 1). The first appearance of EGFP-Ago2 at SGs (arrowheads) occurred between 5 and 6 min after addition of 1 μM hippuristanol (HIPP). (b) Fluorescence recovery after photobleaching (FRAP) analyses of EGFP-Ago2 at single PBs (orange; n = 5) and SGs (purple; n = 3) and the intensities at respective structures relative to their initial intensities were compared over time. (c and d) PAGFP-Dcp1a and PAGFP-Ago2 were photoactivated at single PB labeled by mRFP-Dcp1a for 1 s, and the photoactivated cells were imaged over the next 13 min. (c) The intensities of PAGFP-Dcp1a (orange; n = 3) and PAGFP-Ago2 (blue; n = 3) at the photoactivated PBs were compared over time with their respective initial intensities after photoactivation. (d) Representative images of PAGFP-Ago2 (Upper) and PAGFP-Dcp1a (Lower) at t = 00:00 and 12:54. The intensity of PAGFP-Dcp1a decreased over time at the photoactivated PBs (arrows), whereas the one of PAGFP-Ago2 remained relatively constant over time. Also, it was observed that the intensities of PAGFP-Dcp1a gradually increased over time at the neighboring PBs (broken arrows; see also Fig. 7e), suggesting continuous exchange of Dcp1a between PBs and the cytoplasm. (e) Addition of emetine reduced the accumulation of EGFP-Ago2 at SGs labeled by TIA1 (red) even in the presence of continual oxidative stress (250 μM arsenite for 90 min). In this experiment, 10 μg/ml emetine (Right) or DMSO (Left) were added 30 min after the arsenite treatment and left for 60 min further before fixing the cell for immunolabeling with SG marker TIA1 and PB marker Dcp1a (data not shown). (Scale bars, 5 μm.)