Fig. 2. FRAP and photoactivation of labeled RNPs. (A) FRAP performed on cells cotransfected with pTet-On and YFP-MS2 and induced for 30 to 60 min with doxycycline. Cells either remained untreated or were ATP depleted for 30 min. Recovery of the bleached signal is shown. (B) Comparison of the calculated diffusion rates (D) and fixed fractions (gamma) for free YFP-NLS, free YFP-MS2, mRNPs at different temperatures, and mRNPs in ATP-depleted cells. (C to E) A cell in which the photoactivatable MS2-GFP (MS2-paGFP) gene was stably integrated (2-3-6-PA) was cotransfected with RFP-lac repressor and pTet-On. Transcription was induced for 30 min by doxycycline. The locus was detected (red) prior to photoactivation (C), and the image in GFP before activation was recorded (D). The 405-nm laser was directed at the boxed region of interest (yellow), and the MS2-paGFP was detected at the transcription site 1.635 s after activation (E). Bar, 2 µm. (F) The RNP signal emanating from the transcription site was followed for 262 s (bar, 2 µm) [Movie S11]. (G) To quantify the movement of the mRNPs, we measured concentric arcs at various distances (r) from the transcription site, as indicated in red, and plotted the normalized (to preactivation value) average intensities per pixel for each arc. The three plots represent three examples of time points (green, 1.635 s; black, 21.255 s; blue, 31.065 s). The distances between the peaks of the signal wave (Δr) were calculated over time (Δt) and the diffusion coefficients determined.