FIG. 4.

Simultaneous FLIP-FRAP of telomere-bound GFP-TRF1 and GFP-TRF2. (A) FLIP-FRAP on living HeLa cells expressing GFP-TRF1. Cells are photobleached over a region covering about one-half of the nucleus (indicated by a white box). The images were acquired before bleaching and at 20-s intervals after bleaching, starting at 2 s. The pink circles in the bleached area and blue circles in the unbleached area indicate the regions that are used to calculate fluorescence redistribution. Scale bar, 5 μm. (B and C) Quantitative analysis of redistribution of GFP-TRF1 (B) and GFP-TRF2 (C) at telomeres separately in bleached (pink) and unbleached (blue) half of the nucleus. Values are means ± the SEM from at least 40 cells. (D) Difference (Δ) in telomere intensity in bleached and unbleached part of cell, calculated from the data shown in panels B (TRF1) and C (TRF2). (E) A fitting analysis of the experimental data in panel D to the equation ΔI_rel(t) = f₁e^−k₁t + f₂e^−k₂t indicated a good fit with the single binding kinetics of GFP-TRF1 (green line). In contrast, GFP-TRF2 redistribution does not fit with single binding kinetics (blue dashed line) but does fit with dual binding kinetics (red lines). Note the similarity between the fitted curves of the fast fraction of TRF2 and of TRF1. For percentages of fractions and association constants, see Table 1.