1. The FRAP mobility of EGFP-UAP56, but not its K95N mutant, is ATP-dependent.
The FRAP mobility of EGFP-UAP56, but not its K95N mutant, is ATP-dependent. HeLa cells were transfected with EGFP-UAP56 wt or EGFP-UAP56 K95N. After 24 hours, a nuclear region of interest (white square) was photobleached for 3 seconds using maximum laser intensity at 488 nm. The fluorescence recovery of EGFP-UAP56 or its K95N mutant in the bleached zone was recorded. In live cells, the fluorescence of both proteins recovered after photobleaching, showing that UAP56 was exchanging on binding sites at speckled domains. After digitonin permeabilization, the FRAP recovery of EGFP-UAP56 stopped. By contrast, after digitonin permeabilization, EGFP-UAP56 K95N recovered after photobleaching, showing that its exchange at speckled-domain binding sites continued. Addition of 1 mM ATP restored FRAP recovery to EGFP-UAP56, showing that the FRAP mobility, that is the exchange at speckled-domain binding sites, is ATP-dependent. (A) A single cell for each experiment is shown before and after the photobleach, and then at one recovery time point. Scale bars: 10 μm. (B) The calculated recovery curves for all cells in each experiment are shown, with the number of cells (n) noted on each graph. Means are plotted with error bars for standard deviations.
Krishna P. Kota et al. J Cell Sci 2008;121:
© The Company of Biologists Limited 2008