Figure 5
Nuclear diffusion and binding of recombinant CB(SC). Fluorescence was extinguished by TPM within distinct nuclear regions and the diffusion and binding characteristics of GFP-tagged constructs were determined. A. The approach is described by means of the GFP-tagged histone-construct H2A-EGFP. In the continuous photobleaching experiment a 2 × 2 μ m2 nuclear region was scanned consecutively and the loss of fluorescence caused by the irradiation was monitored simultaneously (see enlarged section). In the FRAP experiment, a region of same dimensions was bleached by continuous irradiation. A time series was grabbed subsequentially from a larger detail of the nucleus (first and last scan are shown). The fluorescence within the bleached region as well as in an untreated control region was measured and normalized according to equation (1). B. Continuous bleaching curves of CB(SC)-EGFP, ECFP-CB(SC)-EYFP as well as of the control proteins EGFP, H2A-EGFP, and TIF1A-EGFP (n = 2 or 3) are plotted as a function of time. The fitting function is composed of two partial terms and matches the values sufficiently enough and more precise than a simple exponential function. The term meets the fact that there are both bound and freely diffusing fluorochrome labelled fractions. The first subterm describes the bleaching of the bound and the second one the bleaching of the diffusible component. While the graphs for EGFP and TIF1A-EGFP support free diffusion, the H2A-EGFP-population exists mostly in a bound state. Both CB(SC) constructs show an intermediate behaviour which points to bound as well as mobile fractions. C. FRAP curves of the same set of fusion proteins corroborate the findings above.