FRAP of GFP-H3 fusion proteins.

(A) Digital fluorescent micrographs of the nucleus of a cell expressing GFP-H3.3 before and after photobleaching. Vero cells were transfected with plasmids expressing GFP-H3.3. A region passing across the long nuclear axis was photobleached, and the fluorescence recovery in the photobleached region was evaluated. The photobleached region was selected independently of the presence or absence of replication compartments, and thus includes nuclear domains containing cellular and viral DNA. Fluorescence in the photobleached region recovers as the photobleached GFP-histones within this region exchange with the non-photobleached GFP-histones from outside of this region. FRAP was evaluated for only 100 s; therefore, potential contributions from newly synthesized GFP-histones to fluorescence recovery are negligible. The enlargements in the lower panel highlight the photobleached region. (B) Line graph of a representative GFP-H3.3 FRAP. The fluorescence intensity of the photobleached region at a given time is normalized to the fluorescence of the entire nucleus at that same time, expressed as a ratio of the normalized fluorescence prior to photobleaching, and plotted against time. The fluorescence intensity is therefore independent of GFP-H3 expression levels. The first data point after photobleaching is a surrogate measure for the levels of free GFP-H3. The subsequent fluorescence recovery is biphasic. The initial faster phase represents those histones that are weakly bound in chromatin and undergoing faster chromatin exchange. As a surrogate measure for this population, we calculated the initial rate of normalized fluorescence recovery (the slope between the normalized fluorescence at the first and second data points after photobleaching; shown in the inset). The second slower phase of fluorescence recovery represents those histones that are more stably bound in chromatin and undergoing slower chromatin exchange.