In vivo Study of the Histone Chaperone Activity of Nucleolin by FRAP

Figure 6

Schematic of histone exchange after photobleaching a subregion of a cell nucleus. Fluorescence recovery of histone-eGFP after photobleaching requires several molecular events. Timewise, the first step prior to observing histone fluorescence recovery is the disassembly of bleached histones from the chromatin in the FRAP region (step 1). The second step is their replacement by fluorescent histones coming from the pool of free nuclear histones (step 2), which can diffuse freely and rapidly, in the range of few seconds to cross the nucleus. The source of free nuclear histones comes from either “old” previously synthesized histones which have been disassembled from chromatin (“Old” histone pool) or from newly synthesized histones (“New” histone pool) imported in the nucleus. Thus, except in S-phase, when histone synthesis takes place, the pool of unbound histone is mostly nourished by the source of histone disassembly (“Old’’ histone pool). During S-phase, the pool of unbound nuclear histones will also be replenished by import of newly synthesized histones coming from the cytoplasm. After photobleaching, fluorescence recovery is the consequence of histones reassembly with free fluorescent histones coming from the nuclear pool of free histones.