An in silico approach to analyzing nuclear FRAP protein recovery curves using an extension of the Gillespie algorithm

摘要

Proper recruitment of repair proteins to the foci of DNA double-stranded breaks represent one instance of a fundamental reaction diffusion process in which the geometry and topology within an environment are critically important. Here present a novel computational method to analyze protein diffusion. Our method is based on a previously published extension of the Gillespie algorithm that includes diffusion in 3-D volumes. In particular, our method helps analyze FRAP (fluorescence recovery after photo-bleaching) recovery curves. Simulations are run in idealized nuclei with different geometries (i.e., spheres and cubes). Each nucleus is subdivided into cubic elements (about 64 000 in total) used to model diffusion. The program has 2 adjustable parameters: the initial average concentration of proteins, and the diffusion coefficient. As an illustration, we simulated the bleaching of regions of varying dimensions. Protein molecules of negligible volume were uniformly distributed throughout the simulatednucleus. Proteins had 2 states: fluorescent and bleached. At time 0, only fluorescent molecules were present. At the time of exposure, proteins in the selected region changed states (from, fluorescent to bleached) and the recovery of fluorescence owing to diffusion in the bleached region was measured. Our results are in agreement with previous studies and show that varying the initial nuclear protein concentration, the diffusion coefficients, or the geometry of bleached regions changes the FRAP recovery curve. Our study represents an initial step towards better understanding the impact of geometric elements, e.g., chro-matin fibers, on proteins as they diffuse into the bleached regions. This research is partially supported by the SCORE National Institutes of Health program grant No. 2S06GM52588-12 and the San Francisco State University Center for Computing in the Life Sciences.