Tropical – software for quantitative analysis of FRAP experiments : identifying the dynamics of linker histone H1°

Abstract

Fluorescence recovery after photobleaching (FRAP) experiments using laser scanning microscopes to follow the in vivo dynamics of proteins tagged to fluorescent markers like the green fluorescent protein (GFP) has become a standard method in cell biology. FRAP perturbs the fluorescence distribution by photobleaching GFPtagged proteins in a specific area of a cell and monitors the fluorescence redistribution. Adequate methods to quantify the results of FRAP experiments have recently became available. Those methods allow the extraction of diffusion coefficients and dissociation constants of proteins diffusing inside distinct cellular compartments and undergoing dynamic binding and dissociation with immobile or mobile binding sites. However, software incorporating such methods was not available until now. Therefore I developed Tropical, a software for simulation and parameter estimation of reaction–diffusion models. Based on spatio-temporal microscopy images, Tropical estimates reaction and diffusion coefficients for user-defined models. Tropical allows the investigation of systems with an inhomogeneous distribution of molecules, making it well suited for quantitative analyses of microscopy experiments such as FRAP. Tropical was used in this thesis to analyze the dynamic behavior of linker histone H1°, which plays a crucial role in the dynamic organization of chromatin by stabilizing the nucleosome, a structure involved in DNA packing in eucaryotic cells. FRAP experiments were performed using three forms of linker histone H1°, the wild type, and two forms with mutated sites, that are likely to play a major role in DNA binding. Diffusion coefficients on the three forms were estimated with Tropical by fitting a pure diffusion model, assuming binding to happen instantaneously. The model showed a very good fit to the experimental data. It could be shown that lysine 52 significantly influences the DNA binding properties of H1° and its mutation resulted in a 3-fold enhanced diffusion coefficient. The H1° form containing six point mutations however showed an even higher diffusion coefficient, about 15 times higher than the one of the wild type histone, revealing a much larger contribution to DNA binding of these six mutated sites. Using Tropical to estimate the diffusion coefficients of linker histone H1° was another proof for the power and functionality of Tropical, besides the recently published one (Ulrich et al. 2006). Tropicals’ main advantages are (1) that it directly operates on microscopy images, (2) an inhomogeneous distribution of binding partners can be considered and (3) the obtained result can directly be verified. This thesis will first line out the current knowledge of eucaryotic chromatin organization, to clarify the role of linker histone H1. I will then give an overview of microscopy techniques available to reveal protein dynamics and their quantitative analysis using mathematical models. The next part will explain Tropical and its implemented methods in detail. Finally I will present the results obtained on the dynamics of H1° and critically discuss the applicability of Tropical to analyze FRAP data and FRAP as a method to reveal protein dynamics.