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Free keywords:
FRAP; polycomb group protein; PcG proteins
Abstract:
Fluorescence recovery after photobleaching (FRAP) microscopy was used to determine the kinetic properties of Polycomb group proteins in whole living Drosophila organisms (embryos) and tissues (wing imaginal discs and salivary glands). These are the first photobleaching experiments performed in whole embryos and tissues.
Polycomb group (PcG) genes are essential genes in higher eukaryotes responsible for the maintenance of the spatially distinct repression of developmentally important regulators such as the homeotic genes. Their absence, as well as overexpression, causes transformations in the axial organization of the body. Although protein complexes have been isolated in vitro, little is known about their stability or exact mechanism of repression in vivo.
I determined the translational diffusion coefficients of PcG proteins, dissociation constants and residence times for complexes in vivo at different developmental stages. In polytene nuclei the rate constants suggest heterogeneity of the complexes. Computer simulations with new models for spatially distributed protein complexes were performed in systems showing both diffusion and binding equilibria and the results compared with the experimental data. I was able to determine forward and reverse rate constants for complex formation. Complexes exchanged within a period of one to ten minutes, more than an order of magnitude faster than the cell cycle time, ruling out models of repression in which access of transcription activators to the chromatin is limited and demonstrating that longterm repression primarily reflects mass-action chemical equilibria. With the help of computer programs built to simulate diffusion in boundary conditions and binding kinetics of proteins to localized binding sites in the genome I describe in detail the observed biophysical processes underlying FRAP and offer guidance for a better setup, optimization and interpretation of photobleaching experiments.
