Measuring protein diffusion in living cells by Raster Image Correlation Spectroscopy (RICS)

Time-lapse fluorescence imaging has revolutionized studies of biology in the last 15 years. In addition to the now routine tracking of bulk fluorescence, for instance of a protein moving into the nucleus in response to an extracellular signal, technologies are now emerging that enable much more sophisticated analysis of the motion and interactions of proteins within living cells. The potential of these approaches to elucidate biological processes is clear, but they have not yet been developed and validated for broad use by biologists. This thesis describes the adaptation of a recently introduced method, Raster Image Correlation Spectroscopy (RICS). RICS is a novel approach to assess the dynamic properties of fluorescent macromolecules in solutions and within living cells by confocal laser scanning microscopy. Based on RICS theory, we developed novel software with which to analyse confocal images and to measure diffusion coefficients of the fluorophores. This new software has several advantages compared with published RICS software, and its ability to give accurate diffusion coefficient values was characterized under a range of settings. Once a RICS routine was established, it was applied to measure the diffusion coefficient of PAK-interacting exchange factor (βPIX) within living fibroblast cells as a paradigm for RICS analysis. The interaction between βPIX and the adaptor protein, Scribble, plays a critical role in cell polarity and actin polymerization. These preliminary measurements indicate the potential of RICS in elucidating the dynamics of proteins within living cells, and demonstrate how the use of RICS will open new opportunities in the cell biology research.