Studying protein dynamics in living cells

The cloning of green fluorescent protein (GFP), engineering of chimeric fusion proteins, and advances in fluorescence imaging methods have made it possible for researchers to follow the dynamics and interactions of proteins in living cells. In this review, we describe the application of biophysical microscopy-based techniques in combination with GFP-chimaeras to characterize protein dynamics in living cells. The photobleaching technique FRAP (fluorescence recovery after photobleaching)) has been used since the mid-1970s to determine the diffusion constant of fluorescent antibody-labelled proteins on the plasma membrane of cells. Photobleaching of GFP-chimeric proteins localized throughout the cell has been recently exploited to determine the viscosities of different cellular environments and to reveal the diffusional mobilities of various proteins. Variations of FRAP, including selective photobleaching and FLIP (fluorescence loss in photobleaching), can reveal the continuities and discontinuities of intracellular organelles and compartments. In addition, FLIP has been used to characterize the kinetics of protein binding and release in living cells. FRET (fluorescence resonance energy transfer) is a property of certain pairs of fluorophores, in which a high energy fluorophore can excite a lower energy fluorophore when the two fluorophores are in extremely close proximity. FRET microscopy has been used to determine whether proteins that co-localize are physically interacting in fixed and living cells. We describe several recent applications and variations of FRET in the review. The technique of FCS (fluorescence correlation spectroscopy) has recently become accessible to cell biologists with commercially available microscopes. FCS can be used to measure diffusion constants for multiple populations and ratios of bound and free proteins, allowing for sensitive measurements of protein–protein interactions in cells. Ongoing development of GFP variants, unusual properties of GFP, alternatives to GFP in living cells, and new microscopy techniques hold much promise for future studies of protein dynamics in living cells.