Fluorescent Correlation Spectroscopy

REVIEWS

STUDYING PROTEIN DYNAMICS IN LIVING CELLS
Jennifer Lippincott-Schwartz, Erik Snapp and Anne Kenworthy
Since the advent of the green fluorescent protein, the subcellular localization, mobility, transport routes and binding interactions of proteins can be studied in living cells. Live cell imaging, in combination with photobleaching, energy transfer or fluorescence correlationspectroscopy are providing unprecedented insights into the movement of proteins and their interactions with cellular components. Remarkably, these powerful techniques are accessible to non-specialists using commercially available microscope systems.

GREEN FLUORESCENT PROTEIN

Fluorescent protein cloned from the jellyfish Aequoria victoria. The most frequently used mutant, EGFP, is excited at 488 nmand has an emission maximum at 510 nm.
RED FLUORESCENT PROTEIN

Fluorescent protein cloned from the sea anemone Discosoma striata with an excitation maximum of 558 nm and emission maximum at 583 nm.

Cell Biology and Metabolism Branch, 18 Library Drive, NICHD, NIH Bethesda, Maryland 20892-5430 USA. Correspondence to J.L-S. e-mail: jlippin@helix.nih.gov

With the genome sequences of manyorganisms now complete, research is turning to the study of protein function. Proteins are essential for most biological processes, but understanding their function is often difficult because proteins inside cells are not merely objects with chemically reactive surfaces. They localize to specific environments (that is, membranes, cytosol, organelle lumen or nucleoplasm), undergo diffusive or directedmovement, and often have mechanical parts, the actions of which are coupled to chemical events. The fine tuning of geography, movement and chemistry gives proteins their extraordinary capability to regulate virtually all dynamic processes in living cells. The discovery and development of GREEN FLUORESCENT PROTEIN (GFP) from the jellyfish Aequorea victoria, and more recently RED FLUORESCENT PROTEIN(DsRed) from the sea anemone Discosoma striata1, have revolutionized our ability to study protein localization, dynamics and interactions in living cells2. In so doing, these fluorescent proteins have allowed protein function to be investigated within the complex environment of the cell. Virtually any protein can be tagged with GFP, a β-barrel-shaped protein that contains an amino-acid triplet(Ser-Tyr-Gly) that undergoes a chemical rearrangement to form a fluorophore3. The resulting chimaera often retains parent-protein targeting and function when expressed in cells2, and therefore can be used as a fluorescent reporter to study protein dynamics. Advances in GFP biology, most notably the molecular engineering of the GFP-coding

sequence, have resulted in optimized expression of GFP indifferent cell types, as well as the generation of GFP variants with more favourable spectral properties, including increased brightness, relative resistance to the effects of pH variation on fluorescence, and photostability. BOX 1 summarizes properties of GFP and DsRed protein that are relevant for live cell imaging studies. Paralleling the developments in GFP biology have been advances influorescence imaging methods and microscope systems that make it easy to visualize the localization of GFP fusion proteins, to quantitate their abundance and to probe their mobility and interactions. Imaging methods such as fluorescence recovery after photobleaching (FRAP), fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) have been modified so that they can bedone on user-friendly, commercially available laser scanning microscopes, replacing the need for custombuilt microscopes. Cost-effective computing resources have become available to handle large amounts of data, and powerful software packages are easily obtainable for analysing digital information. The combined advances in GFP biology, imaging methods and technical equipment are providing a...