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 correlation spectroscopy 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 With the genome sequences of many organisms now sequence, have resulted in optimized expression of Fluorescent protein cloned complete, research is turning to the study of protein GFP in different cell types, as well as the generation of from the jellyfish Aequoria function. Proteins are essential for most biological GFP variants with more favourable spectral proper- victoria. The most frequently processes, but understanding their function is often dif- ties, including increased brightness, relative resistance used mutant, EGFP,is excited at 488 nm and has an emission ficult because proteins inside cells are not merely to the effects of pH variation on fluorescence, and maximum at 510 nm. objects with chemically reactive surfaces. They localize photostability. BOX 1 summarizes properties of GFP to specific environments (that is, membranes, cytosol, and DsRed protein that are relevant for live cell imag- RED FLUORESCENT PROTEIN organelle lumen or nucleoplasm), undergo diffusive or ing studies. Fluorescent protein cloned from the sea anemone directed movement, and often have mechanical parts, Paralleling the developments in GFP biology have Discosoma striata with an the actions of which are coupled to chemical events. been advances in fluorescence imaging methods and excitation maximum of 558 nm The fine tuning of geography, movement and chemistry microscope systems that make it easy to visualize the and emission maximum at gives proteins their extraordinary capability to regulate localization of GFP fusion proteins, to quantitate their 583 nm. virtually all dynamic processes in living cells. abundance and to probe their mobility and interac- The discovery and development of GREEN FLUORES- tions. Imaging methods such as fluorescence recovery CENT PROTEIN (GFP) from the jellyfish Aequorea victoria, after photobleaching (FRAP), fluorescence resonance and more recently RED FLUORESCENT PROTEIN (DsRed) energy transfer (FRET) and fluorescence correlation from the sea anemone Discosoma striata1,have revolu- spectroscopy (FCS) have been modified so that they can tionized our ability to study protein localization, be done on user-friendly, commercially available laser dynamics and interactions in living cells2. In so doing, scanning microscopes, replacing the need for custom- these fluorescent proteins have allowed protein func- built microscopes. Cost-effective computing resources tion to be investigated within the complex environ- have become available to handle large amounts of data, ment of the cell. Virtually any protein can be tagged and powerful software packages are easily obtainable for with GFP,a β-barrel-shaped protein that contains an analysing digital information. The combined advances amino-acid triplet (Ser-Tyr-Gly) that undergoes a in GFP biology, imaging methods and technical equip- chemical rearrangement to form a fluorophore3.The ment are providing a tremendous stimulus for investi- Cell Biology and Metabolism resulting chimaera often retains parent-protein target- gating the kinetic properties of proteins in living cells. In Branch, 18 Library Drive, ing and function when expressed in cells2, and there- this review, we discuss some of these advances, focusing NICHD, NIH Bethesda, Maryland 20892-5430 USA. fore can be used as a fluorescent reporter to study pro- on the fluorescent imaging techniques that are being Correspondence to J.L-S. tein dynamics. Advances in GFP biology, most notably used to analyse protein movement and interactions in e-mail: [email protected] the molecular engineering of the GFP-coding living cells. 444 | JUNE 2001 | VOLUME 2 www.nature.com/reviews/molcellbio © 2001 Macmillan Magazines Ltd REVIEWS fluorescent molecules in a small region of the cell are Box 1 | Fluorescent and chemical properties of GFP and DsRed irreversibly photobleached using a high-powered laser Green fluorescent protein (GFP) beam and subsequent movement of surrounding non- • Fluorophore forms by chemical rearrangement of amino-acid triplet3,7. bleached fluorescent molecules into the photobleached • Various spectral variants available that can be used for fluorescence resonance energy area is recorded at low laser power. GFP fusion proteins transfer (FRET) and dual-colour imaging2. are ideal for use in FRAP studies because they can be 2 • High fluorescence yield . bleached without detectable damage to the cell5. This is 96 • Resistant to photobleaching at low illumination . presumably because the compact barrel-like structure of • Readily and irreversibly photobleached at high illumination2. 4 GFP shields the external environment from the damag- • Can partially reversibly photobleach over very short (millisecond) timescales . ing effects that are caused by reactive intermediates gen- • Fluorescence is relatively insensitive to environment2. erated by photobleaching6,7. • Optical shifting/blinking observed in single molecules84,85. Two kinetic parameters of a protein can be discerned • Photoconverts under anaerobic conditions82. from quantitative studies that use FRAP: the mobile • Complex photophysical states7. fraction, M , which is the fraction of fluorescent proteins Red fluorescent protein (DsRed) f 1 that can diffuse into the bleached region during the time • Red-shifted fluorescence compared with GFP . course of the experiment, and the diffusion constant, D, • Predicted to be good FRET acceptor for EGFP or EYFP47. which is a measure of the rate of protein movement in •Forms tetramers97. the absence of flow or active transport5,8,9. D reflects the • Difficult to photobleach97. • Can take several days to convert from green to red fluorescence97. mean squared displacement that a protein explores through a random walk over time and has units of area per time (usually cm2 s–1 or µm2 s–1). All proteins under- Assessing the kinetic properties of a protein go this type of diffusive movement if they are not Proteins inside cells localize to two fundamentally dif- immobilized or experiencing active transport. Diffusion ferent environments: they are either embedded in, or theory and the characteristics of a protein that underlie peripherally associated with, membranes; or they are its D are discussed in BOX 2. in an aqueous phase, such as the cytoplasm, nucleo- plasm or organelle lumen. Within these environments, a protein can freely diffuse, be immobilized to a scaf- Box 2 | Diffusion theory fold, or be actively transported. These dynamic prop- The diffusion constant for a particle in a free volume is erties have crucial roles in determining what function described by the Stokes–Einstein formula (EQN 1): a protein serves within the cell. GFP fusion proteins are ideal for studying these properties of proteins. Not D = kT (1) πη only does the GFP fluorophore have a high fluores- 6 R cence yield, which makes it bright, but also it is resis- where D is the diffusion constant, T is the absolute 4 tant at low illumination to photobleaching — the temperature, η is the viscosity of the solution, k is the photo-induced alteration of a fluorophore that extin- Boltzmann constant and R is the hydrodynamic radius guishes its fluorescence. These characteristics of the of the particle. Because absolute temperature is usually GFP fluorophore allow GFP chimaeras expressed constant within cells, the most important factors within cells to be imaged with low light intensities over underlying D are the size of a protein (or radius) and the many hours, allowing a protein’s steady-state distribu- viscosity of the medium within which it is diffusing. tion and life history to be studied5. Because with high Membranes have a much higher viscosity than illumination levels the GFP fluorophore can be photo- cytoplasm, so the lateral diffusion of a protein bleached, GFP chimaeras can also be used in photo- assembled within a membrane is considerably slower bleaching experiments to study movement of non- than that of a soluble protein, and this is reflected by a bleached GFP chimaeras into a photobleached area. lower D value. When the viscosity is constant, the D Results from this type of experiment can provide value of a protein is mainly determined by its radius or important insights into a protein’s diffusional proper- size. For a soluble spherical protein, an eightfold ties and its movement between compartments within increase in size will lead to a twofold decrease in D.But cells. Photobleaching can also be used to reduce fluo- this relationship does not hold for transmembrane proteins. Owing to the higher viscosity of membrane, rescence from background noise, so faint populations the radius of the transmembrane segment dominates of fluorescent proteins can be visualized. In the next the D value of a membrane protein, whereas the aqueous sections, we discuss how photobleaching, which tradi- portion usually does not significantly contribute to the tionally has been viewed as something to avoid