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.
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.
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We thank members of the Lippincott-Schwartz laboratory for helpful comments on the manuscript. We also thank Gregoire Bonnet for useful discussions on fluorescence correlation spectroscopy. Anne Kenworthy was supported by a National Research Council–NICHD Research Associateship. Erik Snapp was supported by a PRAT Fellowship.
ENCYCLOPEDIA OF LIFE SCIENCES
- GREEN FLUORESCENT PROTEIN
Fluorescent protein cloned from the jellyfish Aequoria victoria. The most frequently used mutant, EGFP, is excited at 488 nm and 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.
- ULTRAVIOLET-LIGHT-INDUCED DNA DAMAGE
Ultraviolet light promotes a covalent linkage of two adjacent pyrimidine bases (most often two thymines) in DNA.
- NUCLEAR ENVELOPE
Double membrane that surrounds the nucleus. The outer nuclear membrane is continuous with the endoplasmic reticulum. The outer nuclear membrane is connected to the inner nuclear membrane at nuclear pores.
- NUCLEAR LAMINA
Electron-dense layer lying on the nucleoplasmic side of the inner membrane of a nucleus.
An antibiotic that inhibits the glycosylation of asparagine residues yielding carbohydrate-poor glycoproteins.
Small GTPase that regulates the assembly of coats and vesicle budding.
One of seven subunits of the COPI coatomer complex.
Component of the COPII coat complex.
Large multisubunit protease complex that selectively degrades intracellular proteins. Targeting to proteasomes most often occurs through attachment of multi-ubiquitin tags.
- CYAN FLUORESCENT PROTEIN
S65A, Y66W, S72A, N1461I, M153T, V163A mutant of green fluorescent protein with excitation peak of 434 nm and an emission maximum at 477 nm.
- YELLOW FLUORESCENT PROTEIN
S65G, V68L, S72A, T203Y mutant of green fluorescent protein with an excitation peak of 514 nm and an emission maximum at 527 nm.
- QUANTUM YIELD
The probability of luminescence occurring in given conditions, expressed by the ratio of the number of photons (the quanta of light) emitted by the luminescing species to the number absorbed.
Fluorescent dye with an excitation maximum of 492 nm and an emission maximum of 520 nm.
Fluorescent dye with an excitation maximum at 550 nm and an emission maximum at 590 nm.
Fluorescent cynanine dye with an excitation maximum at 550 nm and an emission maximum at 570 nm.
Fluorescent cynanine dye with an excitation maximum at 650 nm and an emission maximum at 670 nm.
- REPORTER CONSTRUCTS
Artificial proteins engineered to act as intracellular sensors. Often consist of a pair of GFP mutants that act as a FRET pair linked by a peptide that undergoes conformational changes or is physically altered in response to the intracellular environment or enzyme activity.
Fluorescent cynanine dye with an excitation maximum at 580 nm and an emission maximum at 590 nm.
- AUTOCORRELATION FUNCTION
Mathematical function that is used to extract statistical properties of time-dependent noise. Used to analyse time-dependent fluctuations of fluorescence intensity in an FCS experiment to find similarities within the signal — for example, a correlation time reflecting diffusion of a fluorescent protein through a sample volume.
- TWO-PHOTON MICROSCOPY
A form of multiphoton microscopy.
A membrane-permeable fluorophore (fluorescein arsenical helix binder) that specifically, non-covalently, and reversibly binds a recombinant protein motif containing four cysteines at the i, i+1, i+4, and i+5 positions.
- SINGLE-CHAIN ANTIBODIES
Peptides derived from immunoglobulins (which usually consist of two heavy chains and two light chains). These peptides do not oligomerize and have specific affinity for an antigen.
- TOTAL INTERNAL REFLECTION MICROSCOPY
Fluorescence microscopy technique with significant depth discrimination, that can selectively excite only those fluorescent molecules within 100 nm of the interface between a cell and a coverslip.
- MULTIPHOTON MICROSCOPY
Microscopy technique that uses the simultaneous absorbance of two or more photons of low energy (long wavelength) to excite fluorophores normally excited with single photons of shorter wavelengths. The technique reduces photodamage and permits imaging of much thicker samples.
- IMAGE CORRELATION SPECTROSCOPY
Technique that measures the density and degree of aggregation of fluorescent particles using autocorrelation analysis of images from laser scanning confocal microscopy. Can be used, for example, to measure quantitatively the state of aggregation of receptors on the cell surface.
- ATOMIC FORCE MICROSCOPY
A microscope that nondestructively measures the forces (at the atomic level) between a sharp probing tip (which is attached to a cantilever spring) and a sample surface. The microscope images structures at the resolution of individual atoms.
- 4-PI MICROSCOPE
A microscope that combines the wavefronts produced by two opposed high-aperture lenses and a two-photon excitation laser to allow three-dimensional imaging of transparent biological specimens with an axial resolution in the 100–140-nm range.
- STIMULATED EMISSION MICROSCOPE
(Also referred to as ultrafast-dynamics microscope). A light microscope that increases the spatial resolution of a fluorescent sample by exciting the fluorophore with a femtosecond laser pulse followed by a quenching time-delayed red-shifted femtosecond laser pulse that depletes fluorescence at the focal rim surrounding the focal volume.
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Lippincott-Schwartz, J., Snapp, E. & Kenworthy, A. Studying protein dynamics in living cells. Nat Rev Mol Cell Biol 2, 444–456 (2001). https://doi.org/10.1038/35073068
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