Key Points
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In the past 10 years, there has been great progress in the development of fluorescent proteins (FPs) to study protein movement and protein interactions. Green FP (GFP)-like proteins have been mutated to be monomers as useful tags for analysing protein movement.
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FPs have been used to study bulk protein movement, primarily through photobleaching or photoactivation techniques, which allow the determination of protein diffusion and protein binding kinetics.
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FPs have also been used to track single proteins within the cell. The technique may vary depending on properties of the protein; for example, whether it is soluble.
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By studying movement, insights have been obtained on protein–protein interactions. Fluorescence cross-correlation spectroscopy (FCCS) has been used to detect the synchronous movement of two proteins fused to different FPs. Bimolecular fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) have both been used to study protein proximity, an indicator of protein interactions.
Abstract
Proteins are always on the move, and this may occur through diffusion or active transport. The realization that the regulation of signal transduction is highly dynamic in space and time has stimulated intense interest in the movement of proteins. Over the past decade, numerous new technologies using fluorescent proteins have been developed, allowing us to observe the spatiotemporal dynamics of proteins in living cells. These technologies have greatly advanced our understanding of protein dynamics, including protein movement and protein interactions.
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Acknowledgements
I thank S. Shimozono and R. Ando for the images in figures 2d and 2h; H. Sakurai, V. Lakshmanam and T. Fukano for general assistance; and S. Trowbridge, D. Mou, V. Verkhusha and S. Manley for valuable comments.
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Supplementary information S2 (figure) | Molecular mechanisms for the irreversible photoactivation of PA-GFP and photoconversion of Kaede. (PDF 317 kb)
Glossary
- Site-directed mutagenesis
-
An in vitro mutagenesis procedure that is often carried out using a polymerase chain reaction in which specific mutations are introduced into a DNA molecule.
- Hydrodynamic radius
-
The effective size of the molecule as detected by its diffusion.
- Fractal model
-
A model in which diffusing molecules and complexes encounter the same obstructions regardless of their size.
- Dendritic spines
-
Small membranous protrusions from dendritic shafts that usually receive excitatory input.
- Pyramidal neurons
-
The predominant type of neuron in the neocortex. They are named after their triangular cell bodies.
- Barrel cortex
-
The dark-staining regions of layer 4 of the somatosensory cortex, where somatosensory inputs from the contralateral side of the body come in from the thalamus.
- Axial dimension
-
The dimension along the optical axis of an objective lens.
- Highly inclined and laminated optical sheet
-
The illumination light for single-molecule imaging inside cells. The light is generated by positioning the incident beam to propagate near the objective edge.
- Multiplexing
-
A method that allows the simultaneous imaging of multiple events in a single cell.
- Diffraction limit
-
Although an ideal optical system would image an object point perfectly as a point, diffraction occurs owing to the wave-like nature of radiation, and the result is that the image of a point is a blur. Thus, the ability of an imaging system to resolve detail is ultimately limited by diffraction.
- Stochastic optical reconstruction microscopy
-
A high-resolution fluorescence microscopy method based on high-accuracy localization of photoswitchable fluorophores.
- Pair correlation function
-
A function that shows the probability of finding the centre of a particle that is located a given distance from the centre of another particle.
- Euchromatin
-
A form of chromatin that is lightly packed and often transcriptionally active during interphase.
- Heterochromatin
-
A condensed form of chromatin in which the degree of compaction is similar to that of mitotic chromosomes. It is usually found around the centromere.
- Stokes shifts
-
The energy difference between the emitted photon and the absorbed photon. The Stokes shift is usually expressed as the difference between positions of the band maxima of the absorption and emission spectra.
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Miyawaki, A. Proteins on the move: insights gained from fluorescent protein technologies. Nat Rev Mol Cell Biol 12, 656–668 (2011). https://doi.org/10.1038/nrm3199
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DOI: https://doi.org/10.1038/nrm3199
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