M. ormö et al., Science 273:1392–1395, 1996. With permission from aaas.)
Figure 9–24 GFP-tagged proteins. this living cell from a tobacco plant is expressing high levels of green fluorescent protein, fused to a protein that is targeted to mitochondria, which accordingly appear green. the mitochondria are seen to cluster around the chloroplasts, whose chlorophyll autofluorescence marks them out in red. (Courtesy of olivier grandjean.) transfer, both abbreviated FRET. In this technique, two molecules of interest are each labeled with a different fluorochrome, chosen so that the emission spectrum of one fluorochrome, the donor, overlaps with the absorption spectrum of the other, the acceptor. If the two proteins bind so as to bring their fluorochromes into very close proximity (closer than about 5 nm), one fluorochrome, when excited, can transfer energy from the absorbed light directly (by resonance, nonradiatively) to the other. Thus, when the complex is illuminated at the excitation wavelength of the first fluorochrome, fluorescent light is produced at the emission wavelength of the second. This method can be used with two different spectral variants of GFP as fluorochromes to monitor processes such as the interaction of signaling molecules with their receptors, or proteins in macromolecular complexes at specific locations inside living cells (Figure 9–26). The FRET can be measured by quantifying the reduction of the donor fluorescence in the presence of the acceptor.
A second example of a fluorescencetagging technique that allows detailed observations of proteins within cells involves synthesizing an inactive form of the fluorescent molecule of interest, introducing it into the cell, and then activating it suddenly at a chosen site in the cell by focusing a spot of light on it. This process is referred to as photoactivation. Many inactive photosensitive precursors of this type, often called caged molecules, have been made based on a variety of fluorescent molecules. A microscope can be used to focus a strong pulse of light from a laser on any tiny region of the cell, so that the experimenter can control exactly where and when the fluorescent molecule is photoactivated. The technique allows us to follow complex and rapid intracellular processes, such as the actions of signaling molecules or the movements of cytoskeletal proteins.
When a photoactivatable fluorescent tag is attached to a purified protein, it is important that the modified protein remain biologically active: labeling with a caged fluorescent dye adds a bulky group to the surface of a protein, which can easily change the protein’s properties. A satisfactory labeling protocol is usually found by trial and error. Once a biologically active labeled protein has been produced, it needs to be introduced into the living cell where its behavior can be followed. Tubulin labeled with caged fluorescein, for example, can be injected into a dividing cell, where it is incorporated into microtubules of the mitotic spindle. When a small region of the spindle is illuminated with a laser, the labeled tubulin
Figure 9–25 Dynamics of GFP tagging. this sequence of micrographs shows a set of three-dimensional images of a living nucleus taken over the course of 135 minutes. tobacco cells have been stably transformed with gFp fused to a spliceosomal protein that is concentrated in small nuclear bodies called Cajal bodies (see Figure 6–46). the fluorescent Cajal bodies, easily visible in a living cell with confocal microscopy, are dynamic structures that move around within the nucleus. (Courtesy of kurt Boudonck, Liam Dolan, and peter shaw.) becomes fluorescent, so that its movement along the spindle microtubules can be readily followed (Figure 9–27).