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Techniques developed for single-molecule spectroscopy and imaging are now helping unravel the mysteries surrounding cellular processes. Thanks to sensitive fluorescence microscopy and spectroscopy and a vast spectrum of fluorescent labels to tag proteins, scientists can now study how cells ingest large molecules or particles and move them around within the cell.
There's no one "best" tag. VFPs have the advantage of being genetically encoded and expressed by the cell itself, so there are no delivery issues. However, VFPs can lose their fluorescence over time through photobleaching. Plus, the potential overlap of their relatively wide emission bands can limit the number that can be used simultaneously.
In contrast, quantum dots are conjugated to the proteins through a high-affinity interaction such as that between biotin and streptavidin. The main advantage of quantum dots for cellular imaging is their photostability, which translates into a long fluorescence lifetime even at high illumination intensities. Another advantage of quantum dots is their narrow emission bandwidth, which allows the use of multiple quantum dots to label a variety of species.
"The single-molecule spectroscopy field has branched out much more broadly than people thought a few years ago," said Shuming Nie, a professor of biomedical engineering, chemistry, hematology, and oncology at Emory University and Georgia Institute of Technology. "We now see that some of these highly sensitive imaging technologies for single molecules can be used to study processes inside living cells."
Nie was one of the organizers of a weeklong symposium on single-molecule spectroscopy and imaging sponsored by the Division of Physical Chemistry at the American Chemical Society national meeting in Philadelphia in August. Although symposia on single-molecule spectroscopy have been held at previous ACS meetings, this symposium was the first to incorporate sessions on live-cell imaging.
Thomas M. Jovin, a molecular biologist at Max Planck Institute for Biophysical Chemistry, Göttingen, Germany, was one of the speakers. The Jovin lab and collaborators at the University of Buenos Aires use a variety of fluorescent probes, including VFPs and quantum dots, to study endocytosis--the process by which cells take up large molecules--and what happens to the resulting endosomes--the organelles that carry the ingested material--in the cells.
IN PARTICULAR, the Jovin lab has focused on endocytosis mediated by a family of receptor tyrosine kinases (RTKs) that respond to epidermal growth factor and related ligands. These receptors and their ligands participate in cellular signaling. Various fluorescent probes are used simultaneously to label different components mediating the interaction between the cell surface receptor (the RTK) and the growth factor. The RTK is expressed as a fusion protein with a VFP, and the growth factor is labeled with quantum dots. Currently, the VFP is located at the end of the receptor, which is inside the cell, because that's the easiest and most convenient thing to do.
"What's eluded us so far has been the outside labeling," Jovin said. "That would be nice because then we could follow the interactions at the first level, where the actual binding takes place and the receptors presumably change their structure in a dramatic way. That has not been measured or demonstrated on living cells as yet. We're very anxious to show that."
In the future, they would even like to place multiple labels on different parts of the receptor protein so that they can track what happens to them during endocytosis. "Each label would be specific," Jovin said. "By looking at all of them at the same time, you could say quite a bit about structural changes, association, and so forth."
In labeling the growth factor with quantum dots, they expected nothing more than a way to look at the binding of the growth factor to the cell surface. "We would have been satisfied with that," Jovin said.
Instead, they got more than they hoped for: They anticipated that the size of the quantum dots might interfere with endocytosis, but the process appears to proceed normally. "There's a lot of biochemistry involved with the endocytosis mechanism, but the quantum dot doesn't seem to affect it in any significant way that we've been able to establish so far," Jovin said.
Using these different probes, Jovin and coworkers have been able to follow endosomes in the cell as they move to their destination. They have found that extensive fusion of multiple endosomes into a larger structure occurs earlier in the process than originally thought. However, the researchers have been unable to follow the late stages of the process to demonstrate where the molecules in the endosome ultimately end up, whether in proteasomes or lysosomes (both structures are involved in degrading unwanted cellular components) or back on the cell surface.
"We're still scouting for the probes that we need to establish those kinds of things," Jovin said. "It's also the question of what happens to quantum dots in the cell. The cell fills up with these things, and it has to sequester them in some way because they aren't degradable biomolecules."
FOR SOME experiments, Jovin and his colleagues turn the size of quantum dots to their advantage. Even after growth factors are attached to the quantum dots, the nanocrystals still have plenty of room to bind other ligands that may be available in the medium. If these ligands have spectral characteristics matching those of the quantum dot, when they bind, they change the quantum dot's emission wavelength through a process known as FRET (fluorescence resonance energy transfer). Because the ligands are placed in the medium outside the cell, the FRET-induced color change can occur only if the quantum dot is still outside the cell. Using this method, Jovin and his colleagues are unraveling the mechanisms of transport along cellular projections known as filopodia. These are bundles of the protein actin surrounded by a plasma membrane, and they are involved in cell movement. The researchers want to know whether the growth factor, which is bound to the quantum dot, is inside or outside the cell as the receptor moves along the filopodia toward the main cell body. When they add the ligands, they find that the quantum dots do indeed change colors, indicating that the receptor is outside the cell as it moves. "Endocytosis does not take place on these filopodia," Jovin explained, "but the receptor somehow gets dragged along by a mechanism that we're trying to elucidate."
Another scientist studying endocytosis is biophysicist Xiaowei Zhuang, an assistant professor in the departments of chemistry and chemical biology and physics at Harvard University. She studies endocytosis in the context of how influenza viruses enter cells.
She tracks the flu virus by decorating the virus with fluorescent dyes that spontaneously associate with the viral membrane. Because the dyes are incorporated so efficiently into the membrane, many dye molecules can be added. However, because so many dye molecules associate with the membrane, they quench each other's fluorescence.
This quenching makes it possible to track when the viral membrane fuses with the endosomal membrane. The endosomal membrane is much larger than that of the virus, so the dye molecules are effectively diluted and no longer quench each other after fusion. The dramatic increase in the fluorescence serves as a marker for successful entry of virus into the cell.
Several endocytic pathways are available to influenza. The dominant route, accounting for about two-thirds of influenza entry, involves the association of the virus with features at the cell membrane known as clathrin-coated pits. (Clathrin is the protein that encases so-called coated vesicles.) The virus doesn't seem to exploit existing pits, Zhuang said. Instead, new pits seem to form around the virus. The rate at which these pits form at the virus binding sites appears to be much higher than elsewhere, as if the virus actively tries to enter the cell. One hypothesis is that the virus induces curvature in the cell membrane that triggers the formation of the pits [Nat. Struct. Mol. Biol., 11, 567 (2004)]. "Now that we can directly see it happening, we can sort out these different pathways much more quantitatively than before," Zhuang said.
However, endocytosis is not all it takes for successful viral infection. Trafficking is also important, and Zhuang is using sensitive fluorescent microscopy methods to figure out what happens to the viruses once they have been taken up in the endosome.
For this purpose, her group labels early, or newly formed, endosomes with fusion proteins composed of VFP and the protein called Rab5 and labels late, or mature, endosomes with fusion proteins made of VFP and the protein Rab7. "We found that viruses are targeted to a small population of rapidly maturing endosomes, placing themselves in a great position to achieve fusion. Currently, we are curious about how this happens," Zhuang said.
Such experiments require a number of labels, one each for Rab5, Rab7, and the virus. "We can relatively comfortably do three colors when we do this kind of experiment to track single particles in living cells," Zhuang said. However, some experiments require even more labels, and the probes aren't available. "There are a number of experiments where we want to use five labels," she added. "That becomes a problem because each one will have substantial overlap" with the others.
Chemistry remains the biggest challenge, Zhuang said. "How do you actually label the object you want to see intelligently to tell you the right stuff?" she asked. "You want to label the virus in a way that you're looking at the relevant information."
Although their narrow emission bands and brightness would seem to make quantum dots well suited to such tasks, Zhuang hesitates to use them to label viruses. "A lot of the things we're interested in are, if not smaller, just as big as a quantum dot," she said.
For example, she never considered labeling the viral genetic material, which consists of ribonucleoproteins (RNA-protein complexes), with quantum dots, even though the small size of a viral ribonucleoprotein makes it difficult to achieve large signals with dyes while maintaining its healthy behavior.
"In order for the genetic material to do its own thing, we have to put very few dyes per particle," Zhuang said. "That makes tracking very difficult, but we never even thought about using quantum dots." She worries that ribonucleoproteins labeled with quantum dots would be unable to squeeze into the nucleus.
Robert H. Singer, a professor in the department of anatomy and structural biology at Albert Einstein College of Medicine, New York City, also described single-molecule methods to study ribonucleoproteins. Using fusion proteins of yellow fluorescent protein and an mRNA-binding protein known as MS2 to track movements of individual ribonucleoproteins in cells, he and his coworkers have found that the movement of the mRNA-protein complexes is a diffusive, rather than an energy-driven, process [Science, 304, 1797 (2004)].
FURTHER DEVELOPMENT of probes for live-cell imaging is still needed. Fluorescence lifetime and intensity are the characteristics that most need improvement. "A successful probe should be bright, allow single-molecule sensitivity, be very stable so you can follow the movement of the probe over a long period of time, and be relatively small so that it does not perturb the biological functions you want to study," Nie said. "Of course, you will never get it perfect."
Jovin pointed out that improvements are also needed in the instruments and microscopes used for cell imaging. "We have to exploit the better microscopes, the ones that have higher temporal and spatial resolution," he said. "Our group is involved in microscopy development, and the newest group at our institute is developing superresolution techniques, which allow one to get far below the limit of resolution that the optical microscope classically imposes. We're getting down to molecular dimensions with the light microscope."
These needs haven't been lost on funding agencies. The National Institutes of Health Roadmap for Medical Research includes an emphasis on imaging probes (C&EN, Oct. 6, 2003, page 10). At the symposium, Catherine D. Lewis from the National Institute of General Medical Sciences at NIH pointed out that cellular imaging is the focus of the first initiative under the road map. NIH recently awarded nine grants--several to people who spoke at the symposium--to establish centers that will develop imaging probes.
"Everybody recognizes that the development of imaging probes is still a major challenge," Nie said.
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