Embargoed by Science for 2 pm EDT Thursday, April 18, 2002

New Post-Genomic Technique Provides Real-Time,
Multiscale Look At Protein Life Cycles in Living Cells

Scientists have developed a new molecular-tagging technique to chronicle the development, movement and interactions of proteins as they do their work in living cells.

The results are published in the April 19, 2002 issue of the journal Science by University of California, San Diego (UCSD) School of Medicine and National Center for Microscopy and Imaging Research (NCMIR) researchers at the UCSD campus in La Jolla.

"Now that the human genome has been sequenced, the next big push is to determine the properties of proteins associated with those genes," said Mark Ellisman, Ph.D., UCSD professor of neurosciences and bioengineering, director of NCMIR and senior author of the study published in Science. "The challenge is the development of technologies and methodologies to mark and observe those proteins in living cells, to see how they fit together into teams or groups and to determine precisely where these teams are located. The technology we have developed delivers important new capabilities at these next levels of scientific research."

Optical section of double-labeled (green and red) gap junctions in HeLa cells. The tubulin network is shown in white, the cell nuclei in blue."

Like Lego pieces that are used to build objects and structures, proteins are the building blocks for cellular activity and the development of tissues and organisms. Proteins are constantly added to and removed from the cellular building.

"If we want to follow this frenetic activity as it takes place, we need comparably dynamic experimental approaches," Ellisman said. "Furthermore, we need techniques that allow us to view both the single protein and the final structure while they are being produced, assembled, modified and, finally, degraded."

To date, scientists have used marking techniques involving intrinsically fluorescent structures to tag the protein of interest. While extremely useful to monitor the distribution of the protein in living specimens and to witness some of its interactions with other cellular components, these techniques don't allow for discrimination between the different, time-separated stages of development and degradation of the protein. In addition, the fluorescent proteins are often larger than many of the proteins they are attached to. And, they don't allow researchers to explore the dynamics of the protein at different resolution levels, from the larger cellular building down to the macromolecular level of the individual protein complexes.

With funding primarily from the National Institutes of Health and the Howard Hughes Medical Institutes (HHMI), the UCSD team combined advanced microscopic capabilities and molecular biology from the NCMIR with chemistry and biochemistry, primarily from the research team of Roger Tsien, Ph.D., professor of pharmacology, chemistry and biochemistry, and an HHMI investigator.

The result was a powerful, integrated and innovative multiscale molecular tagging technology that lets researchers genetically tag a protein with a small binding area called a domain (six to 20 amino-acids long), that then interacts with a variety of other compounds. The UCSD team employed two of these compounds, FlAsH and ReAsH, in the work published in Science. Both compounds are capable of crossing the plasma membrane of living cells to quickly reach the protein genetically engineered to contain the small binding domain. Then, upon binding, the compounds turn brightly fluorescent. FlAsH fluoresces green, whereas ReAsH glows red.

To distinguish old from new proteins, the researchers first used the green FlAsH. Then, they applied ReAsH after new proteins were developed. By administering FlAsH and ReAsH at different times, the UCSD team was able to distinguish young from old copies of the target protein.

An important advantage of the new technique is its application for electron microscopy. Most molecular tagging techniques currently used for monitoring protein distribution and fate in living cells are applicable only to light microscopy, which doesn't provide enough power to allow the exploration of fine structural details of macromolecular structures. These older techniques are not transferable to electron microscopic evaluation, which is 1,000 to 10,000 times more powerful than the light microscope, and is able to locate the precise position of individual protein complexes.

One of the compounds employed by the UCSD team, ReAsH, can be used in both light and electron microscopy. After identifying temporally separated pools of the target protein at the light microscope in living cells, the researchers were able to identify the ReAsH-labeled pool at the electron microscope in cells which had been stopped, using a chemical reaction called photo-oxidation.

Theoretically, this technique could be applied to any protein and would allow scientists to follow the dynamics of assembly and disassembly of intracellular structures containing the tagged protein. The experiments presented in the Science article followed the trafficking and turnover of connexins, the protein constituent of gap junctions, which are tissue membranes formed by the association of thousands of proteins which cross the plasma membrane of two adjacent cells. Through their nano-scale sized pores, cells in the gap junction establish a cytoplasmic continuity that enables them to communicate with one another by passage of signaling molecules, ions and metabolites.

Using the new tagging technology, the research team was able to elucidate some aspects of gap junction assembly and turnover in the living cell. For example, they showed that newly synthesized connexins were transported to the plasma membrane in small, 100 to 150 nanometer vesicles and incorporated at the periphery of pre-existing gap junction plaques. Older connexins were instead removed from the center of the plaque and transported into degradative vesicles of various sizes.

Although the findings on gap junction refurbishing were of great interest, the researchers were most excited about the possibility of generalizing their technique to study the life cycle of virtually any protein system and being able to visualize these proteins in the cell.

In addition to Ellisman and Tsien, the UCSD team was formed by first author Guido Gaietta, Ph.D., project scientist; Tom Deerinck, research associate; James Bouwer, graduate student; and Gina Sosinsky, Ph.D., professor of neurosciences, all with the NCMIR; Stephen Adams, Ph.D., project scientist; and Oded Tour, Ph.D., post-doctoral fellow, who work with Tsien in the UCSD Departments of Pharmacology, Chemistry and Biochemistry. Also contributing to the work was Dale Laird, Ph.D., Department of Anatomy and Cell Biology, University of Western Ontario, London, Canada.

The National Science Foundation and the Canadian Institutes of Health Research also provided funding for the study.

# # #