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 issue of 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. The research is funded in part by the National Science Foundation (NSF).
“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, neuroscientist and bioengineer, director of NCMIR, and co-author of the study.
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.
The UCSD team combined advanced microscopic capabilities and molecular biology from the NCMIR with chemistry and biochemistry to create 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.
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.
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.
“This is a great example of how multidisciplinary teams, composed of talented individuals who each bring their own special expertise into the mix, can address modern biological problems in ways that none alone could do,” said Eve Barak, program director in NSF’s division of molecular and cellular biosciences.
The Canadian Institutes of Health Research, the U.S. National Institutes of Health, and the Howard Hughes Medical Institutes also provided funding for the study.
Materials provided by National Science Foundation. Note: Content may be edited for style and length.
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