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By turning their instruments toward the nuclear pore complex, a huge cluster of proteins that serves as a gateway to a cell's nucleus, the scientists say they have filled in the gaps left by other techniques and made important new discoveries about how the complex works.
"Our new technique allows us to measure how components of large protein complexes are arranged in relation to one another," says Sandy Simon, head of the Laboratory of Cellular Biophysics. "This has the potential to give us important new information about how the nuclear pore complex functions, but we believe it can also be applied to other multi-protein complexes such as those involved in DNA transcription, protein synthesis or viral replication."
Although researchers have spent years studying the workings of the nuclear pore complex, there is still much that has remained mysterious. One problem is that there is a "resolution gap" between the two techniques primarily used to visualize protein complexes. Electron microscopy can reveal the broad outlines of a large protein complex, but it can't show details. X-ray crystallography, meanwhile, can show minute detail but only of a small piece of the complex; it can't say how the individual pieces fit together. To further complicate matters, both techniques require fixed samples – while they can give you an idea of what something looks like at a moment in time, they can't tell you how its pieces might move.
The new technique takes advantage of the properties of polarized light to show how specific proteins are aligned in relation to one another. After genetically attaching fluorescent markers to individual components of the nuclear pore complex, the scientists replaced the cell's own copy of the gene that encodes the protein with the new form that has the fluorescent tag. Then, they used customized microscopes to measure the orientation of the waves of light the fluorescently tagged proteins emitted. By combining these measurements with known data about the structure of the complex, the scientists can confirm or deny the accuracy of previously suggested models.
The scientists used the technique to study nuclear pore complexes in both budding yeast and human cells. In the case of the human cells, their new data shows that multiple copies of a key building block of the nuclear pore complex, the Y-shaped subcomplex, are arranged head-to-tail, rather than like fence posts.
COMPAMED.de; Source: Rockefeller University