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Polarized Microscopy Shows Protein Position

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NEW YORK, April 21, 2011 — A new polarization-based technique can help deduce the orientation of specific proteins within a cell. By turning their instruments toward the nuclear pore complex — a huge cluster of proteins that serves as a gateway to a cell's nucleus — scientists at Rockefeller University have filled the gaps left by other techniques and made important discoveries about how the complex works.

Although researchers have spent years studying the workings of the nuclear pore complex, much 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, on the other hand, 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 and so can’t tell you how cell components move.

Researchers at Rockefeller University have developed a technique that uses polarized light microscopy to help answer questions about the orientation of proteins within cells. The results demonstrate that the Y-shaped subcomplexes are arranged head to tail (left) and that a “fence post” model proposed earlier was not correct. Here, Sandy Simon and Claire Atkinson review data from their experiments. (Image: Zach Veilleux, Rockefeller University)

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. They used customized microscopes to measure the orientation of the waves of light emitted by the fluorescently tagged proteins. 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 method was developed by Sandy Simon, head of the university’s cellular biophysics laboratory, along with Alexa Mattheyses, Claire Atkinson and Martin Kampmann, a former member of Günter Blobel’s cell biology lab who is now at the University of California, San Francisco.

“Our experimental approach to the structure is synergistic with other studies being conducted at Rockefeller, including analysis with x-ray crystallography in Günter’s lab and electron microscopy and computer analysis in Mike Rout’s lab," Simon said. “By utilizing multiple techniques, we are able to get a more precise picture of these complexes than has ever been possible before.”

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, confirming a model proposed by Blobel in 2007.

Eventually, the scientists say, their technique could go even further. Because the proteins’ fluorescence can be measured while the cells are still alive, it could give scientists new insights into how protein complexes react to varying environmental conditions, and how their configurations change over time.

“What happens when other proteins pass through the nuclear pore? Does the orientation of the nucleoporins change? With this technique, we can find out not only what the pore looks like when it’s sitting still, but what happens to it when it’s active,” Simon said. Their first characterization of the dynamics of the nuclear pore proteins was published recently in The Biophysical Journal.

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Apr 2011
x-ray crystallography
The study of the arrangement of atoms in a crystal by means of x-rays.
Alexa MattheysesAmericasBiophotonicsCaliforniacell biologycell nucleusClaire Atkinsonelectron microscopyGünter BlobelimagingMartin KampmannMicroscopyNew Yorknuclear pore complexpolarized light microscopyproteinsResearch & TechnologyRockefeller UniversitySan FranciscoSandy SimonThe Biophysical Journalthe University of Californiax-ray crystallographyY-shaped complex

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