Microscopy shows subcellular protein position

Compiled by BioPhotonics staff

A new polarization-based technique can help researchers 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 have filled in 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 a mystery. One hurdle they faced was a “resolution gap” between two techniques used primarily to visualize protein complexes. Even though electron microscopy can reveal the broad outlines of a large protein complex, it cannot show details. X-ray crystallography can show minute detail but only of a small piece of the complex; it can’t say how the individual pieces fit together. Further complicating matters, both techniques require fixed samples, making it difficult to tell how cell components move.

The new technique, developed by scientists at Rockefeller University, takes advantage of the properties of polarized light to show how specific proteins are aligned in relation to one another. They genetically attached fluorescent markers to individual components of the nuclear pore complex and then replaced the cell’s own copy of the gene that encodes the protein with a new form that has the fluorescent tag. Next, they used customized microscopes to measure the orientation of the waves of light emitted by the fluorescently tagged proteins. By combining the measurements with known data about the structure of the complex, the researchers were able to confirm or deny the accuracy of previously suggested models.

The technique, detailed in the Feb. 2, 2011, issue of Biophysical Journal (doi: 10.1016/j.bpj.2010.12.967), enabled the scientists to study nuclear pore complexes in both budding yeast and human cells. The data gathered in the human cell experiments has shown 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 Günter Blobel in 2007.

The researchers said their technique could eventually go further. Because the proteins’ fluorescence can be measured while the cells are still alive, the data could provide scientists new insights into how protein complexes react to varying environmental conditions, as well as into how their configurations change over time.