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Microscopy shows subcellular protein position

BioPhotonics
Jul 2011
Compiled by BioPhotonics staff

NEW YORK – 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.


GLOSSARY
microscope
An instrument consisting essentially of a tube 160 mm long, with an objective lens at the distant end and an eyepiece at the near end. The objective forms a real aerial image of the object in the focal plane of the eyepiece where it is observed by the eye. The overall magnifying power is equal to the linear magnification of the objective multiplied by the magnifying power of the eyepiece. The eyepiece can be replaced by a film to photograph the primary image, or a positive or negative relay...
polarization
With respect to light radiation, the restriction of the vibrations of the magnetic or electric field vector to a single plane. In a beam of electromagnetic radiation, the polarization direction is the direction of the electric field vector (with no distinction between positive and negative as the field oscillates back and forth). The polarization vector is always in the plane at right angles to the beam direction. Near some given stationary point in space the polarization direction in the beam...
x-ray crystallography
The study of the arrangement of atoms in a crystal by means of x-rays.
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