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STED microscopy reveals how immune system attacks infected cells

BioPhotonics
Nov 2011
Ashley N. Paddock, ashley.paddock@photonics.com

PHILADELPHIA – A new stimulated emission depletion (STED) microscope has yielded unprecedented views of the immune system in action.

The superresolution microscope shows how granules from natural killer (NK) cells pass through openings in the dynamic cell skeleton to destroy their targets: tumor and virus-infected cells. Understanding these biological events better may soon allow researchers to devise more effective treatments for inherited diseases that leave the immune system compromised.

“From a biological perspective, the work defines a previously unappreciated regulatory hurdle that an NK cell must overcome in order to access essential host defense functions,” said Dr. Jordan S. Orange of the Children’s Hospital of Philadelphia. “This of course presents opportunities for exploiting this process in order to obtain more or less of this particular variety of secretion. This could have very relevant clinical and therapeutic implications.”


Superresolution stimulated emission depletion (STED) fluorescence micrograph of an activated human natural killer cell demonstrating actin filaments (green) and perforin-containing lytic granules (red). Courtesy of Dr. Emily Mace.


Previously, conventional fluorescence light microscopes could not resolve objects smaller than 200 nm. The new STED technique uses a unique arrangement of lasers and fluorescence to image fine structures such as protein filaments smaller than 60 nm.

Orange has long researched the biology of NK cells at the immunological synapse – the site where the NK cell attaches to its target cell and delivers cell-killing molecules. A crucial component of this highly regulated process is filamentous actin (F-actin), a protein in NK cells that forms a dense network through which cell-killing molecules move into the synapse.

It was thought that F-actin is not present at the center of the network, where the granules fuse with the cell surface. The current study reveals under superresolution, however, that F-actin pervades the synapse but leaves openings just large enough to allow granules to pass through. The scientists observed that F-actin appeared to be dynamically interacting with the granules to move them toward their targets.


Platinum rotary electron micrograph of a natural killer cell cortex colorized to depict actin filaments (blue) and a single intercalated lytic granule (yellow). Courtesy of Dr. Jordan Orange.


Orange compared the F-actin filaments to the rails of a roller coaster that quickly rearranges itself to guide a rider through a narrow tunnel. He explained that further studies of NK function will investigate energy use and biological mechanisms that allow the lytic granules to navigate the immunological synapse.

“The patterning and coordination of the pervasive actin network is telling a story – one that is likely to underscore how the critical host defense function mediated by NK cells is accessed,” Orange said. “We certainly need to sort this out as well as what underlies the interaction between NK cell lytic organelles and hypodense regions within the pervasive actin at the synaptic interface.”

The study was published Sept. 13 in the online open-access journal PLoS Biology (doi: 10.1371/journal.pbio.1001151).


GLOSSARY
fluorescence
The emission of light or other electromagnetic radiation of longer wavelengths by a substance as a result of the absorption of some other radiation of shorter wavelengths, provided the emission continues only as long as the stimulus producing it is maintained. In other words, fluorescence is the luminescence that persists for less than about 10-8 s after excitation.
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