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Glowing Proteins See Cells

Photonics.com
Jan 2009
BRONX, N.Y., Jan. 27, 2009 – New fluorescent proteins have been discovered that allow scientists to noninvasively visualize the structures and processes in living cells at the molecular level.

Researchers at Gruss Lipper Biophotonics Center at Albert Einstein College of Medicine of Yeshiva University discovered the tools, which are photoactivatable fluorescent proteins (PAFPs) and other advanced fluorescent proteins (FPs).

The fluorescent proteins now can follow cancer cells as they seek out blood vessels and spread throughout the body or can watch how cells manage intracellular debris, preventing premature aging.

They add considerably to the biomedical imaging revolution started by the 1992 discovery that the gene for a green fluorescent protein (GFP) found in a jellyfish could be fused to any gene in a living cell. GFP fluoresces when the target gene is expressed, creating a visual marker of gene expression and protein localization via light, or optical, microscopy.
 
Three scientists won the 2008 Nobel Prize in chemistry for their GFP-related discoveries. Fluorescent proteins of other colors have since been found in marine organisms such as corals.

Although this form of imaging is invaluable, it is limited by the inherent nature of optical microscopy, which cannot image details of objects smaller than 200 nm or so. However, many cellular structures, which could hold the key to managing or curing disease, are a fraction of that size – just a few nanometers or more.

Using a sophisticated combination of lasers, computers and highly sensitive digital cameras, scientists have surmounted the barriers of optical imaging. The first generation of these new imaging devices, collectively known as superresolution fluorescence microscopes, captured images as small as 15 to 20 nm – the scale of single molecules. But this could be done only in nonliving cells. The addition of PAFPs, more versatile versions of FPs, made it possible to do real-time superresolution fluorescence microscopy in living cells.

Vladislav Verkhusha, associate professor of anatomy and structural biology at Einstein and a member of the Biophotonics Center, has developed a variety of PAFPs and FPs for use in imaging mammalian cells, expanding the applications of fluorescence microscopy. Among these are PAFPs that can be turned on and off with a pulse of light, and FPs that can fluoresce in different colors and that have better resolution for deep-tissue imaging.

Most recently, Verkhusha developed a red PAFP called PAmCherry1, which has faster photoactivation, improved contrast and better stability compared with other PAFPs of its type.

“PAmCherry1 will allow improvements in several imaging techniques, notably two-color superresolution fluorescence microscopy, in which two different molecules or two biological processes can be viewed simultaneously in a single cell,” Verkhusha explained.

Several studies have employed Verkhusha’s PAFPs, revealing new insights into a variety of biological processes. For example, one of his PAFPs was used to capture the first nanoscale images of the orientation of molecules within biological structures.

“Such images could be useful in studying protein-protein interactions, the growth and collapse of intracellular structures and many other biological questions,” Verkhusha said.

He also contributed a novel PAFP to a new method of viewing individual breast cancer cells for several days at a time, providing details on how cancer cells invade surrounding tissue and reach blood vessels, a process called metastasis.

“Mapping the fate of tumor cells in different regions of a tumor was not possible before the development of the photoswitching technology,” said John Condeelis, co-chairman and professor of anatomy and structural biology and co-director of the Biophotonics Center.

In addition, Verkhusha has developed new types of fluorescent proteins for use in conventional fluorescence microscopy. These new proteins, called fluorescent timers (FTs), can change color from blue to red over a matter of hours.

“These FTs will enable scientists to study the trafficking of cellular proteins and to provide accurate insight into the timing of intracellular processes, such as activation or inhibition of gene expression or protein synthesis,” Verkhusha said.

Together with another Einstein scientist, Ana Maria Cuervo, associate professor of developmental and molecular biology, anatomy and structural biology, and medicine, Verkhusha employed the FTs to demonstrate for the first time how a protein called LAMP-2A, which scavenges cellular debris, is transported to intracellular organelles called lysosomes, where the debris is digested.

Understanding this process, which maintains the health of cells and organs, could lead to treatments to keep elderly people’s organs in prime condition.

For more information, visit: www.aecom.yu.edu



GLOSSARY
photonics
The technology of generating and harnessing light and other forms of radiant energy whose quantum unit is the photon. The science includes light emission, transmission, deflection, amplification and detection by optical components and instruments, lasers and other light sources, fiber optics, electro-optical instrumentation, related hardware and electronics, and sophisticated systems. The range of applications of photonics extends from energy generation to detection to communications and...
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