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New dye directly reveals activated proteins in living cells

CHAPEL HILL -- A series of experiments reported on this week in the journal Science shows for the first time that novel biosensor dyes can directly reveal activation of proteins in individual living cells.

The research, led by Dr. Klaus M. Hahn, professor of pharmacology at the University of North Carolina at Chapel Hill's School of Medicine, demonstrated that at least one of the dyes Hahn developed makes it possible to dramatically visualize the changing activation and intracellular location of the protein Cdc42.

The novel dyes open new possibilities for screening the molecular effects of drugs within the living cell. Currently, automated "high throughput" drug assays are conducted on thousands of cells at a time, but in vitro, in laboratory test tubes.

Cdc42, a member of the Rho family of proteins, regulates multiple and sometimes opposite functions within the cell: movement, proliferation, cell death and shape.

Injected into connective tissue cells, the dye "I-SO" displayed a bright green-colored fluorescence as Cdc42 activation and interaction with other proteins occurred. In addition, the dye proved highly sensitive, enabling detection of protein activation at low levels, unlike current fluorescence methods that require protein over-expression for detection.

"For the first time we saw native Cdc42 activity in living cells," Hahn said. "But perhaps the most important aspect of the paper is that we demonstrated a new approach: We showed we can look at endogenous molecules and their activation using novel dyes."

Unlike other protein visualization methods, "you're looking directly at the fluorescence from this dye, which means it's much brighter and more sensitive," Hahn said.

Also differing from current methods, the new approach does not require making modifications to the protein in question.

"Many proteins occur in small amounts, so if you put in exogenous material you change everything," Hahn said.
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Contact: L. H. Lang
llang@med.unc.edu
919-843-9687
University of North Carolina School of Medicine
10-Sep-2004


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