New Dye Directly Reveals Activated Proteins In 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.

Among the reasons Hahn and co-authors at Scripps Research Institute in La Jolla, Calif., decided to study the Rho proteins was that different members of the protein family each control a different aspect of cellular movement of extension and retraction. One family controls extension of the edge, another the formation of fibers, and still another controls tail retraction.

"And the key to understanding this mechanism is to see where in time and space each of these is turned on and how it's all coordinated," Hahn said. "So there's a really good reason to look at this in live cells. You can't understand spatio-temporal control if you look at this in a test tube."

Another reason to study Rho proteins is that their activation is necessary to induce essentially opposite behaviors. "They're activated for proliferation and for cell death (apoptosis), also for motility. So it may be that this spatio-temporal control is what's producing these differences."

Some of the study's biological findings in that latter regard were tantalizing. Cdc42 induced formation of cell extensions called filopodia when it was activated around the filopodia base and not within the lengths, Hahn said.

"When we looked at extension and retraction, we found that Cdc42 activation was remarkably correlated with both. It was activated at exact locations relative to cell extensions and was turned off in exact parallel with retraction."

Further experiments showed that this coordination was produced by "upstream signals" regulating both retraction and extension.

"The use of fluorescent labeling of molecules in live cells was pioneered over a decade ago here at UNC," Hahn said. "My work with these new dyes is an extension of that work, it grew out of that."

This research was funded by the National Institute of General Medical Sciences, a component of the National Institutes of Health.