Fluorescence Technique Traces Protein Activation

Researchers have used a combination of fluorescent labeling and time-lapse microscopy to localize activated neuronal Wiskott-Aldrich syndrome protein (N-WASP) in tissue culture cells. Members of the WASP/WAVE family of proteins are key regulators of cytoskeletal reorganization across a diverse range of cellular processes.

Investigators at Washington University School of Medicine (St. Louis, MO, USA) labeled each end of the N-WASP molecule with a different colored fluorescent probe. They employed a technique called fluorescence resonance energy transfer to show that when the molecule became activated and folded in half, the fluorescent light increased in intensity.

They reported in the January 19, 2004, online edition of the Proceedings of the [U.S.] National Academy of Sciences that time-lapse microscopy revealed that N-WASP was activated inside extending filopodia and also seemed to play a role in regulating membrane ruffling. Some of the highest levels of N-WASP activity were in the nucleus, which was unexpected, since the protein's main functions are in cell movement occurring in the periphery of the cell.

"To our knowledge this is the first probe of its kind that allows us to actually see in a living system where, when, and how proteins are activated,” explained first author Michael E. Ward, a graduate student in anatomy and neurobiology at Washington University School of Medicine. "Because we were able to visualize where N-WASP is activated, we were able to show it is activated in certain unexpected cellular compartments. Now that we have demonstrated this technique is effective, we hope to further examine this protein's activity and also to see whether similar probes can help us visualize other folding proteins.”





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