New dye reveals proteins in living cells
visualization has been developed. The technique, which uses a novel
set of biosensor dyes, promises to dramatically increase the
accuracy and speed of the technique, used in the pharmaceutical
industry for applications such as screening receptor binding
interactions and cell responses.
A series of experiments has directly revealed the activation of proteins in individual living cells, opening up new possibilities for screening the molecular effects of drugs within the living cell. The novel dyes are a viable alternative to the techniques currently used such as automated "high throughput" drug assays, which are conducted on thousands of cells at a time, but in vitro, in laboratory test tubes.
The research, led by Dr. Klaus Hahn, demonstrated that at least one of the dyes he developed make it possible to dramatically visualize the changing activation and intracellular location of the protein Cd42.
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-coloured 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.
Dr Hahn, who is also a professor of pharmacology at the University of North Carolina, said: "Unlike other protein visualization methods, you're looking directly at the fluorescence from this dye, which means it's much brighter and more sensitive."
"Perhaps the most important aspect of the paper is that we demonstrated a new approach. We showed we could look at endogenous molecules and their activation using novel dyes."
One major advantage of this method over current methods is it does not require making modifications to the protein in question. Because many proteins occur in small amounts, so the inclusion of exogenous material can change everything including results.
Fluorescence-based detection depends on the absorption of light by the cell and the subsequent re-emission of this light at a different frequency. Traditional flow cytometers make use of this by employing filters to block the original light source from reaching the detector, while the fluorescence emission is allowed through for detection, something that allows only a very low background of stray light to reach the detector.
In flow cytometry experiments, fluorescence is achieved by deliberate labelling of a cellular component using a fluorescent marker, usually a type of dye. These dyes fluoresce only when light of the appropriate wavelength (the laser being designed to emit light at that wavelength) hits them, causing the emission of secondary light at a different wavelength. Detection of the second wavelength is used as a measure of the presence of the dye on the cell and thus the component it is labelling.
Biomolecules of interest such as antibodies, cancer cells and specific genes are often marked by a fluorescent dye. The brightness of the signal from a sample can indicate whether or not the biomolecular count has increased or decreased in number. This can be used to track events within cells in response to a stimulus, or even indicate the severity of a disease.
