Glowing zebrafish help researchers track role of sugars in the cell

Scientists have known for more than a century how to attach fluorescent dyes to proteins, and have used the technique to study protein trafficking in cell culture and even in whole organisms, though often at the expense of killing the cells or organism. Bertozzi has focused on making it just as easy to study the sugars on cells, in part to investigate their role in such diseases as tuberculosis and influenza. In the latter, the flu virus enters cells by way of hemagglutinin, a sugar-protein complex on the viral surface that attaches to sugars on the surface of host cells. But sugars clearly have roles in cell-to-cell communication that have yet to be discovered.

One technique Bertozzi has developed is to feed cells an artificial sugar that looks so much like the real thing that cells are tricked into incorporating the sugar into their carbohydrate chains. Once the sugar becomes part of the forest of carbohydrates adorning a living cell, she then uses a non-toxic chemical reaction to attach small organic labels to it. Simple, highly selective and non-toxic chemical reactions like this have come to be called click chemistry.

In their work on zebrafish, Baskin, Bertozzi and their colleagues soaked zebrafish embryos in the artificial sugar N-azidoacetylgalactosamine, which the embryo cells then used as a carbohydrate building block to replace the natural sugar N-acetylgalactosamine. The researchers then modified a chemical reaction that is normally toxic to cells to eliminate the toxic copper catalyst and employed this reaction to attach a small fluorescent molecule, a fluorophore, to the "azido" part of the unnatural sugar.

The copper-free click chemistry worked with three separate fluorophores, enabling the researchers to make two-to five-day-old zebrafish cells glow red, green and even near infrared, which is invisible to the eye but can be detected by some microscopes. They were able to observe differences over time in when and where on a single cell the sugar appeared, sugar movement through the cell interior, and in which tissues the sugar showed up.

"We're hoping to extend the technique to other sugars, too," Baskin said, noting that of the nine sugars used by vertebrates to build carbohydrates, Bertozzi's lab has found artificial surrogates for four of them. "We also want to try getting (artificial sugar) to work in different organisms and different disease models, such as cancer models in mice. Basically, we are providing this as a tool for the general community to use."

Amacher, who studies tissue patterning in the very early zebrafish embryo, is anxious to work with the labeling technique, but is waiting until Bertozzi's group gets it to work in hours-old embryos, at a stage when muscles and organs begin to form.

"Once they get the labeling technique to work at very early times, it is going to be an even more exciting collaboration, and hopefully, a continuing one," she said.

The work was supported by the National Institutes of Health.