(Nanowerk News) On-the-spot reporting is now available in live cells.
Synthetic biologists from Rice University and Princeton University have demonstrated “live reporter” technology that can unravel the workings of signaling protein networks in living cells with much higher precision than current methods. The first reporting tool of its kind can show, for example, how quickly signaling networks respond and how their response varies from cell to cell in both space and time.
The researchers created the tool using inconspicuous proteins that support important signaling mechanisms that human cells use to regulate growth, differentiation, migration, inflammation and other processes.
In a recent study in an open access journal eLife (“pYtags enable spatiotemporal measurement of receptor tyrosine kinase signaling in living cells”), the Rice-Princeton team demonstrated its modular approach to tagging receptor tyrosine kinases (RTKs) with reporter proteins that activate green fluorescent proteins whenever their RTK partners are phosphorylated.
Kinases are enzymes that can change the behavior of other proteins by attaching or removing phosphate groups, a process called phosphorylation. RTK is a specialized kinase that itself becomes phosphorylated when it detects incoming signals or stimuli outside the cell, and then regulates vital cell functions.
The Rice-Princeton team demonstrated the “live reporter” system could be used with a microscope to produce video recordings of signaling network activity in living cells. Where a cell glows and how brightly, reveals the location and intensity of the signaling network response, said Caleb Bashor, study co-author and assistant professor of bioengineering and biosciences at Rice.
“Most of the time, when you study things that happen inside cells, like signaling networks or gene networks, you have to destroy the cell to see what’s inside,” Bashor said. “Anytime you can build something where the cells are kept alive, and you can see how the signaling network is working in real time, inside the cell, that’s a huge advantage.”
The researchers dubbed the reporters pYtags, referring to the biochemical nomenclature in which tyrosine is denoted as “Y” and as “pY” when it is phosphorylated.
Bashor and Xiaoyu Yang, Ph.D. students in Bashor’s research group, developed pYtags in collaboration with the research group of Jared Toettcher and Celeste Nelson of Princeton. The study shows the system can record the activity of an RTK called a growth factor receptor in human fibroblast cells.
“We took an engineered protein that is part of a different system — it’s actually part of immune signaling — and we put it into this new context, which is the fibroblast cell that Jared is working with in his lab at Princeton,” said Bashor. “We thought it might not interact with anything else in the cell because it comes from a completely different type of cell. So, it just hangs at the end of the growth factor receptor.”
The pYtag reporter is designed to be activated in conjunction with its RTK counterpart and trigger a proportional amount of fluorescence. So the stronger the RTK response, the brighter the cell glows when viewed through a microscope.
“It can receive phosphorylation signals from growth factor receptors,” says Bashor. “So when the receptor is activated, a green fluorescent protein kicks in, binds to something close to the membrane, and you get what looks like a green ring around the outside of the cell. That tells you, in real time, when the cell is viewing a growth factor and how fast its pathway is firing.
Bashor said pYtags can be used to monitor many types of receptor tyrosine kinases.
“We showed in the paper that this reporter can be attached to several different types of growth factor receptors, and that it can be used as a reporter for all of them,” he said. “This is a window into cellular signaling dynamics that we really didn’t have before.”