New imaging method reveals hidden molecular interactions in cells

Cells are crowded, dynamic places where thousands of molecules interact in tight quarters. Until now, scientists lacked a reliable way to see many of these molecular interactions as they happen.

Researchers at the University of Illinois Chicago have developed a new imaging method that allows scientists to see previously hidden enzyme activities in small regions across the whole cell. The findings, published in the Proceedings of the National Academy of Sciences, open new possibilities for understanding how cells process information.

This could help scientists better understand how drugs work or fail by showing exactly where cellular signals happen in real time. 

We've discovered a cool lens into the very small molecular features used by a cell."

Gary Mo, co-author of the study and associate professor of pharmacology and regenerative medicine and biomedical engineering, UIC

The other authors on the study are UIC's Kriti Srivastava, Kevin P. Schnur and Kay Petruzzi.

Why biosensor imaging sometimes fails

Scientists have long used tools called biosensors, which are fluorescent molecules that sense events in a cell and then report their activity.

"A biosensor is anything that can sense environmental change in the cell," said Srivastava, a research assistant professor in the College of Medicine. If a cell is like a city, and each molecule is one of the city's inhabitants, biosensors are like private investigators following and reporting on what the people are doing. They are the molecular eyes and ears that tell scientists what's happening in a living cell.

Biosensors can be positive or negative, meaning they either "light up" or "go dark" when they sense events in the cell. Unfortunately, negative biosensors have been often unusable for scientists.

The problem, Mo explained, is like wearing green in front of a green screen: important details disappear. "Because these biosensors go dark, some parts of the foreground, where the action is, blend into a background," Mo said. The result is that some regions of high enzyme activity can, falsely, look identical to regions where there is no activity, he explained.

Turning unusable signals into clear images

To tackle this, the UIC team developed a technique they dubbed Fluctuation Increase Negated by Intra-Chain, or FINICI. The approach flips the optical readout of each negative biosensor into a positive, readable one. This allows the researchers to use existing negative biosensors, without having to re-design them from scratch, which can take years.

Using FINICI, the team imaged the activity of three molecules: Src kinase, Syk kinase and cGMP.

For Src kinase, a protein linked to cancer and cell movement, the team found bursts of activity in small areas of the cell membrane, including cholesterol-rich lipid rafts. Some of these active regions appeared briefly before dissolving, while others persisted longer, differences that aren't visible in traditional whole-cell measurements.

The team also found that the signaling molecule cGMP formed small clusters that were quickly overwhelmed as the signal spread through the cell.

In immune cells, the enzyme Syk was most active near the cell's internal scaffolding rather than near the receptors that activate it.

Together, these findings show that critical signals are controlled by where molecules act in the cell - like in real estate, location is hugely important. "You have to be in the room to do the job," Mo said. "If an enzyme isn't in the right place, it doesn't matter if it's active - it can do the work, but it's not going to."

The implications of their findings, Mo and Srivastava said, extend far beyond the lab. Many drugs are designed to target enzymes and signaling pathways, and their efficacy can depend on this issue of location.

"Cell signaling determines how drugs work," said Mo. "Drug molecules directly interact with molecules in your cells, and visualizing these details is a significant step that helps to understand and improve how they work."