Imaging method reveals a “symphony of cellular activity”

Within a single cell, thousands of molecules, such as proteins, ions and other signaling molecules, work together to carry out all types of functions: to absorb nutrients, storing memories and differentiate into specific tissues, among many others.

Deciphering these molecules and all their interactions is a monumental task. Over the past 20 years, scientists have developed fluorescent reporters that they can use to read the dynamics of individual molecules within cells. However, usually only one or two of these signals can be observed at a time, as a microscope cannot distinguish between many fluorescent colors.

MIT researchers have now developed a way to visualize up to five different types of molecules at a time by measuring each signal from random and distinct locations in a cell. This approach could allow scientists to learn much more about the complex signaling networks that control most cellular functions, says Edward Boyden, Y. Eva Tan Professor in Neurotechnology and Professor of Biological Engineering, Media Arts and Sciences, and Brain Sciences. and cognitive at MIT.

“There are thousands of molecules encoded by the genome and they interact in ways we don’t understand. Only by observing them at the same time can we understand their relationships, “says Boyden, who is also a member of the McGovern Institute for Brain Research and the Koch Institute for Integrative Cancer Research at MIT.

In a new study, Boyden and his colleagues used this technique to identify two populations of neurons that respond to calcium signals in different ways, which can affect how they encode long-term memories, the researchers say.

Boyden is the senior author of the study, which appears on Cell today. The paper’s lead authors are MIT postdoc Changyang Linghu and graduate student Shannon Johnson.

Fluorescent clusters

To make molecular activity within a cell visible, scientists typically create reporters by fusing a protein that detects a target molecule to a protein that glows. “This is similar to how a smoke detector detects smoke and then flashes a light,” says Johnson, who is also a fellow at the Yang-Tan Center for Molecular Therapeutics. The most commonly used light protein is green fluorescent protein (GFP), which is based on a molecule originally found in a fluorescent jellyfish.

“Typically a biologist can see one or two colors at the same time on a microscope, and many of the reporters out there are green, because they’re based on the green fluorescent protein,” Boyden says. “What has been missing so far is the ability to see more than a couple of these signals at the same time.”

“Just as hearing the sound of a single instrument from an orchestra is far from enough to fully appreciate a symphony,” says Linghu, “by allowing the observation of multiple cellular signals at the same time, our technology will help us understand the symphony. “of cellular activity.”

To increase the number of signals they could see, the researchers decided to identify the signals by location rather than by color. They modified existing journalists to accumulate them in groups at different locations within a cell. They did this by adding two small peptides to each reporter, which helped the reporters form distinct groups within the cells.

“It’s like having Reporter X tied to a LEGO brick and Reporter Z tied to a K’NEX piece – only the LEGO bricks clip onto other LEGO bricks, causing only Reporter X to be grouped with multiple Reporters X”, Johnson says.

With this technique, each cell ends up with hundreds of groups of fluorescent reporters. After measuring the activity of each cluster under the microscope, based on the variable fluorescence, researchers can identify which molecule was being measured in each cluster while preserving the cell and staining for peptide tags that are unique to each reporter. Peptide tags are invisible in the living cell, but can be stained and seen after performing live imaging. This allows researchers to distinguish the signals for different molecules even though they can all be fluorescent the same color in the living cell.

Using this approach, the researchers showed they could see five different molecular signals in a single cell. To demonstrate the potential utility of this strategy, they measured the activities of three molecules in parallel: calcium, cyclic AMP, and protein kinase A (PKA). These molecules form a signaling network that is involved in many different cellular functions throughout the body. In neurons, it plays an important role in translating short-term input (from upstream neurons) into long-term changes such as strengthening connections between neurons, a process necessary for learning and the formation of new memories.

By applying this imaging technique to pyramidal neurons in the hippocampus, the researchers identified two new subpopulations with different calcium signaling dynamics. One population showed slow responses to calcium. In the other population, neurons had faster responses than calcium. The latter population had larger PKA responses. Researchers believe this heightened response may help support long-lasting changes in neurons.

Image signaling networks

The researchers now intend to try this approach in live animals so that they can study how the activities of the signaling network relate to behavior and also to expand it to other cell types, such as immune cells. This technique could also be useful for comparing the signaling network patterns between healthy and diseased tissue cells.

In this article, the researchers showed they can record five different molecular signals at once, and by tweaking their existing strategy, they believe they can go as high as 16. With additional work, that number could reach into the hundreds, they say.

“This could really help open up some of these difficult questions about how the parts of a cell work together,” says Boyden. “One could imagine an era where we can look at everything that happens in a living cell, or at least the part involved with learning, or with disease, or with treating a disease.”

The research was funded by the Friends of the McGovern Institute Fellowship; the J. Douglas Tan Fellowship; Lisa Yang; the Yang-Tan Center for Molecular Therapeutics; John Doerr; the Open Philanthropy Project; the HHMI-Simons Faculty Scholars program; the Human Frontier science program; the US Army Research Laboratory; the MIT Media Lab; the Picower Institute Innovation Fund; the National Institutes of Health, including the NIH Director’s Pioneer Award; and the National Science Foundation.