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In a single cell, thousands of molecules, such as proteins, ions, and other signaling molecules, work together to perform all kinds of functions – absorbing nutrients, storing memories, and differentiating into specific tissues, among others. .
Deciphering these molecules and all of 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 in cells. Typically, however, only one or two of these signals can be seen at a time, as a microscope cannot distinguish between many fluorescent colors.
MIT researchers have now developed a way to imagine up to five different types of molecules at once, measuring each signal from random, separate locations in a cell. This approach could allow scientists to learn much more about the complex signaling networks that control most cell functions, says Edward Boyden, Y. Eva Tan professor in neurotechnology and professor of biological engineering, arts and media sciences. and brain and cognitive sciences at MIT.
“There are thousands of molecules encoded by the genome, and they interact in ways we don’t understand. It is only by looking at them at the same time that we can understand their relationships, ”says Boyden, who is also a fellow of the McGovern Institute for Brain Research at MIT and the Koch Institute for Integrative Research. about cancer.
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 may influence how they encode long-term memories, the researchers say.
Boyden is the lead author of the study, which appears today in Cell. The main authors of the article are Changyang Linghu, postdoc at MIT, and Shannon Johnson, graduate student.
Fluorescent clusters
To make molecular activity visible in a cell, scientists typically create journalists by fusing a protein that detects a target molecule with a protein that glows. “This is similar to how a smoke detector will detect smoke and then flash a light,” says Johnson, who is also a member of the Yang-Tan Center for Molecular Therapeutics. The most commonly used glowing 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 reporters are green because they are based on the green fluorescent protein,” Boyden explains. “What has been missing so far is the ability to see more than one or two of these signals at a time.”
“Just as listening to the sound of a single instrument from an orchestra is far from sufficient to fully appreciate a symphony,” says Linghu, “by allowing the observation of several cellular signals at the same time, our technology will help us to understand the symphony of “cellular activities.”
To increase the number of signals they could see, the researchers set out to identify signals by location rather than color. They modified the existing journalists to make them accumulate in clusters in different places of a cell. To do this, they added two small peptides to each reporter, which helped reporters form distinct groups within cells.
“It’s like Journalist X is tied to a LEGO brick and Reporter Z is tied to a K’NEX piece – only the LEGO bricks will stick to other LEGO bricks, resulting in the lone Journalist X being regrouped with more X reporter, ”says Johnson. .
With this technique, each cell ends up with hundreds of groups of fluorescent reporters. After measuring the activity of each cluster under a microscope, based on the changing fluorescence, researchers can identify which molecule was measured in each cluster by preserving the cell and staining the peptide markers unique to each reporter. Peptide tags are invisible in the living cell, but they can be stained and seen after live imaging. This allows researchers to distinguish signals from different molecules even though they can all fluoresce the same color in the living cell.
Using this approach, the researchers showed that 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 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 forming new memories. .
By applying this imaging technique to pyramidal neurons in the hippocampus, researchers identified two new subpopulations with different calcium signaling dynamics. One population showed slow calcium responses. In the other population, neurons had faster calcium responses. This latter population had greater PKA responses. Researchers believe this increased response can help maintain lasting changes in neurons.
Imaging of signaling networks
The researchers now plan to try this approach in living animals to study the link between the activities of the signaling network and behavior and to extend it to other cell types, such as immune cells. This technique could also be useful for comparing signaling network patterns between healthy and diseased tissue cells.
In this article, the researchers showed that they could record five different molecular signals at once, and that by changing their existing strategy, they thought they could reach 16. With more work, that number could reach hundreds, they say. .
“It could really help resolve some of these difficult questions about how the parts of a cell work together,” Boyden says. “You could imagine a time when we can look at everything that goes on in a living cell, or at least the part involved in learning, or with disease, or with the treatment of a disease.
The research was funded by the Friends of the McGovern Institute scholarship; the J. Douglas Tan scholarship; Lisa Yang; the Yang-Tan Center for Molecular Therapy; John Doerr; the Open Philanthropy project; the HHMI-Simons Faculty Fellows Program; the science program on human frontiers; the US Army Research Laboratory; the MIT Media Lab; the Picower Institute Innovation Fund; the National Institutes of Health, including an NIH Director’s Pioneer Award; and the National Science Foundation.
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