Scientists use light to control the logic networks of a cell
New technique illuminates role of previously inaccessible proteins involved in health and disease
Proteins are the workhorse molecules of life. Among their many jobs, they carry oxygen, build tissue, copy DNA for the next generation, and coordinate events within and between cells. Now scientists at the University of North Carolina at Chapel Hill have developed a method to control proteins inside live cells with the flick of a switch, giving researchers an unprecedented tool for pinpointing the causes of disease using the simplest of tools: light.
The work, led by Klaus Hahn and Nikolay Dokholyan and spearheaded by Onur Dagliyan, a graduate student in their labs, builds on the breakthrough technology known as optogenetics. The technique, developed in the early 2000s, allowed scientists, for the first time, to use light to activate and deactivate proteins that could turn brain cells on and off, refining ideas of what individual brain circuits do and how they relate to different aspects of behavior and personality.
But the technique has had its limitations. Only a few proteins could be controlled by light; they were put in parts of a cell where they normally didn’t exist; and they had been heavily engineered, losing much of their original ability to detect and respond to their environment.
In their new work, published recently in Science, Hahn, Dokholyan and Dagliyan expand optogenetics to control a wide range of proteins without changing their function, allowing a light-controllable protein to carry out its everyday chores. The proteins can be turned on almost anywhere in the cell, enabling the researchers to see how proteins do very different jobs depending on where they are turned on and off.
“We can take the whole, intact protein, just the way nature made it, and stick this little knob on it that allows us to turn it on and off with light,” said Hahn, Thurman Distinguished Professor of Pharmacology and a UNC Lineberger Comprehensive Cancer Center member. “It’s like a switch.”
The switch that Hahn, Dokholyan and colleagues developed is versatile and fast – they can toggle a protein on or off as fast as they can toggle their light. By changing the intensity of light, they can also control how much of the protein is activated or inactivated. And by controlling the timing of irradiation, they can control exactly how long proteins are activated at different points in the cell.
“A lot of aspects of cell behavior depend on transient, fast changes in protein activity,” said Hahn. “But those changes have to happen in exact locations. The same protein can cause a cell to do different things if it’s active in different places, building flexible logic networks in different parts of the cell, depending on what it is responding to.”
To make their breakthrough, Hahn and Dagliyan used a computational approach to identify which parts of a protein could be modified without changing the protein’s normal operation, and showed that loops of protein structure commonly found on protein surfaces can be readily modified with different ‘knobs’ to control proteins with light, or even to respond to drugs.
Imagine sticking a video camera on a bus; put it on the gas pedal and it will obstruct its function, so the bus will not drive properly. But put it on the hood, and the bus will continue to drive just fine. The new computational approach pointed the researchers toward each protein’s hood.
Because the tools keep the natural protein function intact, the new technique allows scientists to study proteins in living systems, where proteins normally live and work in all their natural complexity. This ability to manipulate proteins in living systems also provides an opportunity to study a wide range of diseases, which often arise from the malfunctioning of a single protein.
“In order to understand what’s happening you need to see the parts moving around,” said Hahn. “It’s that dynamic behavior that you need to know to understand what’s going on.”
New chip could help test drugs for ALS, other neuromuscular disorders.
MIT engineers have developed a microfluidic device that replicates the neuromuscular junction — the vital connection where nerve meets muscle. The device, about the size of a U.S. quarter, contains a single muscle strip and a small set of motor neurons. Researchers can influence and observe the interactions between the two, within a realistic, three-dimensional matrix.
The researchers genetically modified the neurons in the device to respond to light. By shining light directly on the neurons, they can precisely stimulate these cells, which in turn send signals to excite the muscle fiber. The researchers also measured the force the muscle exerts within the device as it twitches or contracts in response.
The team’s results, published online today in Science Advances, may help scientists understand and identify drugs to treat amyotrophic lateral sclerosis (ALS), more commonly known as Lou Gehrig’s disease, as well as other neuromuscular-related conditions.
“The neuromuscular junction is involved in a lot of very incapacitating, sometimes brutal and fatal disorders, for which a lot has yet to be discovered,” says Sebastien Uzel, who led the work as a graduate student in MIT’s Department of Mechanical Engineering. “The hope is, being able to form neuromuscular junctions in vitro will help us understand how certain diseases function.”
Uzel’s coauthors include Roger Kamm, the Cecil and Ida Green Distinguished Professor of Mechanical and Biological Engineering at MIT, along with former graduate student and now postdoc Randall Platt, research scientist Vidya Subramanian, former undergraduate researcher Taylor Pearl, senior postdoc Christopher Rowlands, former postdoc Vincent Chan, associate professor of biology Laurie Boyer, and professor of mechanical engineering and biological engineering Peter So.
Closing in on a counterpart
Since the 1970s, researchers have come up with numerous ways to simulate the neuromuscular junction in the lab. Most of these experiments involve growing muscle and nerve cells in shallow Petri dishes or on small glass substrates. But such environments are a far cry from the body, where muscles and neurons live in complex, three-dimensional environments, often separated over long distances.
“Think of a giraffe,” says Uzel, who is now a postdoc at the Wyss Institute at Harvard University. “Neurons that live in the spinal cord send axons across very large distances to connect with muscles in the leg.”
To recreate more realistic in vitro neuromuscular junctions, Uzel and his colleagues fabricated a microfluidic device with two important features: a three-dimensional environment, and compartments that separate muscles from nerves to mimic their natural separation in the human body. The researchers suspended muscle and neuron cells in the millimeter-sized compartments, which they then filled with gel to mimic a three-dimensional environment.
A flash and a twitch
To grow a muscle fiber, the team used muscle precursor cells obtained from mice, which they then differentiated into muscle cells. They injected the cells into the microfluidic compartment, where the cells grew and fused to form a single muscle strip. Similarly, they differentiated motor neurons from a cluster of stem cells, and placed the resulting aggregate of neural cells in the second compartment. Before differentiating both cell types, the researchers genetically modified the neural cells to respond to light, using a now-common technique known as optogenetics.
Kamm says light “gives you pinpoint control of what cells you want to activate,” as opposed to using electrodes, which, in such a confined space, can inadvertently stimulate cells other than the targeted neural cells.
Finally, the researchers added one more feature to the device: force sensing. To measure muscle contraction, they fabricated two tiny, flexible pillars within the muscle cells’ compartment, around which the growing muscle fiber could wrap. As the muscle contracts, the pillars squeeze together, creating a displacement that researchers can measure and convert to mechanical force.
In experiments to test the device, Uzel and his colleagues first observed neurons extending axons toward the muscle fiber within the three-dimensional region. Once they observed that an axon had made a connection, they stimulated the neuron with a tiny burst of blue light and instantly observed a muscle contraction.
“You flash a light, you get a twitch,” Kamm says.
Judging from these experiments, Kamm says the microfluidic device may serve as a fruitful testing ground for drugs to treat neuromuscular disorders, and could even be tailored to individual patients.
“You could potentially take pluripotent cells from an ALS patient, differentiate them into muscle and nerve cells, and make the whole system for that particular patient,” Kamm says. “Then you could replicate it as many times as you want, and try different drugs or combinations of therapies to see which is most effective in improving the connection between nerves and muscles.”
On the flip side, he says the device may be useful in “modeling exercise protocols.” For instance, by stimulating muscle fibers at varying frequencies, scientists can study how repeated stress affects muscle performance.
“Now with all these new microfluidic approaches people are developing, you can start to model more complex systems with neurons and muscles,” Kamm says. “The neuromuscular junction is another unit people can now incorporate into those testing modalities.”
Device shown capable of reading and writing neural signals at the spatial and temporal scale of natural brain activity, could eventually serve as “Rosetta Stone” to crack the code on how brains work
For the first time, researchers have developed a microscope capable of observing—and manipulating—neural activity in the brains of live animals at the scale of a single cell with millisecond precision. By allowing scientists to directly control the firing of individual neurons within complex brain circuits, the device could ultimately revolutionize how neuroscience is done and lead to new insights about healthy brain functioning and neurological disorders.
“With this new microscope, we believe we will soon be able to treat the brain as the keyboard of a piano, so to speak, and write in a sequence of activity that is needed to understand or correct brain function,” said Hillel Adesnik, Ph.D., assistant professor of neurobiology at the University of California, Berkeley, who led the research team. “After more refinements, this instrument may be able to function as a sort of Rosetta Stone to help us crack the neural code.”
Adesnik will present this research at the American Association of Anatomists Annual Meeting duringExperimental Biology 2016. He has been awarded the American Association of Anatomists 2016 C.J. Herrick Award in Neuroanatomy.
To process inputs, store information and issue commands, the brain’s neurons communicate with each other through on-off electrical signals akin to the ones and zeroes used to encode information in computer programming. Although scientists have long been able to observe these signals with various imaging techniques, without understanding the “syntax” of how that digital code translates into information, the brain’s communication system has been essentially indecipherable.
“If you want to learn a language, you need a dictionary, and if you want to understand how a machine works, you need to know its parts,” said Adesnik. “We wanted to develop a technology that can offer a general approach to understand the basic syntax of neural signals, so that we can begin to understand what a given brain circuit is doing and perhaps what’s gone wrong with that in the case of a disease.”
The best way to learn that syntax, Adesnik said, is to not simply read the information, but to actually write it by making small tweaks in the code, inputting the new code back into the brain and seeing how it alters a perception or behavior. The new microscope, which Adesnik’s team developed by combining and building upon several existing technologies developed by other researchers, is the first to be able to handle and transmit information at a spatial and temporal scale that is truly relevant to manipulating brain activity.
“The brain is an enormous collection of single cells, and cells right next to each other could be doing entirely different things,” Adesnik said. “The resolution of our technique is key, because if you aren’t looking at a single cell you could be scrambling your code, so to speak, and you won’t be able to correctly interpret it. By overcoming the last technological hurdles to get to that single cell resolution, and at the same time getting to the temporal scale that cells operate at, we have developed a prototype microscope that achieves the level of detail needed to actually understand the neural code.”
The tool they have devised is essentially a microscope that points into the brain of a live mouse, zooms in on a few thousand cells and uses sophisticated lasers to manipulate electrical signals between individual neurons.
Since the lasers can penetrate brain tissue but not skull, the research team implanted small glass windows into the skulls of the mice used to test the instrument. When positioned atop the window, the microscope uses two different types of high-powered infrared lasers to create a 3-dimensional holographic pattern in a specific area of interest within the brain. Because the research is done in mice genetically modified to have neurons that respond to light—a technique called optogenetics—the hologram induces the neurons to send electrical signals in a specific pattern that is pre-determined by the researchers.
“We’re adapting holographic display technology, optogenetics and sensory biology and behavior into one complete system that allows an all-optical approach to image and manipulate the nervous system,” said Adesnik. “We’ve essentially put a lot of disparate existing pieces together to achieve something nobody had yet achieved.”
So far, the team has conducted preliminary tests of the instrument by mapping the effects of small perturbations, such as wiggling a whisker, and then creating holograms that induce the neurons to fire in the same—or slightly different—patterns. In a series of tests that are still underway, they are working with mice trained to push a specific lever when they see a certain shape in order to develop holograms that “trick” the mouse into seeing, for example, a circle where none exists, or to make the mouse perceive a square as a circle. In the near future, the team hopes to apply the microscope to studies of memory formation.
Once it is further tested and refined, the most immediate applications for the microscope are likely to be in basic research, but Adesnik said it is conceivable that its core technology could one day be adapted for therapeutic use, for example, to correct neurological problems in a high-tech form of brain surgery. Such an application is still a long way off, however, and applying the device in human beings would require overcoming a whole new set of technological challenges.