Exploring the unknown

View the interactive version of this story in our Summer 2021 issue of BrainScan.

 

McGovern Investigator Ed Boyden.

McGovern Investigator Ed Boyden says his lab’s vision is clear.

“We want to understand how our brains take our sensory inputs, generate emotions and memories and decisions, and ultimately result in motor outputs. We want to be able to see the building blocks of life, and how they go into disarray in brain diseases. We want to be able to control the signals of the brain, so we can repair it,” Boyden says.

To get there, he and his team are exploring the brain’s complexity at every scale, from the function and architecture of its neural networks to the molecules that work together to process information.

And when they don’t have the tools to take them where they want to go, they create them, opening new frontiers for neuroscientists everywhere.

Open to discovery

Boyden’s team is highly interdisciplinary and collaborative. Its specialty, Boyden says, is problem solving. Creativity, adaptability, and deep curiosity are essential, because while many of neuroscience’s challenges are clear, the best way to address them is not. In its search for answers, Boyden’s lab is betting that an important path to discovery begins with finding new ways to explore.

They’ve made that possible with an innovative imaging approach called expansion microscopy (ExM). ExM physically enlarges biological samples so that minute details become visible under a standard laboratory microscope, enabling researchers everywhere to peer into spaces that once went unseen (see video below).

To use the technique, researchers permeate a biological sample with an absorbent gel, then add water, causing the components of the gel to spread apart and the tissue to expand.

This year, postdoctoral researcher Ruixuan Gao and graduate student Chih-Chieh (Jay) Yu made the method more precise, with a new material that anchors a sample’s molecules within a crystal-like lattice, better preserving structure during expansion than the irregular mesh-like composition of the original gel. The advance is an important step toward being able to image expanded samples with single-molecule precision, Gao says.

A revealing look

By opening space within the brain, ExM has let Boyden’s team venture into those spaces in new ways.

Areas of research and brain disorders page
Graduate student Oz Wassie examines expanded brain tissue. Photo: Justin Knight

In work led by Deblina Sarkar (who is now an assistant professor at MIT’s Media Lab), Jinyoung Kang, and Asmamaw (Oz) Wassie, they showed that they can pull apart proteins in densely packed regions like synapses so that it is easier to introduce fluorescent labels, illuminating proteins that were once too crowded to see. The process, called expansion revealing, has made it possible to visualize in intact brain tissue important structures such as ion channels that help transmit signals and fine-scale amyloid clusters in Alzheimer’s model mice.

Another reaction the lab has adapted to the expanded-brain context is RNA sequencing—an important tool for understanding cellular diversity. “Typically, the first thing you do in a sequencing project is you grind up the tissue, and you lose the spatial dimension,” explains Daniel Goodwin, a graduate student in Boyden’s lab. But when sequencing reactions are performed inside cells instead, new information is revealed.

Confocal image showing targeted ExSeq of a 34-panel gene set across a slice of mouse hippocampus. Green indicates YFP, magenta indicates reads identified with ExSeq, and white indicates reads localized within YFP-expressing cells. Image courtesy of the researchers.

Goodwin and fellow Boyden lab members Shahar Alon, Anubhav Sinha, Oz Wassie, and Fei Chen developed expansion sequencing (ExSeq), which copies RNA molecules, nucleotide by nucleotide, directly inside expanded tissue, using fluorescent labels that spell out the molecules’ codes just as they would in a sequencer.

The approach shows researchers which genes are turned on in which cells, as well as where those RNA molecules are—revealing, for example, which genes are active in the neuronal projections that carry out the brain’s communications. A next step, Sinha says, is to integrate expansion sequencing with other technologies to obtain even deeper insights.

That might include combining information revealed with ExSeq with a topographical map of the same cells’ genomes, using a method Boyden’s lab and collaborators Chen (who is now a core member of the Broad Institute) and Jason Buenrostro at Harvard have developed for DNA sequencing. That information is important because the shape of the genome varies across cells and circumstances, and that has consequences for how the genetic code is used.

Using similar techniques to those that make ExSeq possible, graduate students Andrew Payne, Zachary Chiang, and Paul Reginato figured out how to recreate the steps of commercial DNA sequencing within the genome’s natural environment.

By pinpointing the location of specific DNA sequences inside cells, the new method, called in situ genome sequencing (IGS) allows researchers to watch a genome reorganize itself in a developing embryo.

They haven’t yet performed this analysis inside expanded tissue, but Payne says integrating in situ genome sequencing (IGS) with ExM should open up new opportunities to study genomes’ structure.

Signaling clusters

Alongside these efforts, Boyden’s team is working to give researchers better tools to explore how molecules move, change, and interact, including a modular system that lets users assemble sets of sensors into clusters to simultaneously monitor multiple cellular activities.

Molecular sensors use fluorescence to report on certain changes inside cells, such as the calcium that surges into a neuron after it fires. But they come in a limited palette, so in most experiments only one or two things can be seen at once.

Graduate student Shannon Johnson and postdoctoral fellow Changyang Linghu solved this problem by putting different sensors at different points throughout a cell so they can report on different signals. Their technique, called spatial multiplexing, links sensors to molecular scaffolds designed to cling to their own kind. Sensors built on the same scaffold form islands inside cells, so when they light up their signals are distinct from those produced by other sensor islands.

Eventually, as new sensors and scaffolds become available, Johnson says the technique might be used to simultaneously follow dozens of molecular signals in living cells. The more precise information they can help people uncover, the better, Boyden says.

“The brain is so full of surprises, we don’t know where the next big discovery will come from,” he says. With new support from the recently established K. Lisa Yang and Hock E. Tan Center for Molecular Therapeutics in Neuroscience, the Boyden lab is positioned to make these big discoveries.

“My dream would be to image the signaling dynamics of the brain, and then perturb the dynamics, and then use expansion methods to make a map of the brain. If we can get those three data sets—the dynamics, the causality, and the molecular organization—I think stitching those together could potentially yield deep insights into how the brain works, and how we can repair it in disease states.”

Method offers inexpensive imaging at the scale of virus particles

Using an ordinary light microscope, MIT engineers have devised a technique for imaging biological samples with accuracy at the scale of 10 nanometers — which should enable them to image viruses and potentially even single biomolecules, the researchers say.

The new technique builds on expansion microscopy, an approach that involves embedding biological samples in a hydrogel and then expanding them before imaging them with a microscope. For the latest version of the technique, the researchers developed a new type of hydrogel that maintains a more uniform configuration, allowing for greater accuracy in imaging tiny structures.

This degree of accuracy could open the door to studying the basic molecular interactions that make life possible, says Edward Boyden, the Y. Eva Tan Professor in Neurotechnology, a professor of biological engineering and brain and cognitive sciences at MIT, and a member of MIT’s McGovern Institute for Brain Research and Koch Institute for Integrative Cancer Research.

“If you could see individual molecules and identify what kind they are, with single-digit-nanometer accuracy, then you might be able to actually look at the structure of life.”

“And structure, as a century of modern biology has told us, governs function,” says Boyden, who is the senior author of the new study.

The lead authors of the paper, which appears today in Nature Nanotechnology, are MIT Research Scientist Ruixuan Gao and Chih-Chieh “Jay” Yu PhD ’20. Other authors include Linyi Gao PhD ’20; former MIT postdoc Kiryl Piatkevich; Rachael Neve, director of the Gene Technology Core at Massachusetts General Hospital; James Munro, an associate professor of microbiology and physiological systems at University of Massachusetts Medical School; and Srigokul Upadhyayula, a former assistant professor of pediatrics at Harvard Medical School and an assistant professor in residence of cell and developmental biology at the University of California at Berkeley.

Low cost, high resolution

Many labs around the world have begun using expansion microscopy since Boyden’s lab first introduced it in 2015. With this technique, researchers physically enlarge their samples about fourfold in linear dimension before imaging them, allowing them to generate high-resolution images without expensive equipment. Boyden’s lab has also developed methods for labeling proteins, RNA, and other molecules in a sample so that they can be imaged after expansion.

“Hundreds of groups are doing expansion microscopy. There’s clearly pent-up demand for an easy, inexpensive method of nanoimaging,” Boyden says. “Now the question is, how good can we get? Can we get down to single-molecule accuracy? Because in the end, you want to reach a resolution that gets down to the fundamental building blocks of life.”

Other techniques such as electron microscopy and super-resolution imaging offer high resolution, but the equipment required is expensive and not widely accessible. Expansion microscopy, however, enables high-resolution imaging with an ordinary light microscope.

In a 2017 paper, Boyden’s lab demonstrated resolution of around 20 nanometers, using a process in which samples were expanded twice before imaging. This approach, as well as the earlier versions of expansion microscopy, relies on an absorbent polymer made from sodium polyacrylate, assembled using a method called free radical synthesis. These gels swell when exposed to water; however, one limitation of these gels is that they are not completely uniform in structure or density. This irregularity leads to small distortions in the shape of the sample when it’s expanded, limiting the accuracy that can be achieved.

To overcome this, the researchers developed a new gel called tetra-gel, which forms a more predictable structure. By combining tetrahedral PEG molecules with tetrahedral sodium polyacrylates, the researchers were able to create a lattice-like structure that is much more uniform than the free-radical synthesized sodium polyacrylate hydrogels they previously used.

Three-dimensional (3D) rendered movie of envelope proteins of an herpes simplex virus type 1 (HSV-1) virion expanded by tetra-gel (TG)-based three-round iterative expansion. The deconvolved puncta (white), the overlay of the deconvolved puncta (white) and the fitted centroids (red), and the extracted centroids (red) are shown from left to right. Expansion factor, 38.3×. Scale bars, 100 nm.
Credit: Ruixuan Gao and Boyden Lab

The researchers demonstrated the accuracy of this approach by using it to expand particles of herpes simplex virus type 1 (HSV-1), which have a distinctive spherical shape. After expanding the virus particles, the researchers compared the shapes to the shapes obtained by electron microscopy and found that the distortion was lower than that seen with previous versions of expansion microscopy, allowing them to achieve an accuracy of about 10 nanometers.

“We can look at how the arrangements of these proteins change as they are expanded and evaluate how close they are to the spherical shape. That’s how we validated it and determined how faithfully we can preserve the nanostructure of the shapes and the relative spatial arrangements of these molecules,” Ruixuan Gao says.

Single molecules

The researchers also used their new hydrogel to expand cells, including human kidney cells and mouse brain cells. They are now working on ways to improve the accuracy to the point where they can image individual molecules within such cells. One limitation on this degree of accuracy is the size of the antibodies used to label molecules in the cell, which are about 10 to 20 nanometers long. To image individual molecules, the researchers would likely need to create smaller labels or to add the labels after expansion was complete.

Left, HeLa cell with two-color labeling of clathrin-coated pits/vesicles and microtubules, expanded by TG-based two-round iterative expansion. Expansion factor, 15.6×. Scale bar, 10 μm (156 μm). Right, magnified view of the boxed region for each color channel. Scale bars, 1 μm (15.6 μm). Image: Boyden Lab

They are also exploring whether other types of polymers, or modified versions of the tetra-gel polymer, could help them realize greater accuracy.

If they can achieve accuracy down to single molecules, many new frontiers could be explored, Boyden says. For example, scientists could glimpse how different molecules interact with each other, which could shed light on cell signaling pathways, immune response activation, synaptic communication, drug-target interactions, and many other biological phenomena.

“We’d love to look at regions of a cell, like the synapse between two neurons, or other molecules involved in cell-cell signaling, and to figure out how all the parts talk to each other,” he says. “How do they work together and how do they go wrong in diseases?”

The research was funded by Lisa Yang, John Doerr, Open Philanthropy, the National Institutes of Health, the Howard Hughes Medical Institute Simons Faculty Scholars Program, the Intelligence Advanced Research Projects Activity, the U.S. Army Research Laboratory, the US-Israel Binational Science Foundation, the National Science Foundation, the Friends of the McGovern Fellowship, and the Fellows program of the Image and Data Analysis Core at Harvard Medical School.

A high-resolution glimpse of gene expression in cells

Using a novel technique for expanding tissue, MIT and Harvard Medical School researchers have devised a way to label individual molecules of messenger RNA within a tissue sample and then sequence the RNA.

This approach offers a unique snapshot of which genes are being expressed in different parts of a cell, and could allow scientists to learn much more about how gene expression is influenced by a cell’s location or its interactions with nearby cells. The technique could also be useful for mapping cells in the brain or other tissues and classifying them according to their function.

“Gene expression is one of the most fundamental processes in all of biology, and it plays roles in all biological processes, both healthy and disease-related. However, you need to know more than just whether a gene is on or off,” says Ed Boyden, the Y. Eva Tan Professor in Neurotechnology and a professor of biological engineering, media arts and sciences, and brain and cognitive sciences at MIT.

“You want to know where the gene products are located. You care what cell types they’re in, which individual cells they play roles in, and even which parts of cells they work in,” says Boyden.

In a study appearing today in Science, the researchers showed that they could use this technique to locate and then sequence thousands of different messenger RNA molecules within the mouse brain and in human tumor samples.

The senior authors of the study are Boyden, an investigator at the MIT McGovern Institute and the Howard Hughes Medical Institute; George Church, a professor of genetics at Harvard Medical School; and Adam Marblestone, a former MIT research scientist. The paper’s lead authors are Shahar Alon, a former MIT postdoc who is now a senior lecturer at Bar-Ilan University; Daniel Goodwin, an MIT graduate student; Anubhav Sinha ’14 MNG ’15, an MIT graduate student; Asmamaw Wassie ’12, PhD ’19; and Fei Chen PhD ’17, who is an assistant professor of stem cell and regenerative biology at Harvard University and a member of the Broad Institute of MIT and Harvard.

Tissue expansion

The new sequencing technique builds on a method that Boyden’s group devised in 2015 for expanding tissue samples and then imaging them. By embedding water-absorbent polymers into a tissue sample, researchers can swell the tissue sample while keeping its overall organization intact. Using this approach, tissues can be expanded by a factor of 100 or more, allowing scientists to obtain very high-resolution images of the brain or other tissues using a regular light microscope.

In 2014, Church’s lab developed an RNA sequencing technique known as FISSEQ (fluorescent in situ sequencing), which allows thousands of mRNA molecules to be located and sequenced within cells grown in a lab dish. The Boyden and Church labs decided to join forces to combine tissue expansion and in situ RNA sequencing, creating a new technique they call expansion sequencing (ExSeq).

Expanding the tissue before performing RNA sequencing has two main benefits: It offers a higher-resolution look at the RNA in cells, and it makes it easier to sequence those RNA molecules. “When you separate these molecules in the expanding sample, and move them away from each other, that gives you more room to actually perform the chemical reactions of in situ sequencing,” Marblestone says.

Once the tissue is expanded, the researchers can label and sequence thousands of RNA molecules in a sample, at a resolution that allows them to pinpoint the molecules’ locations not only within cells but within specific compartments such as dendrites — the tiny extensions of neurons that receive communications from other neurons.

“We know that the location of RNA in these small regions is important for learning and memory, but until now, we didn’t have any way to measure these locations because they are very small, on the order of nanometers,” Alon says.

Using an “untargeted” version of this technique, meaning that they are not looking for specific RNA sequences, the researchers can turn up thousands of different sequences. They estimate that in a given sample, they can sequence between 20 and 50 percent of all of the genes present.

In the mouse hippocampus, this technique yielded some surprising results. For one, the researchers found mRNA containing introns, which are sections of RNA that are normally edited out of mRNA in the nucleus, in dendrites. They also discovered mRNA molecules encoding transcription factors in the dendrites, which may help with novel forms of dendrite-to-nucleus communication.

“These are just examples of things that we never would have gone looking for intentionally, but now that we can sequence RNA exactly where it is in the neuron, we’re able to explore a lot more biology,” Goodwin says.

Cellular interactions

The researchers also showed that they could explore gene expression in a more targeted way, looking for a specific set of RNA sequences that correspond to genes of interest. In the visual cortex of the mouse, the researchers used this approach to classify neurons into different types based on an analysis of 42 different genes that they express.

In situ sequencing of physically expanded specimens enables multiplexed mapping of RNAs at nanoscale, subcellular resolution throughout intact tissues. Top: schematics of physical expansion and in situ sequencing (left), and image analysis (right). Bottom: characterization of nanoscale transcriptomic compartmentalization in mouse hippocampal neuron dendrites and spines (left, middle), and maps of cell types and states in a metastatic human breast cancer biopsy (right). Image courtesy of the researchers.

This technology could also be useful to analyze many other kinds of tissues, such as tumor biopsies. In this paper, the researchers studied breast cancer metastases, which contain many different cell types, including cancer cells and immune cells. The study revealed that these cell types can behave differently depending on their location within a tumor. For example, the researchers found that B cells that were near tumor cells expressed certain inflammatory genes at a higher level than B cells that were farther from tumor cells.

“The tumor microenvironment has been studied in many different contexts for a long time, but it’s been difficult to study it with any depth,” Sinha says. “A cancer biologist can give you a list of 20 or 30 marker genes that will identify most of the cell types in the tissue. Here, since we interrogated 297 different RNA transcripts in the sample, we can ask and answer more detailed questions about gene expression.”

The researchers now plan to further study the interactions between cancer cells and immune cells, as well as gene expression in the brain in healthy and disease states. They also plan to extend their techniques to allow them to map additional types of biomolecules, such as proteins, alongside RNA.

The research was funded, in part, by the National Institutes of Health and the National Science Foundation, as well as by Lisa Yang, John Doerr, the Open Philanthropy Project, Cancer Research UK, the Chan Zuckerberg Initiative Human Cell Atlas pilot program, and HHMI.

Sequencing inside cells

By bringing DNA sequencing out of the sequencer and directly to cells, MIT scientists have revealed an entirely new view of the genome. With a new method for in situ genome sequencing reported December 31, 2020, in the journal Science, researchers can, for the first time, see exactly how DNA sequences are organized and packed inside cells.

The approach, whose development was led by Ed Boyden, the Y. Eva Tan Professor in Neurotechnology at MIT, and Harvard University Stem Cell and Regenerative Biology faculty members Jason Buenrostro and Fei Chen, integrates DNA sequencing technology with microscopy to pinpoint exactly where specific DNA sequences are located inside intact cells.

While alternative methods allow scientists to reconstruct structural information about the genome, this is the first sequencing technology to give scientists a direct look.

The technology creates new opportunities to investigate a broad range of biology, from fundamental questions about how DNA’s three-dimensional organization affects its function to the structural changes and chromosomal rearrangements associated with aging, cancer, brain disorders, and other diseases.

Seeing is believing

“How structure yields function is one of the core themes of biology,” says Boyden, who is also an investigator at the McGovern Institute and the Howard Hughes Medical Institute.“And the history of biology tells us that when you can actually see something, you can make lots of advances.” Seeing how an organism’s genome is packed inside its cells could help explain how different cell types in the brain interpret the genetic code, or reveal structural patterns that mean the difference between health and disease, he says. Additionally, the researchers note, the technique also makes it possible to directly see how proteins and other factors interact with specific parts of the genome.

The new method builds on work underway in Boyden and Chen’s laboratories focused on sequencing RNA inside cells. Buenrostro collaborated with Boyden and Chen, who is also a core member of the Broad Institute, to adapt the technique for use with DNA. “It was clear the technology they had developed would be an extraordinary opportunity to have a new perspective on cells’ genomes,” Boyden says.

Their approach begins by fixing cells onto a glass surface to preserve their structure. Then, after inserting small DNA adapters into the genome, thousands of short segments of DNA—about 20 letters of code apiece—are amplified and sequenced in their original locations inside the cells. Finally, the samples are ground up and put into a sequencer, which sequences all of the cells’ DNA about 300 letters at a time. By finding the location-identified short sequences within those longer segments, the method pinpoints each one’s position within the three-dimensional structure of the cell.

Sequencing inside the cells is done more or less the same way DNA is sequenced inside a standard next-generation sequencer, Boyden explains, by watching under a microscope as a DNA strand is copied using fluorescently labeled building blocks. As in a traditional sequencer, each of DNA’s four building blocks, or nucleotides, is tagged with a different color so that they can be visually identified as they are added to a growing DNA strand.

A collaborative effort

Boyden, Buenrostro, and Chen, who began their collaboration several years ago, say the new technology represents a heroic effort on the part of MIT and Harvard graduate students Andrew Payne, Zachary Chiang, and Paul Reginato, who took the lead in developing and integrating its many technical steps and computational analyses. That involved both recapitulating the methods used in commercial sequencers and introducing several key innovations. “Some advances on the technology side have taken this from impossible to do to being possible,” Chen says.

The team has already used the method to visualize a genome as it reorganizes itself during the earliest moments of life. Brightly colored representations of DNA that they sequenced inside a mouse embryo show how genetic information inherited from each parent remains distinct and compartmentalized immediately after fertilization, then gradually intertwines as development progresses. Their sequencing also reveals how patterns of genome organization, which very early in life vary from cell to cell, are passed on as cells divide, generating a memory of each cell’s developmental origins. Being able to watch these processes unfold across entire cells instead of piecing them together through less direct means offered a dramatic new view of development, the researchers say.

While the team continues to improve the spatial resolution of the technique and adapt it to a broader range of cell types, they have made their method and associated software freely available to other labs. The researchers hope this new approach to DNA sequencing will change the way people think about studying the structure of the genome and will help illuminate patterns and consequences of genome organization across a variety of contexts.

Storytelling brings MIT neuroscience community together

When the coronavirus pandemic shut down offices, labs, and classrooms across the MIT campus last spring, many members of the MIT community found it challenging to remain connected to one another in meaningful ways. Motivated by a desire to bring the neuroscience community back together, the McGovern Institute hosted a virtual storytelling competition featuring a selection of postdocs, grad students, and staff from across the institute.

“This has been an unprecedented year for us all,” says McGovern Institute Director Robert Desimone. “It has been twenty years since Pat and Lore McGovern founded the McGovern Institute, and despite the challenges this anniversary year has brought to our community, I have been inspired by the strength and perseverance demonstrated by our faculty, postdocs, students and staff. The resilience of this neuroscience community – and MIT as a whole – is indeed something to celebrate.”

The McGovern Institute had initially planned to hold a large 20th anniversary celebration in the atrium of Building 46 in the fall of 2020, but the pandemic made a gathering of this size impossible. The institute instead held a series of virtual events, including the November 12 story slam on the theme of resilience.

Imaging method reveals a “symphony of cellular activities”

Within 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 many others.

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

MIT researchers have now developed a way to image up to five different molecule types at a time, by measuring each signal from random, distinct locations throughout a cell.

This approach could allow scientists to learn much more about the complex signaling networks that control most cell functions, says Edward Boyden, the Y. Eva Tan Professor in Neurotechnology and a professor of biological engineering, media arts and sciences, and brain and cognitive sciences at MIT.

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

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

Fluorescent clusters

Shannon Johnson is a graduate fellow in the fellow in the Yang-Tan Center for Molecular Therapeutics.

To make molecular activity visible within a cell, scientists typically create reporters by fusing a protein that senses a target molecule to a protein that glows. “This is similar to how a smoke detector will sense smoke and then flash a light,” says Johnson, who is also a fellow in 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 of the reporters out there are green, because they’re based on the green fluorescent protein,” Boyden says. “What has been lacking until now is the ability to see more than a couple of these signals at once.”

“Just like listening to the sound of a single instrument from an orchestra is far from enough to fully appreciate a symphony,” Linghu says, “by enabling observations of multiple cellular signals at the same time, our technology will help us understand the ‘symphony’ of cellular activities.”

To boost the number of signals they could see, the researchers set out to identify signals by location instead of by color. They modified existing reporters to cause them to accumulate in clusters at different locations within a cell. They did this by adding two small peptides to each reporter, which helped the reporters form distinct clusters within cells.

“It’s like having reporter X be tethered to a LEGO brick, and reporter Z tethered to a K’NEX piece — only LEGO bricks will snap to other LEGO bricks, causing only reporter X to be clustered with more of reporter X,” Johnson says.

Changyang Linghu is the J. Douglas Tan Postdoctoral Fellow in the Hock E. Tan and K. Lisa Yang Center for Autism Research.

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

Using this approach, the researchers showed that they could see five different molecular signals in a single cell. To demonstrate the potential usefulness 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 with many different cellular functions throughout the body. In neurons, it plays an important role in translating a short-term input (from upstream neurons) into long-term changes such as strengthening the connections between neurons — a process that is necessary for learning and forming new memories.

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

Imaging signaling networks

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

In this paper, the researchers showed they could record five different molecular signals at once, and by modifying their existing strategy, they believe they could get up to 16. With additional work, that number could reach into the hundreds, they say.

“That really might help crack open some of these tough questions about how the parts of a cell work together,” Boyden says. “One might imagine an era when we can watch everything going on in a living cell, or at least the part involved with learning, or with disease, or with the treatment of 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 U.S. 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.

RNA “ticker tape” records gene activity over time

As cells grow, divide, and respond to their environment,  their gene expression changes; one gene may be transcribed into more RNA at one time point and less at another time when it’s no longer needed. Now, researchers at the McGovern Institute, Harvard, and the Broad Institute of MIT and Harvard have developed a way to determine when specific RNA molecules are produced in cells.  The method, described today in Nature Biotechnology, allows scientists to more easily study how a cell’s gene expression fluctuates over time.

“Biology is very dynamic but most of the tools we use in biology are static; you get a fixed snapshot of what’s happening in a cell at a given moment,” said Fei Chen, a core institute member at the Broad, an assistant professor at Harvard University, and a co-senior author of the new work. “This will now allow us to record what’s happening over hours or days.”

To find out the level of RNA a cell is transcribing, researchers typically extract genetic material from the cell—destroying the cell in the process—and use RNA sequencing technology to determine which genes are being transcribed into RNA, and how much. Although researchers can sample cells at various times, they can’t easily measure gene expression at multiple time points.

To create a more precise timestamp, the team added strings of repetitive DNA bases to genes of interest in cultured human cells. These strings caused the cell to add repetitive regions of adenosine molecules—one of four building blocks of RNA — to the ends of RNA when the RNA was transcribed from these genes. The researchers also introduced an engineered version of an enzyme called adenosine deaminase acting on RNA (ADAR2cd), which slowly changed the adenosine molecules to a related molecule, inosine, at a predictable rate in the RNA. By measuring the ratio of inosines to adenosines in the timestamped section of any given RNA molecule, the researchers could elucidate when it was first produced, while keeping cells intact.

“It was pretty surprising to see how well this worked as a timestamp,” said Sam Rodriques, a co-first author of the new paper and former MIT graduate student who is now founding the Applied Biotechnology Laboratory at the Crick Institute in London. “And the more molecules you look at, the better your temporal resolution.”

Using their method, the researchers could estimate the age of a single timestamped RNA molecule to within 2.7 hours. But when they looked simultaneously at four RNA molecules, they could estimate the age of the molecules to within 1.5 hours. Looking at 200 molecules at once allowed the scientists to correctly sort RNA molecules into groups based on their age, or order them along a timeline with 86 percent accuracy.

“Extremely interesting biology, such as immune responses and development, occurs over a timescale of hours,” said co-first author of the paper Linlin Chen of the Broad. “Now we have the opportunity to better probe what’s happening on this timescale.”

The researchers found that the approach, with some small tweaks, worked well on various cell types — neurons, fibroblasts and embryonic kidney cells. They’re planning to now use the method to study how levels of gene activity related to learning and memory change in the hours after a neuron fires.

The current system allows researchers to record changes in gene expression over half a day. The team is now expanding the time range over which they can record gene activity, making the method more precise, and adding the ability to track several different genes at a time.

“Gene expression is constantly changing in response to the environment,” said co-senior author Edward Boyden of MIT, the McGovern Institute for Brain Research, and the Howard Hughes Medical Institute. “Tools like this will help us eavesdrop on how cells evolve over time, and help us pinpoint new targets for treating diseases.”

Support for the research was provided by the National Institutes of Health, the Schmidt Fellows Program at Broad Institute, the Burroughs Wellcome Fund, John Doerr, the Open Philanthropy Project, the HHMI-Simons Faculty Scholars Program, the U. S. Army Research Laboratory and the U. S. Army Research Office, the MIT Media Lab, Lisa Yang, the Hertz Graduate Fellowship and the National Science Foundation Graduate Research Fellowship Program.

New molecular therapeutics center established at MIT’s McGovern Institute

More than one million Americans are diagnosed with a chronic brain disorder each year, yet effective treatments for most complex brain disorders are inadequate or even nonexistent.

A major new research effort at MIT’s McGovern Institute aims to change how we treat brain disorders by developing innovative molecular tools that precisely target dysfunctional genetic, molecular, and circuit pathways.

The K. Lisa Yang and Hock E. Tan Center for Molecular Therapeutics in Neuroscience was established at MIT through a $28 million gift from philanthropist Lisa Yang and MIT alumnus Hock Tan ’75. Yang is a former investment banker who has devoted much of her time to advocacy for individuals with disabilities and autism spectrum disorders. Tan is President and CEO of Broadcom, a global technology infrastructure company. This latest gift brings Yang and Tan’s total philanthropy to MIT to more than $72 million.

Lisa Yang (center) and MIT alumnus Hock Tan ’75 with their daughter Eva (far left) pictured at the opening of the Hock E. Tan and K. Lisa Yang Center for Autism Research in 2017. Photo: Justin Knight

“In the best MIT spirit, Lisa and Hock have always focused their generosity on insights that lead to real impact,” says MIT President L. Rafael Reif. “Scientifically, we stand at a moment when the tools and insights to make progress against major brain disorders are finally within reach. By accelerating the development of promising treatments, the new center opens the door to a hopeful new future for all those who suffer from these disorders and those who love them. I am deeply grateful to Lisa and Hock for making MIT the home of this pivotal research.”

Engineering with precision

Research at the K. Lisa Yang and Hock E. Tan Center for Molecular Therapeutics in Neuroscience will initially focus on three major lines of investigation: genetic engineering using CRISPR tools, delivery of genetic and molecular cargo across the blood-brain barrier, and the translation of basic research into the clinical setting. The center will serve as a hub for researchers with backgrounds ranging from biological engineering and genetics to computer science and medicine.

“Developing the next generation of molecular therapeutics demands collaboration among researchers with diverse backgrounds,” says Robert Desimone, McGovern Institute Director and Doris and Don Berkey Professor of Neuroscience at MIT. “I am confident that the multidisciplinary expertise convened by this center will revolutionize how we improve our health and fight disease in the coming decade. Although our initial focus will be on the brain and its relationship to the body, many of the new therapies could have other health applications.”

There are an estimated 19,000 to 22,000 genes in the human genome and a third of those genes are active in the brain–the highest proportion of genes expressed in any part of the body.

Variations in genetic code have been linked to many complex brain disorders, including depression and Parkinson’s. Emerging genetic technologies, such as the CRISPR gene editing platform pioneered by McGovern Investigator Feng Zhang, hold great potential in both targeting and fixing these errant genes. But the safe and effective delivery of this genetic cargo to the brain remains a challenge.

Researchers within the new Yang-Tan Center will improve and fine-tune CRISPR gene therapies and develop innovative ways of delivering gene therapy cargo into the brain and other organs. In addition, the center will leverage newly developed single cell analysis technologies that are revealing cellular targets for modulating brain functions with unprecedented precision, opening the door for noninvasive neuromodulation as well as the development of medicines. The center will also focus on developing novel engineering approaches to delivering small molecules and proteins from the bloodstream into the brain. Desimone will direct the center and some of the initial research initiatives will be led by Associate Professor of Materials Science and Engineering Polina Anikeeva; Ed Boyden, the Y. Eva Tan Professor in Neurotechnology at MIT; Guoping Feng, the James W. (1963) and Patricia T. Poitras Professor of Brain and Cognitive Sciences at MIT; and Feng Zhang, James and Patricia Poitras Professor of Neuroscience at MIT.

Building a research hub

“My goal in creating this center is to cement the Cambridge and Boston region as the global epicenter of next-generation therapeutics research. The novel ideas I have seen undertaken at MIT’s McGovern Institute and Broad Institute of MIT and Harvard leave no doubt in my mind that major therapeutic breakthroughs for mental illness, neurodegenerative disease, autism and epilepsy are just around the corner,” says Yang.

Center funding will also be earmarked to create the Y. Eva Tan Fellows program, named for Tan and Yang’s daughter Eva, which will support fellowships for young neuroscientists and engineers eager to design revolutionary treatments for human diseases.

“We want to build a strong pipeline for tomorrow’s scientists and neuroengineers,” explains Hock Tan. “We depend on the next generation of bright young minds to help improve the lives of people suffering from chronic illnesses, and I can think of no better place to provide the very best education and training than MIT.”

The molecular therapeutics center is the second research center established by Yang and Tan at MIT. In 2017, they launched the Hock E. Tan and K. Lisa Yang Center for Autism Research, and, two years later, they created a sister center at Harvard Medical School, with the unique strengths of each institution converging toward a shared goal: understanding the basic biology of autism and how genetic and environmental influences converge to give rise to the condition, then translating those insights into novel treatment approaches.

All tools developed at the molecular therapeutics center will be shared globally with academic and clinical researchers with the goal of bringing one or more novel molecular tools to human clinical trials by 2025.

“We are hopeful that our centers, located in the heart of the Cambridge-Boston biotech ecosystem, will spur further innovation and fuel critical new insights to our understanding of health and disease,” says Yang.

 

A focused approach to imaging neural activity in the brain

When neurons fire an electrical impulse, they also experience a surge of calcium ions. By measuring those surges, researchers can indirectly monitor neuron activity, helping them to study the role of individual neurons in many different brain functions.

One drawback to this technique is the crosstalk generated by the axons and dendrites that extend from neighboring neurons, which makes it harder to get a distinctive signal from the neuron being studied. MIT engineers have now developed a way to overcome that issue, by creating calcium indicators, or sensors, that accumulate only in the body of a neuron.

“People are using calcium indicators for monitoring neural activity in many parts of the brain,” says Edward Boyden, the Y. Eva Tan Professor in Neurotechnology and a professor of biological engineering and of brain and cognitive sciences at MIT. “Now they can get better results, obtaining more accurate neural recordings that are less contaminated by crosstalk.”

To achieve this, the researchers fused a commonly used calcium indicator called GCaMP to a short peptide that targets it to the cell body. The new molecule, which the researchers call SomaGCaMP, can be easily incorporated into existing workflows for calcium imaging, the researchers say.

Boyden is the senior author of the study, which appears today in Neuron. The paper’s lead authors are Research Scientist Or Shemesh, postdoc Changyang Linghu, and former postdoc Kiryl Piatkevich.

Molecular focus

The GCaMP calcium indicator consists of a fluorescent protein attached to a calcium-binding protein called calmodulin, and a calmodulin-binding protein called M13 peptide. GCaMP fluoresces when it binds to calcium ions in the brain, allowing researchers to indirectly measure neuron activity.

“Calcium is easy to image, because it goes from a very low concentration inside the cell to a very high concentration when a neuron is active,” says Boyden, who is also a member of MIT’s McGovern Institute for Brain Research, Media Lab, and Koch Institute for Integrative Cancer Research.

The simplest way to detect these fluorescent signals is with a type of imaging called one-photon microscopy. This is a relatively inexpensive technique that can image large brain samples at high speed, but the downside is that it picks up crosstalk between neighboring neurons. GCaMP goes into all parts of a neuron, so signals from the axons of one neuron can appear as if they are coming from the cell body of a neighbor, making the signal less accurate.

A more expensive technique called two-photon microscopy can partly overcome this by focusing light very narrowly onto individual neurons, but this approach requires specialized equipment and is also slower.

Boyden’s lab decided to take a different approach, by modifying the indicator itself, rather than the imaging equipment.

“We thought, rather than optically focusing light, what if we molecularly focused the indicator?” he says. “A lot of people use hardware, such as two-photon microscopes, to clean up the imaging. We’re trying to build a molecular version of what other people do with hardware.”

In a related paper that was published last year, Boyden and his colleagues used a similar approach to reduce crosstalk between fluorescent probes that directly image neurons’ membrane voltage. In parallel, they decided to try a similar approach with calcium imaging, which is a much more widely used technique.

To target GCaMP exclusively to cell bodies of neurons, the researchers tried fusing GCaMP to many different proteins. They explored two types of candidates — naturally occurring proteins that are known to accumulate in the cell body, and human-designed peptides — working with MIT biology Professor Amy Keating, who is also an author of the paper. These synthetic proteins are coiled-coil proteins, which have a distinctive structure in which multiple helices of the proteins coil together.

Less crosstalk

The researchers screened about 30 candidates in neurons grown in lab dishes, and then chose two — one artificial coiled-coil and one naturally occurring peptide — to test in animals. Working with Misha Ahrens, who studies zebrafish at the Janelia Research Campus, they found that both proteins offered significant improvements over the original version of GCaMP. The signal-to-noise ratio — a measure of the strength of the signal compared to background activity — went up, and activity between adjacent neurons showed reduced correlation.

In studies of mice, performed in the lab of Xue Han at Boston University, the researchers also found that the new indicators reduced the correlations between activity of neighboring neurons. Additional studies using a miniature microscope (called a microendoscope), performed in the lab of Kay Tye at the Salk Institute for Biological Studies, revealed a significant increase in signal-to-noise ratio with the new indicators.

“Our new indicator makes the signals more accurate. This suggests that the signals that people are measuring with regular GCaMP could include crosstalk,” Boyden says. “There’s the possibility of artifactual synchrony between the cells.”

In all of the animal studies, they found that the artificial, coiled-coil protein produced a brighter signal than the naturally occurring peptide that they tested. Boyden says it’s unclear why the coiled-coil proteins work so well, but one possibility is that they bind to each other, making them less likely to travel very far within the cell.

Boyden hopes to use the new molecules to try to image the entire brains of small animals such as worms and fish, and his lab is also making the new indicators available to any researchers who want to use them.

“It should be very easy to implement, and in fact many groups are already using it,” Boyden says. “They can just use the regular microscopes that they already are using for calcium imaging, but instead of using the regular GCaMP molecule, they can substitute our new version.”

The research was primarily funded by the National Institute of Mental Health and the National Institute of Drug Abuse, as well as a Director’s Pioneer Award from the National Institutes of Health, and by Lisa Yang, John Doerr, the HHMI-Simons Faculty Scholars Program, and the Human Frontier Science Program.

Ed Boyden wins prestigious entrepreneurial science award

The Austrian Association of Entrepreneurs announced today that Edward S. Boyden, the Y. Eva Tan Professor in Neurotechnology at MIT, has been awarded the 2020 Wilhelm Exner Medal.

Named after Austrian businessman Wilhelm Exner, the medal has been awarded annually since 1921 to scientists, inventors, and designers that are “promoting the economy directly or indirectly in an outstanding manner.” Past honorees include 22 Nobel laureates.

“It’s a great honor to receive this award, which recognizes not only the basic science impact of our group’s work, but the impact of the work in the industrial and startup worlds,” says Boyden, who is a professor of biological engineering and of brain and cognitive sciences at MIT.

Boyden is a leading scientist whose work is widely used in industry, both in his own startup companies and in existing companies. Boyden is also a member of MIT’s McGovern Institute for Brain Research, Media Lab, and Koch Institute for Integrative Cancer Research.

“I am so thrilled that Ed has received this honor,” says Robert Desimone, director of the McGovern Institute. “Ed’s work has transformed neuroscience, through optogenetics, expansion microscopy, and other findings that are pushing biotechnology forward too.”

He is interested in understanding the brain as a computational system, and builds and applies tools for the analysis of neural circuit structure and dynamics, in behavioral and disease contexts. He played a critical role in the development of optogenetics, a revolutionary tool where the activity of neurons can be controlled using light. Boyden also led the team that invented expansion microscopy, which gives an unprecedented view of the nanoscale structures of cells, even in the absence of special super resolution microscopy equipment. Exner Medal laureates include notable luminaries of science, including Robert Langer of MIT. In addition, Boyden has founded a number of companies based on his inventions in the busy biotech hub of Kendall Square, Cambridge. These include a startup that is seeking to apply expansion microscopy to medical problems.

Boyden will deliver his prize lecture at the Exner symposium in November 2020, during which economists and scientists come together to hear about the winner’s research.