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New findings reveal how neurons build and maintain their capacity to communicate

Nerve cells regulate and routinely refresh the collection of calcium channels that enable them to send messages across circuit connections.

David Orenstein | Picower Institute for Learning and Memory
July 21, 2022

The nervous system works because neurons communicate across connections called synapses. They “talk” when calcium ions flow through channels into “active zones” that are loaded with vesicles carrying molecular messages. The electrically charged calcium causes vesicles to “fuse” to the outer membrane of presynaptic neurons, releasing their communicative chemical cargo to the postsynaptic cell. In a new study, scientists at The Picower Institute for Learning and Memory at MIT provide several revelations about how neurons set up and sustain this vital infrastructure.

“Calcium channels are the major determinant of calcium influx, which then triggers vesicle fusion, so it is a critical component of the engine on the presynaptic side that converts electrical signals to chemical synaptic transmission,” says Troy Littleton, senior author of the new study in eLife and Menicon Professor of Neuroscience in MIT’s departments of Biology and Brain and Cognitive Sciences. “How they accumulate at active zones was really unclear. Our study reveals clues into how active zones accumulate and regulate the abundance of calcium channels.”

Neuroscientists have wanted these clues. One reason is that understanding this process can help reveal how neurons change how they communicate, an ability called “plasticity” that underlies learning and memory and other important brain functions. Another is that drugs such as gabapentin, which treats conditions as diverse as epilepsy, anxiety, and nerve pain, binds a protein called alpha2delta that is closely associated with calcium channels. By revealing more about alpha2delta’s exact function, the study better explains what those treatments affect.

“Modulation of the function of presynaptic calcium channels is known to have very important clinical effects,” Littleton says. “Understanding the baseline of how these channels are regulated is really important.”

MIT postdoc Karen Cunningham led the study, which was her doctoral thesis work in Littleton’s lab. Using the model system of fruit fly motor neurons, she employed a wide variety of techniques and experiments to show for the first time the step-by-step process that accounts for the distribution and upkeep of calcium channels at active zones.

A cap on Cac

Cunningham’s first question was whether calcium channels are necessary for active zones to develop in larvae. The fly calcium channel gene (called “cacophony,” or Cac) is so important, flies literally can’t live without it. So rather than knocking out Cac across the fly, Cunningham used a technique to knock it out in just one population of neurons. By doing so, she was able to show that even without Cac, active zones grow and mature normally.

Using another technique that artificially prolongs the larval stage of the fly she was also able to see that given extra time the active zone will continue to build up its structure with a protein called BRP, but that Cac accumulation ceases after the normal six days. Cunningham also found that moderate increases or decreases in the supply of available Cac in the neuron did not affect how much Cac ended up at each active zone. Even more curious, she found that while Cac amount did scale with each active zone’s size, it barely budged if she took away a lot of the BRP in the active zone. Indeed, for each active zone, the neuron seemed to enforce a consistent cap on the amount of Cac present.

“It was revealing that the neuron had very different rules for the structural proteins at the active zone like BRP that continued to accumulate over time, versus the calcium channel that was tightly regulated and had its abundance capped” Cunningham says.

Regular refresh

The findings showed there must be factors other than Cac supply or changes in BRP that regulate Cac levels so tightly. Cunningham turned to alpha2delta. When she genetically manipulated how much of that was expressed, she found that alpha2delta levels directly determined how much Cac accumulated at active zones.

In further experiments, Cunningham was also able to show that alpha2delta’s ability to maintain Cac levels depended on the neuron’s overall Cac supply. That finding suggested that rather than controlling Cac amount at active zones by stabilizing it, alpha2delta likely functioned upstream, during Cac trafficking, to supply and resupply Cac to active zones.

Cunningham used two different techniques to watch that resupply happen, producing measurements of its extent and its timing. She chose a moment after a few days of development to image active zones and measure Cac abundance to ascertain the landscape. Then she bleached out that Cac fluorescence to erase it. After 24 hours, she visualized Cac fluorescence anew to highlight only the new Cac that was delivered to active zones over that 24 hours. She saw that over that day there was Cac delivery across virtually all active zones, but that one day’s work was indeed only a fraction compared to what had built up over several days before. Moreover, she could see that the larger active zones accrued more Cac than smaller ones. And in flies with mutated alpha2delta, there was very little new Cac delivery at all.

If Cac channels were indeed constantly being resupplied, then Cunningham wanted to know at what pace Cac channels are removed from active zones. To determine that, she used a staining technology with a photoconvertible protein called Maple tagged to the Cac protein that allowed her to change the color with a flash of light at the time of her choosing. That way she could first see how much Cac accumulated by a certain time (shown in green) and then flash the light to turn that Cac red. When she checked back five days later, about 30 percent of the red Cac had been replaced with new green Cac, suggesting 30 percent turnover. When she reduced Cac delivery levels by mutating alpha2 delta or reducing Cac biosynthesis, Cac turnover stopped. That means a significant amount of Cac is turned over each day at active zones and that the turnover is prompted by new Cac delivery.

Littleton says his lab is eager to build on these results. Now that the rules of calcium channel abundance and replenishment are clear, he wants to know how they differ when neurons undergo plasticity — for instance, when new incoming information requires neurons to adjust their communication to scale up or down synaptic communication. He says he is also eager to track individual calcium channels as they are made in the cell body and then move down the neural axon to the active zones, and he wants to determine what other genes may affect Cac abundance.

In addition to Cunningham and Littleton, the paper’s other authors are Chad Sauvola and Sara Tavana.

The National Institutes of Health and the JPB Foundation provided support for the research.

Yiyin Erin Chen and Sam Chunte Peng named as core members of Broad Institute and MIT
Broad Communications
July 12, 2022

Broad Institute of MIT and Harvard has named Erin Chen, a dermatologist and microbiologist, and Sam Peng, a biophysicist and physical chemist with expertise in single-molecule imaging, as core institute members.

Chen will join in January 2023 and will also serve as an assistant professor in the Department of Biology at MIT and an attending dermatologist at Massachusetts General Hospital. Peng joined in July 2022 and will serve as an assistant professor in the Department of Chemistry at MIT.

Chen’s lab will study the communication between the immune system and the diverse microbes that colonize every surface of the human body, with a focus on the human body’s largest organ, the skin.

Peng’s lab will develop novel probes and microscopy techniques to visualize the dynamics of individual molecules in living cells, which will improve the understanding of molecular mechanisms underlying human diseases.

“We are delighted to welcome Sam and Erin to the Broad community,” said Todd Golub, director of the Broad. “These creative scientists are each taking inventive approaches to understand the molecular signals and interactions that underlie biological processes in health and disease. These insights will help further the Broad’s mission of advancing the understanding and treatment of human disease.”

Erin Chen.
Erin Chen

Erin Chen earned her BA in biology from the University of Chicago, her PhD from MIT, and her MD from Harvard Medical School. Prior to joining the Broad, Chen was a Howard Hughes Medical Institute Hanna Gray Postdoctoral Fellow at Stanford University, in the lab of Michael Fischbach. She was also an attending dermatologist at the University of California San Francisco and at the San Francisco VA Medical Center. During her postdoctoral research, Chen developed genetic methods to study harmless commensal skin bacteria. She engineered these bacteria to generate anti-tumor immunity, pioneering a novel approach to vaccination and cancer immunotherapy.

At the Broad, members of the Chen lab will continue to employ microbial genetics, immunologic approaches, and mouse models to dissect the molecular signals used by commensal microbes to educate the immune system. Ultimately, Chen aims to harness these microbe-host interactions to engineer novel therapeutics for human disease.

“I’m excited to join the collaborative scientific community at the Broad and MIT, including those who have pioneered novel tools for examining biological mechanisms at higher spatial resolution,” said Chen. “The biology I study is quite basic, but I’m motivated by the potential impact it could have on patients. Figuring out how commensal skin bacteria are captured by the immune system could unlock a whole new therapeutic toolbox.”

Sam Peng
Sam Peng

Sam Peng earned his BS in chemistry from the University of California, Berkeley, and his PhD from MIT in physical chemistry. He completed his postdoctoral research at Stanford University as an NIH K99 Pathway to Independence scholar in the lab of Steve Chu. During his postdoctoral research, he developed long-term single molecule imaging in live cells using a novel class of nanoprobes. He applied this new technique to study axonal transport in neurons and the molecular dynamics of dynein — a motor protein involved in transporting cargo in cells.

At the Broad, the Peng group will aim to elucidate the molecular mechanisms underlying human diseases. Lab members will develop and integrate a diverse toolbox spanning single-molecule microscopy, super-resolution microscopy, spectroscopy, nanomaterial engineering, biophysics, chemical biology, and quantitative modeling to uncover previously unexplored biological processes. With bright and photostable probes, lab members will have unprecedented capability to record ultra-long-term “molecular movies” in living systems with high spatiotemporal resolutions and to reveal molecular interactions that drive biological functions. Peng’s group will focus on studying molecular dynamics, protein-protein interactions, and cellular heterogeneity involved in neurobiology and cancer biology. Their long-term goal is to translate these mechanistic insights into drug discovery.

“Because my research is so multi-disciplinary, joining the Broad and MIT communities allows us to integrate a range of experimental tools and to collaborate with colleagues and students from diverse backgrounds,” said Peng. “I’m excited to see how our techniques can enable discoveries for a variety of cellular processes, including those underlying complex brain functions and dysfunctions. Many problems that previously seemed inaccessible now appear to be within reach in the foreseeable future.”

Investigating how cell orientation drives tissue growth during development

In a new study published July 7, 2022 in eLife, Adam Martin’s lab at the MIT Department of Biology identified the mechanical forces and molecular cues that help the spindles in cells located in the portion of the embryo destined to become the fly’s head to assume the same orientation.

Raleigh McElvery
July 7, 2022

Raleigh McElvery

During development, virtually all multicellular creatures must build themselves up from a ball of cells to a multilayered, fully-functioning organism. In the case of a fruit fly embryo, in order go from blob to organism, the cells must coordinate their divisions along the same axis to drive tissue growth in a specific direction — eventually going on to form the three germ layers known as the endoderm, ectoderm, and mesoderm.

Scientists have long studied how cells orient themselves to divide in a specific direction. It’s clear that this orientation is determined by a bundle of tiny fibers inside the cell called the spindle, which helps segregate the chromosomes so they can be distributed between the two daughter cells as the parent cell splits. When the spindles in neighboring cells are parallel with one another, then the cells will divide along the same axis.

In a new study published in eLife on July 7, 2022, Adam Martin’s lab at the MIT Department of Biology identified the mechanical forces and molecular cues that help the spindles in cells located in the portion of the embryo destined to become the fly’s head to assume the same orientation.

According to Martin, an associate professor of biology and the study’s senior author, his lab was among a handful of labs to take interest in this coordinated spindle orientation in the early fruit fly embryo — and identified the molecular, mechanical, and molecular cues that orient the spindle in a living organism.

“Cell divisions in the fly embryo was thought to be regulated independently of cell shape changes and morphogenetic movements,” Martin says. “However, we found that forces associated with cell invagination actually oriented cell divisions through a novel mechanism that we describe.” First author Jaclyn Camuglia, he explains, spearheaded the project from start to finish.

Prior to Camuglia’s work, researchers knew that spindle orientation was controlled by a complex of multiple proteins, including one protein in fruit flies called Pins (known as LGN in vertebrates). The entire protein complex, including Pins, is coupled to motor proteins that help to rotate the spindle. However, it was still unclear what mechanical forces were orienting Pins to dictate spindle rotation.

“It’s a very striking phenomenon when you look into the microscope at a fly embryo and see all the spindles orienting together,” Camuglia says. “We wanted to know: How are these spindles oriented and why is that directionality important?”

The fly is an ideal organism for probing cell division, because it has a very consistent and predictable cell cycle. During development, cells divide a set number of times, pause briefly, and then start up again in very discrete pockets across the embryo. The researchers wanted to know how the cells in a subset of those pockets in the fly’s head were coordinating their divisions.

Camuglia found that, in healthy embryos, Pins was always recruited to the end of the cell along the anterior-posterior axis (from the fly’s head to rear). By cutting the embryo with a laser, treating it with chemical inhibitors, disrupting the connections between cells, and depleting certain transcription factor proteins, she was able to characterize the mechanical forces that directed Pins to the anterior-posterior axis and thus rotated the spindle in the proper direction. As it turns out, these forces stem from other large-scale tissue movements that occur at the same time to force the embryo to fold in on itself and eventually form the fly’s muscles.

The researchers don’t yet know the role that these coordinated divisions along the anterior-posterior axis play in the developing fruit fly head — or what might happen during development if the divisions were to go awry. But Martin and Camuglia suspect this process facilitates tissue elongation, and helps compensate for any cells that are lost from the tissue during the division process as the embryo folds in on itself.

“This study is unique because it connects mechanical forces to the specific molecular cues like Pins that coordinate cell division,” Camuglia explains. “The factors that shape a developing embryo are critical to understand, because all organisms — from fruit flies to humans — experience these driving forces.” This intersection between physics and biology, she says, is what makes the study so exciting.”

Video: Groups of cells divide in a coordinated and oriented manner in the fruit fly embryo.
Top Image: Enrichment of cortical cues at one end of the cell coordinates the orientation of the mitotic spindle. Credit: Jaclyn Camuglia

Citation:
“Morphogenetic forces planar polarize LGN/Pins in the embryonic head during Drosophila gastrulation”
eLife, online 07/07/2022, DOI: https://doi.org/10.7554/eLife.78779
Jaclyn Camuglia, Soline Chanet, and Adam C Martin

Lab-grown fat cells help scientists understand type 2 diabetes
Eva Frederick | Whitehead Institute
June 16, 2022

In research published June 17 in the journal Science Advances, researchers in the lab of Whitehead Institute Founding Member Rudolf Jaenisch present a way to create fat cells that can be modified to display different levels of insulin sensitivity.

The cells accurately model healthy insulin metabolism, as well as insulin resistance, one of the key hallmarks of type 2 diabetes. “This system, I think, will be really useful for studying the mechanisms of this disease,” said Jaenisch, who is also a professor of biology at the Massachusetts Institute of Technology (MIT).

“It’s really exciting,” said Max Friesen, a postdoctoral researcher in Jaenisch’s lab and a first author of the study. “This is the first time that you can actually use a human stem cell-derived [fat cell] to show a real insulin response.”

Body fat — also known as adipose tissue — is essential for regulating your body’s metabolism and plays an important role in the storage and release of energy. When fat cells called adipocytes encounter the hormone insulin, they suck up sugar from the blood and store it for future use.

But over many years, factors such as genetics, stress, certain diets, or polluted air or water can cause this process to go awry, leading to type 2 diabetes. In this disease, adipocytes, as well as cells in the muscles and liver, become resistant to insulin and therefore unable to regulate the levels of sugar in the blood.

Tools to model diabetes in the lab generally rely on mice or on cells in a petri dish or test tube. Both these systems have their own problems. Mice, although they are comparable with humans in some respects, have a completely different metabolism and do not experience human diabetes comorbidities like heart attacks. And cell culture has, in the past, failed to replicate key markers of diabetes in a way that is comparable to human tissues.

That’s why Friesen and Andrew Khalil, another postdoc in Jaenisch’s lab, set out to create a new model. The researchers started with human pluripotent stem cells. These cells are the shapeshifters of the body — given the right conditions, they can assume the specific characteristics of almost any human cell type. The Jaenisch Lab has used them in the past to replicate liver cells, brain cells, and even cancerous tumors.

They decided to try to optimize an existing method for differentiating pluripotent cells into fat cells. The protocol created cells that looked like adipocytes, but these cells did not recreate the conditions of healthy insulin signaling or insulin resistance seen in the human body in type 2 diabetes. When healthy adipocytes encounter insulin in the human body, they respond by taking up glucose out of the bloodstream. These lab-made fat cells weren’t doing that, unless the researchers cranked up insulin levels to a thousand times higher than levels ever seen in humans. “Taking up glucose [in response to normal levels on insulin] is really the main function of an adipocyte, so if the model fails to do that, anything downstream in terms of disease research is not going to work either,” Friesen said.

Friesen and Khalil wondered if the lab-grown adipocytes’ low sensitivity to insulin could be a product of the conditions in which they grew. “We thought that maybe this happens because we’re feeding them an artificial culture medium, with all kinds of extra supplements that might be inhibiting their metabolic response,” Friesen said.

Friesen and Khalil decided to use a method called the Design of Experiments approach, which allows researchers to tease out the contributions of different factors to a specific outcome. Informed by this approach, they created nearly 30 different media compositions, each with slightly different levels of key ingredients such as glucose, insulin, the growth factor IGF-1, and albumin, a protein found in blood serum.

The medium that worked best had concentrations of insulin and glucose that were similar to the levels in the human body. When grown in this new medium, the cells responded to much lower concentrations of insulin, just like cells in the body. “So this is our healthy adipocyte,” Friesen said. “Next we wanted to see if we could make a disease model out of this — to make it an insulin-resistant adipocyte like you would see in the progression to type 2 diabetes.”

To desensitize the cells, they flooded the media with insulin for a short period of time. This caused the cells to become less sensitive to the hormone, and respond similarly to diabetic or pre-diabetic fat cells in a living person.

The researchers could then study how the cells responded to the change — such as what genes the insulin resistant cells expressed that healthy cells did not — in order to tease out the underlying genetics of insulin resistance. “We saw small changes in a lot of genes that are metabolism regulated, so that seems to be pointing to a deficiency of the metabolism or mitochondria of the insulin-resistant cells,” Friesen said. “That’s one thing we want to pursue in the future — figure out what is wrong with their metabolism, and then hopefully how to fix it.”

Now that they have created this new model for studying insulin resistance in fat cells, the researchers hope to develop similar procedures for other cells affected in diabetes.  “It seems that with some modifications, we can apply this method to other tissues as well,” Friesen said. “In the future, this will hopefully lead to a unified system for all stem cell-derived tissues, including liver, skeletal muscle, and other cell types, to get a really robust insulin response.”

Ankur Jain Named as Pew Scholar in Biomedical Sciences
Merrill Meadow | Whitehead Institute
June 13, 2022

The Pew Charitable Trusts has selected Whitehead Institute Member Ankur Jain to be a 2022 Pew Scholar in the Biomedical Sciences. The Pew program provides funding to young investigators of outstanding promise who work in areas of science relevant to the advancement of human health.

Jain, who joined the Whitehead Institute faculty in 2019, is one of 22 scientists selected to receive this year’s honor, chosen from among 197 nominations submitted by leading U.S. academic and research institutions. “I am grateful to the Pew Trusts for funding our work, and thrilled to be a part of the Pew community,” says Jain, who is also an assistant professor of biology and the Thomas D. and Virginia W. Cabot Career Development Professor at Massachusetts Institute of Technology.

The Pew award will provide research support for the next four years, enabling him to study the role of evolutionarily ancient metabolites called polyamines, which are essential for cell growth and survival.

“Polyamine concentrations within cells are carefully regulated, and disruptions in polyamine production are known to be associated with conditions ranging from cancer and aging to neurological disorders such as Parkinson’s disease” Jain explains. “But, despite being studied for more than a century, the specific role polyamines play in both healthy and diseased cells remains obscure. This is due, in part, to a lack of technologies effective in probing polyamines.”

Jain’s lab will harness the cell’s own polyamine detection machinery to build new tools to inspect polyamines. Those tools will allow his team to measure and track polyamines in individual cells, study how cells maintain their polyamine content, and explore how changing polyamine levels affect cellular functions. “Ultimately, this work could provide the basis for novel strategies for treating cancer or promoting healthy aging,” Jain observes.

Previously, Jain received a 2017 NIH Pathway to Independence Award and was named a 2019 Packard Fellow for Science and Engineering. He is the third current Whitehead Member to be named a Pew Scholar, following in the steps of Mary Gehring (2010) and Jing-Ke Weng (2014). Former Whitehead Fellow Fernando Camargo, now professor of stem cell and regenerative biology at Harvard University, also became a Pew Scholar in 2010.

Launched in 1985, the Pew Scholars in the Biomedical Sciences program supports top U.S. scientists at the assistant professor level and has, since inception, provided nearly 1000 young investigators with  funding for research projects that, though seemingly risky, have the potential to benefit human health. Pew Scholars are selected by a national advisory committee of eminent scientists, who evaluate candidates on the basis of proven creativity.

More information about Jain’s selection, the 2022 class of Pew Scholars, and the Pew Scholars program is available here.

MIT announces 2022 Bose grants for ambitious ideas

Tenth anniversary of the program rewards three innovative projects.

Aaron Braddock | Office of the Provost
June 13, 2022

MIT Provost Cynthia Barnhart has announced three Professor Amar G. Bose Research Grants to support bold research projects across diverse areas of study including biology, engineering, and the humanities.

The three grants honor the visionary and bold thinking in the winning proposals of the following nine researchers: John J. and Dorothy Wilson Professor of Health Sciences and Technology and Electrical Engineering and Computer Science Sangeeta Bhatia; Carl Richard Soderberg Professor of Power Engineering Gang Chen; professor of biology Jianzhu Chen; associate professor of biology Michael Hemann; professor of anthropology and Margaret MacVicar Faculty Fellow Graham Jones; Latham Family Career Development Professor Sebastian Lourido; assistant professor of computer science Arvind Satayanaryan; Howard Hughes Medical Institute Professor Graham Walker; and David H. Koch Professor in Science Michael Yaffe;

“Innovation is born when a unique vision drives daring researchers to take on risky and adventurous projects, a notion that Amar Bose understood well,” says Barnhart. “With support and recognition from this program, these nine talented and forward-thinking faculty have the freedom to explore and study areas not typically backed by conventional funding sources.”

The program was named for the visionary founder of the Bose Corporation and MIT alumnus, Amar G. Bose ’51, SM ’52, ScD ’56. After gaining admission to MIT, Bose became a top math student and a Fulbright Scholarship recipient. He spent 46 years as a professor at MIT, led innovations in sound design, and founded the Bose Corporation in 1964. MIT launched the program a decade ago.

“The legendary explorations and innovations of Professor Amar Bose inspire the Bose Research Grant program,” says President Emerita and Professor Susan Hockfield. “The grants support projects that reach beyond the horizon and so would not receive funding from standard sources. Since its inception, the program has supported 49 MIT faculty to pursue their most compelling ideas and, in doing so, to join the Bose Fellows community of like-minded adventurers.”

The program, which has honored 35 projects to date, is a tribute to the legacy of Bose, who believed that passion and curiosity drive innovation. With that spirit in mind, the projects typically supported by the program are original, cross-disciplinary, and high-risk. The program has encouraged collaborative projects, as reflected in this year’s winners.

This year’s recipients are:

Gang Chen of the Department of Mechanical Engineering. With his proposal, “Photomolecular Effect and Clouds Thinning,” Chen will advance research into his discovery of a way in which photons can be absorbed by cleaving off water clusters from the water-air surface, significantly impacting technologies related to energy and water and climate models.

Graham Jones of the Anthropology Section and Arvind Satayanaryan of the Department of Electrical Engineering and Computer Science (EECS). Their “Magical Data Visualization” proposal uses performance magic to create new visualizations that are responsive to the users’ intent, potentially impacting how misinformation spreads.

Graham Walker, Michael Hemann, Michael Yaffe, Sebastian Lourido, Jianzhu Chen of the Department of Biology and Sangeeta Bhatia of EECS and the Institute of Medical Engineering and Science. Their proposal, “Addressing Critical Human Health Problems with a Special Heme-binding Peptide,” uses a recently discovered plant peptide that binds and sequesters a molecule critical in hemoglobin oxygen binding in a new way, which has significant implications on many health issues.

“This year, more than a dozen faculty members from departments across all five schools and the college participated in the evaluations,” says Chancellor for Academic Advancement Eric Grimson. “Their diverse perspectives were critical in assessing what was a very strong field of interesting proposals. We are grateful for their generous commitment of time and energy and the thoughtfulness with which they approached the selection process.”

The program explores out-of-the-box ideas that would face difficulty in acquiring funding through traditional means but have the potential for strong impacts on the scientific community. Any member of the faculty in any discipline in MIT’s five schools and college is eligible to submit a proposal for a Bose Research Grant, which provides funding over three years.

New CRISPR-based map ties every human gene to its function

Jonathan Weissman and collaborators used their single-cell sequencing tool Perturb-seq on every expressed gene in the human genome, linking each to its job in the cell.

Eva Frederick | Whitehead Institute
June 9, 2022

The Human Genome Project was an ambitious initiative to sequence every piece of human DNA. The project drew together collaborators from research institutions around the world, including MIT’s Whitehead Institute for Biomedical Research, and was finally completed in 2003. Now, over two decades later, MIT Professor Jonathan Weissman and colleagues have gone beyond the sequence to present the first comprehensive functional map of genes that are expressed in human cells. The data from this project, published online June 9 in Cell, ties each gene to its job in the cell, and is the culmination of years of collaboration on the single-cell sequencing method Perturb-seq.

The data are available for other scientists to use. “It’s a big resource in the way the human genome is a big resource, in that you can go in and do discovery-based research,” says Weissman, who is also a member of the Whitehead Institute and an investigator with the Howard Hughes Medical Institute. “Rather than defining ahead of time what biology you’re going to be looking at, you have this map of the genotype-phenotype relationships and you can go in and screen the database without having to do any experiments.”

The screen allowed the researchers to delve into diverse biological questions. They used it to explore the cellular effects of genes with unknown functions, to investigate the response of mitochondria to stress, and to screen for genes that cause chromosomes to be lost or gained, a phenotype that has proved difficult to study in the past. “I think this dataset is going to enable all sorts of analyses that we haven’t even thought up yet by people who come from other parts of biology, and suddenly they just have this available to draw on,” says former Weissman Lab postdoc Tom Norman, a co-senior author of the paper.

Pioneering Perturb-seq

The project takes advantage of the Perturb-seq approach that makes it possible to follow the impact of turning on or off genes with unprecedented depth. This method was first published in 2016 by a group of researchers including Weissman and fellow MIT professor Aviv Regev, but could only be used on small sets of genes and at great expense.

The massive Perturb-seq map was made possible by foundational work from Joseph Replogle, an MD-PhD student in Weissman’s lab and co-first author of the present paper. Replogle, in collaboration with Norman, who now leads a lab at Memorial Sloan Kettering Cancer Center; Britt Adamson, an assistant professor in the Department of Molecular Biology at Princeton University; and a group at 10x Genomics, set out to create a new version of Perturb-seq that could be scaled up. The researchers published a proof-of-concept paper in Nature Biotechnology in 2020.

The Perturb-seq method uses CRISPR-Cas9 genome editing to introduce genetic changes into cells, and then uses single-cell RNA sequencing to capture information about the RNAs that are expressed resulting from a given genetic change. Because RNAs control all aspects of how cells behave, this method can help decode the many cellular effects of genetic changes.

Since their initial proof-of-concept paper, Weissman, Regev, and others have used this sequencing method on smaller scales. For example, the researchers used Perturb-seq in 2021 to explore how human and viral genes interact over the course of an infection with HCMV, a common herpesvirus.

In the new study, Replogle and collaborators including Reuben Saunders, a graduate student in Weissman’s lab and co-first author of the paper, scaled up the method to the entire genome. Using human blood cancer cell lines as well noncancerous cells derived from the retina, he performed Perturb-seq across more than 2.5 million cells, and used the data to build a comprehensive map tying genotypes to phenotypes.

Delving into the data

Upon completing the screen, the researchers decided to put their new dataset to use and examine a few biological questions. “The advantage of Perturb-seq is it lets you get a big dataset in an unbiased way,” says Tom Norman. “No one knows entirely what the limits are of what you can get out of that kind of dataset. Now, the question is, what do you actually do with it?”

The first, most obvious application was to look into genes with unknown functions. Because the screen also read out phenotypes of many known genes, the researchers could use the data to compare unknown genes to known ones and look for similar transcriptional outcomes, which could suggest the gene products worked together as part of a larger complex.

The mutation of one gene called C7orf26 in particular stood out. Researchers noticed that genes whose removal led to a similar phenotype were part of a protein complex called Integrator that played a role in creating small nuclear RNAs. The Integrator complex is made up of many smaller subunits — previous studies had suggested 14 individual proteins — and the researchers were able to confirm that C7orf26 made up a 15th component of the complex.

They also discovered that the 15 subunits worked together in smaller modules to perform specific functions within the Integrator complex. “Absent this thousand-foot-high view of the situation, it was not so clear that these different modules were so functionally distinct,” says Saunders.

Another perk of Perturb-seq is that because the assay focuses on single cells, the researchers could use the data to look at more complex phenotypes that become muddied when they are studied together with data from other cells. “We often take all the cells where ‘gene X’ is knocked down and average them together to look at how they changed,” Weissman says. “But sometimes when you knock down a gene, different cells that are losing that same gene behave differently, and that behavior may be missed by the average.”

The researchers found that a subset of genes whose removal led to different outcomes from cell to cell were responsible for chromosome segregation. Their removal was causing cells to lose a chromosome or pick up an extra one, a condition known as aneuploidy. “You couldn’t predict what the transcriptional response to losing this gene was because it depended on the secondary effect of what chromosome you gained or lost,” Weissman says. “We realized we could then turn this around and create this composite phenotype looking for signatures of chromosomes being gained and lost. In this way, we’ve done the first genome-wide screen for factors that are required for the correct segregation of DNA.”

“I think the aneuploidy study is the most interesting application of this data so far,” Norman says. “It captures a phenotype that you can only get using a single-cell readout. You can’t go after it any other way.”

The researchers also used their dataset to study how mitochondria responded to stress. Mitochondria, which evolved from free-living bacteria, carry 13 genes in their genomes. Within the nuclear DNA, around 1,000 genes are somehow related to mitochondrial function. “People have been interested for a long time in how nuclear and mitochondrial DNA are coordinated and regulated in different cellular conditions, especially when a cell is stressed,” Replogle says.

The researchers found that when they perturbed different mitochondria-related genes, the nuclear genome responded similarly to many different genetic changes. However, the mitochondrial genome responses were much more variable.

“There’s still an open question of why mitochondria still have their own DNA,” said Replogle. “A big-picture takeaway from our work is that one benefit of having a separate mitochondrial genome might be having localized or very specific genetic regulation in response to different stressors.”

“If you have one mitochondria that’s broken, and another one that is broken in a different way, those mitochondria could be responding differentially,” Weissman says.

In the future, the researchers hope to use Perturb-seq on different types of cells besides the cancer cell line they started in. They also hope to continue to explore their map of gene functions, and hope others will do the same. “This really is the culmination of many years of work by the authors and other collaborators, and I’m really pleased to see it continue to succeed and expand,” says Norman.

Yiyin Erin Chen

Education

  • Graduate: PhD, 2011, MIT; MD, 2013, Harvard Medical School
  • Undergraduate: BA, 2006, Biology, University of Chicago

Research Summary

Diverse commensal microbes colonize every surface of our bodies. We study the constant communication between these microbes and our immune system. We focus on our largest organ: the skin. By employing microbial genetics, immunologic approaches, and mouse models, we can dissect (1) the molecular signals used by microbes to educate our immune system and (2) how different microbial communities alter immune responses. Ultimately, we aim to harness these microbe-host interactions to engineer novel vaccines and therapeutics for human disease.

Awards

  • Howard Hughes Medical Institute Hanna H. Gray Fellow, 2018-2026
  • A.P. Giannini Postdoctoral Research Fellowship, 2018
  • Dermatology Foundation Research Fellowship, 2017