Research Area: Cell Biology

3 Questions: Sara Prescott on the brain-body connection

New faculty member Sara Prescott investigates how sensory input from within the body control mammalian physiology and behavior.

Lillian Eden | Department of Biology
April 26, 2023

Many of our body’s most important functions occur without our conscious knowledge, such as digestion, heartbeat, and breathing. These vital functions depend on the signals generated by the “interoceptive nervous system,” which enables the brain to monitor our internal organs and trigger responses that sometimes save our lives. One second you are breathing normally as you eat your salad and the next, when a vinegar-soaked crouton enters your throat, you are coughing or swallowing to protect and clear your airway. We know our bodies are sensitive to cues like irritants, but we still have a lot to learn about how the interoceptive system works to meet our physiological needs, keep organs safe and healthy, and affect our behavior. We can also learn how chronic insults may lead to organ dysfunction and use what we learn to create therapeutic interventions.

Focusing on the airway, Sara Prescott, a new faculty member in the Department of Biology and Investigator in The Picower Institute for Learning and Memory, seeks to understand the ways our nervous systems detect and respond to stimuli in health and disease.

Q: You’re interested in interoceptive biology. What makes the nervous system of mice a good model for doing that?

A: Our flagship system is the mammalian airway. We use a mouse model and modern molecular neuroscience tools to manipulate various neural pathways and observe what the effects are on respiratory function and animal health.

Neuroscience and mouse work have a reputation for being a little challenging and intense, but I think this is also where we can ask really important questions that are useful for our everyday lives — and the only place where we can fully recapitulate the complexity of nervous system signaling all the way down to our organs, back to our brain, and back to our organs.

It’s a very fun place to do science with lots of open questions.

One of the core discoveries from my postdoctoral work was focusing on the vagus nerve as a major body-to-brain conduit, as it innervates our lungs, heart and gastrointestinal tract. We found that there were about 40 different subtypes of sensory neurons within this small nerve, which is really a remarkable amount of diversity and reflects the massive sensory space within the body. About a dozen of those vagal neurons project to the airways.

We identified a rare neuron type specifically responsible for triggering protective responses like coughing when water or acid entered the airway. We also discovered a separate population of neurons that make us feel and act sick when we get a flu infection. The field now knows what four to five vagal populations of neurons are actually sensing in the airways, but the remaining populations are still a mystery to us; we don’t know what those populations of sensory neurons are detecting, what their anatomy is, and what reflex effects those neurons are evoking.

Looking ahead, there are many exciting directions for the interoceptive biology field. For example, there’s been a lot of focus on characterizing the circuits underlying acute motor reflexes, like rapid responses to visceral stimuli on the timescale of minutes to hours. But we don’t have a lot of information about what happens when these circuits are activated over long periods of time. For example, respiratory tract infections often last for weeks or longer. We know that the airways undergo changes in composition when they’re exposed to different types of infection or stress to better accommodate future threats. One of the hypotheses we’re testing is that chronically activating neural circuits may drive changes in organ composition. We have this idea, which we’re calling reflexive remodeling: neurons may be communicating with stem cells and progenitor cells in the periphery to drive adaptive remodeling responses.

We have the genetic, molecular and circuit scale tools to explore this pheno­­­menon in mice. In parallel, we’re also setting up some in vitro models of the mouse airway mucosa to expedite receptor screening and to explore basic mechanisms of neuron-epithelium crosstalk. We hope this will inform our understanding of how the airway surface senses and responds to different types of irritants or damage. 

Q: Why is understanding the peripheral nervous system important, and what parts of your background are you drawing on for your current research?

A: The lab focuses on really trying to explore the body-brain connection. 

People often think that our mind exists in a vacuum, but in reality, our nervous system is heavily integrated with the rest of the body, and those neural interfaces are important, both for taking information from our body or environment and turning it into an internal representation of the world, and, in reverse, being able to process that information and being able to enact changes throughout the body. That includes things like autonomic reflexes, basic functions of the body like breathing, blood-gas regulation, digestion, and heart rate.

I’ve integrated both my graduate training and postdoctoral training into thinking about biology across multiple scales.

Graduate school for me was quite focused on deep molecular mechanism questions, particularly gene regulation, so I feel like that has been very useful for me in my general approach to neuroscience because I take a very molecular angle to all of this.

It also showed me the power of in vitro models as reductionist tools to explore fundamental aspects of cell biology. During my postdoc, I focused on larger, emergent phenotypes. We were able to manipulate specific circuits and see very impressive behavioral responses in animals. You could stimulate about 100 neurons in a mouse and see that their breathing would just stop until you remove the stimulation, and then the breathing would return to normal.

Both of those experiences inform how we approach a problem in my research. We need to understand how these circuits work, not just their connectivity at the anatomical level but what is driving their changes in sensitivity over time, the receptor expression programs that affect how they sense and signal, how these circuits emerge during development, and their gene expression.

There are still s­o many foundational questions that haven’t been answered that there’s enough to do in the mouse for quite some time.

Q: This all sounds fascinating. Where does it lead?

A: Human health has been my north star for a long time and I’ve taken a long, wandering path to find particular areas where I can scratch whatever intellectual itch that I have.

I originally thought I would be a doctor and then realized that I felt like I could have a more lasting impact by discovering fundamental truths about how our bodies work. I think there are a number of chronic diseases in which autonomic imbalance is actually a huge clinical component of the disorder.

We have a lot of interest in some of these very common airway remodeling diseases, like chronic obstructive pulmonary disorder—COPD—asthma, and potentially lung cancer. We want to ask questions like how autonomic circuits are altered in disease contexts, and when neurons actually drive features of disease. 

Perhaps this research will help us come up with better molecular, cellular or tissue engineering approaches to improve the outcomes for a variety of autonomic diseases. 

It’s very easy for me to imagine how one day not too far from now we can turn these findings into something actionable for human health.

New instrument lets MIT researchers combine previously disparate microscopy techniques

The first Live μ in the country will reveal fleeting sub-cellular events in high resolution

Saima Sidik
February 1, 2023

Inside cells, events can unfold quickly. Sub-cellular compartments constantly re-arrange while proteins move along structural fibers and membranes fuse and divide. By attaching fluorescent tags to sub-cellular structures, researchers can watch events unfold in real time using light microscopes. But to see the finest details of these processes, scientists need to shift from using light microscopy to using beams of electrons to generate even higher resolution images using a technique called electron microscopy. Using these techniques together is a powerful and rapidly growing strategy called correlative light electron microscopy (CLEM). In CLEM, light microscopy images are used to target regions of interest, and then the same sample is interrogated with electron microscopy to see the same areas at higher resolution.

The Peterson (1957) Nanotechnology Materials Core Facility in the Robert A. Swanson (1969) Biotechnology Center at the Koch Institute for Integrative Cancer Research at MIT recently acquired a high pressure freezer called the Live μ that will let researchers do just that. This instrument allows scientists to image the same biological sample using fluorescent light microscopy and electron microscopy in close succession. These two techniques are usually performed on separate samples, but with the Live μ, researchers will be able to identify fleeting sub-cellular events using light microscopy, then preserve cells and observe the same events in high resolution using electron microscopy — a combination that was not previously available to researchers at MIT. In fact, the Live μ, which is sold by the Paris-based company CryoCapCell, will be the first instrument of its kind in the country.

Although high-pressure freezers like the Live μ have been around for decades, integration with a light microscope is what makes the Live μ special. The instrument itself is a washing machine-sized freezing instrument, equipped with an arm to hold a biological sample under a nearby light microscope. When researchers observe an interesting biological event using the light microscope, they can quickly retract the arm and insert the sample into the Live μ’s inner chamber, exposing it to low temperature and high pressure and freezing it in less than two seconds. Cells must be preserved before they can be observed using an electron microscope, and by freezing samples faster than ice crystals can form, the Live μ creates pristine samples that accurately represent the state of cells before preservation. Superimposing pictures taken using the light microscope on top of images from an electron microscope allows researchers to use the fluorescent signals like a “treasure map,” says Abigail Lytton-Jean, the director of the Peterson Facility.

Exocytosis is a vital sub-cellular event that could be studied using the Live μ. In this cellular process, cells use bubble-like vesicles to ferry proteins from the internal compartments where they’re made to the cell’s surface, where they can sense the external environment, attach cells to one another, or carry information to other cells. Exocytosis is important for many aspects of biology, and a variety of scientists, from ecologists to cancer researchers to microbiologists, would benefit from a greater understanding of this process. With the Live μ, researchers may be able to use light microscopy to catch the vesicles that mediate exocytosis when they dock with the cell’s surface, then use electron microscopy to understand the details of this association.

Researchers creating artificial materials to replace human tissues could also benefit from the Live μ, Lytton-Jean says. These materials are thick and contain a lot of water, but the Live μ is capable of freezing them without generating ice crystals that change their structure. Using this instrument, scientists can examine the internal structure of these synthetic materials and assess their similarities to live tissue.

“People who want to use the Live μ are coming from all sorts of labs,” Lytton-Jean says.

The world of biology and electron microscopy is wildly exciting right now, she adds, thanks in part to instruments like this. “People who have worked with electron microscopes for decades have told me that this is the most exciting time they’ve ever lived in.”

The Live μ recently took its place in the back of the Peterson Facility, under a picture of the Eiffel Tower that Lytton-Jean brought back from Paris when she first went to test the Live μ at CryoCapCell’s headquarters years ago. The Live μ is only the latest addition to a vast suite of instrumentation focused on cutting-edge cryo-electron microscopy and CLEM workflows, expanding the facility’s unusually large portfolio of workflows.

“There aren’t many places in the country that can do all of the different workflows we offer, and all in one place,” said Lytton-Jean. “High pressure freezing is the first step in the preservation process, so having this instrument in our lab will further enable many new workflows with our existing instrumentation. Although these workflows are challenging and sophisticated, our team of dedicated scientists are familiar with conducting this work.”

Daniel Lew

Education

  • Graduate: PhD, 1990, Rockefeller University
  • Undergraduate: BA, 1984, Genetics, Cambridge University

Research Summary

Different cells take on an astonishing variety of shapes, which are often critical to be able to perform specialized cell functions like absorbing nutrients or contracting muscles. We study how different cell shapes arise and how cells control the spatial distribution of their internal constituents. We take advantage of the tractability of fungal model systems, and address these questions using approaches from cell biology, genetics, and computational biology to understand molecular mechanisms. 

Honors and Awards

  • Fellow, American Academy of Microbiology, 2008
  • Fellow, American Association for the Advancement of Science, 2010
  • Duke Equity, Diversity, and Inclusion Award, 2019
Enzyme “atlas” helps researchers decipher cellular pathways

Biologists have mapped out more than 300 protein kinases and their targets, which they hope could yield new leads for cancer drugs.

Anne Trafton | MIT News Office
January 11, 2023

One of the most important classes of human enzymes are protein kinases — signaling molecules that regulate nearly all cellular activities, including growth, cell division, and metabolism. Dysfunction in these cellular pathways can lead to a variety of diseases, particularly cancer.

Identifying the protein kinases involved in cellular dysfunction and cancer development could yield many new drug targets, but for the vast majority of these kinases, scientists don’t have a clear picture of which cellular pathways they are involved in, or what their substrates are.

“We have a lot of sequencing data for cancer genomes, but what we’re missing is the large-scale study of signaling pathway and protein kinase activation states in cancer. If we had that information, we would have a much better idea of how to drug particular tumors,” says Michael Yaffe, who is a David H. Koch Professor of Science at MIT, the director of the MIT Center for Precision Cancer Medicine, a member of MIT’s Koch Institute for Integrative Cancer Research, and one of the senior authors of the new study.

Yaffe and other researchers have now created a comprehensive atlas of more than 300 of the protein kinases found in human cells, and identified which proteins they likely target and control. This information could help scientists decipher many cellular signaling pathways, and help them to discover what happens to those pathways when cells become cancerous or are treated with specific drugs.

Lewis Cantley, a professor of cell biology at Harvard Medical School and Dana Farber Cancer Institute, and Benjamin Turk, an associate professor of pharmacology at Yale School of Medicine, are also senior authors of the paper, which appears today in Nature. The paper’s lead authors are Jared Johnson, an instructor in pharmacology at Weill Cornell Medical College, and Tomer Yaron, a graduate student at Weill Cornell Medical College.

“A Rosetta stone”

The human genome includes more than 500 protein kinases, which activate or deactivate other proteins by tagging them with a chemical modification known as a phosphate group. For most of these kinases, the proteins they target are unknown, although research into kinases such as MEK and RAF, which are both involved in cellular pathways that control growth, has led to new cancer drugs that inhibit those kinases.

To identify additional pathways that are dysregulated in cancer cells, researchers rely on phosphoproteomics using mass spectrometry — a technique that separates molecules based on their mass and charge — to discover proteins that are more highly phosphorylated in cancer cells or healthy cells. However, until now, there has been no easy way to interrogate the mass spectrometry data to determine which protein kinases are responsible for phosphorylating those proteins. Because of that, it has remained unknown how those proteins are regulated or misregulated in disease.

“For most of the phosphopeptides that are measured, we don’t know where they fit in a signaling pathway. We don’t have a Rosetta stone that you could use to look at these peptides and say, this is the pathway that the data is telling us about,” Yaffe says. “The reason for this is that for most protein kinases, we don’t know what their substrates are.”

Twenty-five years ago, while a postdoc in Cantley’s lab, Yaffe began studying the role of protein kinases in signaling pathways. Turk joined the lab shortly after, and the three have since spent decades studying these enzymes in their own research groups.

“This is a collaboration that began when Ben and I were in Lew’s lab 25 years ago, and now it’s all finally really coming together, driven in large part by what the lead authors, Jared and Tomer, did,” Yaffe says.

In this study, the researchers analyzed two classes of kinases — serine kinases and threonine kinases, which make up about 85 percent of the protein kinases in the human body — based on what type of structural motif they put phosphate groups onto.

Working with a library of peptides that Cantley and Turk had previously created to search for motifs that kinases interact with, the researchers measured how the peptides interacted with all 303 of the known serine and threonine kinases. Using a computational model to analyze the interactions they observed, the researchers were able to identify the kinases capable of phosphorylating every one of the 90,000 known phosphorylation sites that have been reported in human cells, for those two classes of kinases.

To their surprise, the researchers found that many kinases with very different amino acid sequences have evolved to bind and phosphorylate the same motifs on their substrates. They also showed that about half of the kinases they studied target one of three major classes of motifs, while the remaining half are specific to one of about a dozen smaller classes.

Decoding networks

This new kinase atlas can help researchers identify signaling pathways that differ between normal and cancerous cells, or between treated and untreated cancer cells, Yaffe says.

“This atlas of kinase motifs now lets us decode signaling networks,” he says. “We can look at all those phosphorylated peptides, and we can map them back onto a specific kinase.”

To demonstrate this approach, the researchers analyzed cells treated with an anticancer drug that inhibits a kinase called Plk1, which regulates cell division. When they analyzed the expression of phosphorylated proteins, they found that many of those affected were controlled by Plk1, as they expected. To their surprise, they also discovered that this treatment increased the activity of two kinases that are involved in the cellular response to DNA damage.

Yaffe’s lab is now interested in using this atlas to try to find other dysfunctional signaling pathways that drive cancer development, particularly in certain types of cancer for which no genetic drivers have been found.

“We can now use phosphoproteomics to say, maybe in this patient’s tumor, these pathways are upregulated or these pathways are downregulated,” he says. “It’s likely to identify signaling pathways that drive cancer in conditions where it isn’t obvious what the genetics that drives the cancer are.”

The research was funded by the Leukemia and Lymphoma Society, the National Institutes of Health, Cancer Research UK, the Brain Tumour Charity, the Charles and Marjorie Holloway foundation, the MIT Center for Precision Cancer Medicine, and the Koch Institute Support (core) grant from the National Cancer Institute.

The molecules behind metastasis
Greta Friar | Whitehead Institute
January 4, 2023

Many cancer cells never leave their original tumors. Some cancer cells evolve the ability to migrate to other tissues, but once there cannot manage to form new tumors, and so remain dormant. The deadliest cancer cells are those that can not only migrate to, but also thrive and multiply in distant tissues. These metastatic cancer cells are responsible for most of the deaths associated with cancer. Understanding what enables some cancer cells to metastasize—to spread and form new tumors—is an important goal for researchers, as it will help them develop therapies to prevent or reverse those deadly occurrences.

Past research from Whitehead Institute Member Robert Weinberg and others suggests that cancer cells are best able to form metastatic tumors when the cells are in a particular state called the quasi-mesenchymal (qM) state. New research from Weinberg and Arthur Lambert, once a postdoc in Weinberg’s lab and now an associate director of translational medicine at AstraZeneca, has identified two gene-regulating molecules as important for keeping cancer cells in the qM state. The research, published in the journal Developmental Cell on December 19, shows that these molecules, ΔNp63 and p73, enable breast cancer cells to form new tumors in mice, and illuminates important aspects of how they do so.

Most potent in the middle

Cells enter the qM state by undergoing the epithelial-mesenchymal transition (EMT), a developmental process that can be co-opted by cancer cells. In the EMT, cells transition from an epithelial state through a spectrum of more mesenchymal states, which allows them to become more mobile and aggressive. Cells in the qM state have only transitioned partway through the EMT, becoming more, but not fully, mesenchymal. This middle ground is perfect for metastasis, whereas cells on either end of the spectrum—cells that are excessively epithelial or excessively mesenchymal—lose their metastatic abilities.

Lambert and colleagues wanted to understand more about how cancer stem cells, which can seed metastases and recurrent tumors, remain in a metastasis-prone qM state. They analyzed how gene activity was regulated in those cells and identified two transcription factors—molecules that influence the activity of target genes—as important. One of the transcription factors, ΔNp63, appeared to most directly control cancer stem cells’ ability to maintain a qM state. The other molecule, p73, seemed to have a similar role because it can activate ΔNp63. When either transcription factor was inactivated, the cancer stem cells transitioned to the far end of the EMT spectrum and so were unable to metastasize.

Next, the researchers looked at what genes ΔNp63 regulates in cancer stem cells. They expected to find a pattern of gene regulation resembling what they would see in healthy breast stem cells. Instead they found a pattern closely resembling what one would see in cells involved in wound healing and regeneration. Notably, ΔNp63 stimulates EGFR signaling, which is used in wound healing to promote rapid multiplication of cells.

“Although this is not what we expected to see, it makes a lot of sense because the process of metastasis requires active proliferation,” Lambert says. “Metastatic cancer cells need both the properties of stem cells—such as the ability to self-renew and differentiate into different cell types—and the ability to multiply their numbers to grow new tumors.”

This finding may help to explain why qM cells are so uniquely good at metastasizing. Only in the qM state can the cells strongly stimulate EGFR signaling and so promote their own proliferation.

“This work gives us some mechanistic understanding of what it is about the quasi-mesenchymal state that drives metastatic tumor growth,” says Weinberg, who is also the Daniel K. Ludwig Professor for Cancer Research at the Massachusetts Institute of Technology.

The researchers hope that these insights could eventually contribute to therapies that prevent metastasis. They also hope to pursue further research into the role of ΔNp63. For example, this work illuminated a possible connection between ΔNp63 and the activation of dormant cancer cells, the cells that travel to new tissues but then cannot proliferate after they arrive there. Such dormant cells are viewed as ticking time bombs, as at any point they may reawaken. Lambert hopes that further research may reveal new insights into what causes dormant cancer cells to eventually gain the ability to grow tumors, adding to our understanding of the mechanisms of metastatic cancer.

Notes

Arthur W. Lambert, Christopher Fiore, Yogesh Chutake, Elisha R. Verhaar, Patrick C. Strasser, Mei Wei Chen, Daneyal Farouq, Sunny Das, Xin Li, Elinor Ng Eaton, Yun Zhang, Joana Liu Donaher, Ian Engstrom, Ferenc Reinhardt, Bingbing Yuan, Sumeet Gupta, Bruce Wollison, Matthew Eaton, Brian Bierie, John Carulli, Eric R. Olson, Matthew G. Guenther, Robert A. Weinberg. “ΔNp63/p73 drive metastatic colonization by controlling a regenerative epithelial stem cell program in quasi-mesenchymal cancer stem cells.” Developmental Cell, Volume 57, Issue 24,
2022, 2714-2730.e8, https://doi.org/10.1016/j.devcel.2022.11.015.

Sally Kornbluth

Education

  • Graduate: PhD, 1989, Rockefeller University
  • Undergraduate: BA, 1982, Political Science, Williams College; BS, 1984, Genetics, Cambridge University

Research Summary

Sally Kornbluth is President of MIT. Before she closed her lab to focus on administration, her research focused on the biological signals that tell a cell to start dividing or to self-destruct — processes that are key to understanding cancer as well as various degenerative disorders. She has published extensively on cell proliferation and programmed cell death, studying both phenomena in a variety of organisms. Her research has helped to show how cancer cells evade this programmed death, or apoptosis, and how metabolism regulates the cell death process; her work has also clarified the role of apoptosis in regulating the duration of female fertility in vertebrates.

Honors and Awards

  • Member, American Academy of Arts and Sciences, 2020
  • Member, National Academy of Inventors, 2018
  • Member, National Academy of Medicine, 2013
  • Distinguished Faculty Award, Duke Medical Alumni Association, 2013
  • Basic Science Research Mentoring Award, Duke University School of Medicine, 2012

Previous Administrative Leadership Positions

  • Provost, Duke University, 2014 – 2022
  • Vice Dean for Basic Sciences, Duke University School of Medicine, 2006 – 2014
Scientists unveil the functional landscape of essential genes

Researchers harness new pooled, image-based screening method to probe the functions of over 5,000 essential genes in human cells.

Nicole Davis | Whitehead Institute
November 21, 2022

A team of scientists at the Whitehead Institute for Biomedical Research and the Broad Institute of MIT and Harvard has systematically evaluated the functions of over 5,000 essential human genes using a novel, pooled, imaged-based screening method. Their analysis harnesses CRISPR-Cas9 to knock out gene activity and forms a first-of-its-kind resource for understanding and visualizing gene function in a wide range of cellular processes with both spatial and temporal resolution. The team’s findings span over 31 million individual cells and include quantitative data on hundreds of different parameters that enable predictions about how genes work and operate together. The new study appears in the Nov. 7 online issue of the journal Cell.

“For my entire career, I’ve wanted to see what happens in cells when the function of an essential gene is eliminated,” says MIT Professor Iain Cheeseman, who is a senior author of the study and a member of Whitehead Institute. “Now, we can do that, not just for one gene but for every single gene that matters for a human cell dividing in a dish, and it’s enormously powerful. The resource we’ve created will benefit not just our own lab, but labs around the world.”

Systematically disrupting the function of essential genes is not a new concept, but conventional methods have been limited by various factors, including cost, feasibility, and the ability to fully eliminate the activity of essential genes. Cheeseman, who is the Herman and Margaret Sokol Professor of Biology at MIT, and his colleagues collaborated with MIT Associate Professor Paul Blainey and his team at the Broad Institute to define and realize this ambitious joint goal. The Broad Institute researchers have pioneered a new genetic screening technology that marries two approaches — large-scale, pooled, genetic screens using CRISPR-Cas9 and imaging of cells to reveal both quantitative and qualitative differences. Moreover, the method is inexpensive compared to other methods and is practiced using commercially available equipment.

“We are proud to show the incredible resolution of cellular processes that are accessible with low-cost imaging assays in partnership with Iain’s lab at the Whitehead Institute,” says Blainey, a senior author of the study, an associate professor in the Department of Biological Engineering at MIT, a member of the Koch Institute for Integrative Cancer Research at MIT, and a core institute member at the Broad Institute. “And it’s clear that this is just the tip of the iceberg for our approach. The ability to relate genetic perturbations based on even more detailed phenotypic readouts is imperative, and now accessible, for many areas of research going forward.”

Cheeseman adds, “The ability to do pooled cell biological screening just fundamentally changes the game. You have two cells sitting next to each other and so your ability to make statistically significant calculations about whether they are the same or not is just so much higher, and you can discern very small differences.”

Cheeseman, Blainey, lead authors Luke Funk and Kuan-Chung Su, and their colleagues evaluated the functions of 5,072 essential genes in a human cell line. They analyzed four markers across the cells in their screen — DNA; the DNA damage response, a key cellular pathway that detects and responds to damaged DNA; and two important structural proteins, actin and tubulin. In addition to their primary screen, the scientists also conducted a smaller, follow-up screen focused on some 200 genes involved in cell division (also called “mitosis”). The genes were identified in their initial screen as playing a clear role in mitosis but had not been previously associated with the process. These data, which are made available via a companion website, provide a resource for other scientists to investigate the functions of genes they are interested in.

“There’s a huge amount of information that we collected on these cells. For example, for the cells’ nucleus, it is not just how brightly stained it is, but how large is it, how round is it, are the edges smooth or bumpy?” says Cheeseman. “A computer really can extract a wealth of spatial information.”

Flowing from this rich, multi-dimensional data, the scientists’ work provides a kind of cell biological “fingerprint” for each gene analyzed in the screen. Using sophisticated computational clustering strategies, the researchers can compare these fingerprints to each other and construct potential regulatory relationships among genes. Because the team’s data confirms multiple relationships that are already known, it can be used to confidently make predictions about genes whose functions and/or interactions with other genes are unknown.

There are a multitude of notable discoveries to emerge from the researchers’ screening data, including a surprising one related to ion channels. Two genes, AQP7 and ATP1A1, were identified for their roles in mitosis, specifically the proper segregation of chromosomes. These genes encode membrane-bound proteins that transport ions into and out of the cell. “In all the years I’ve been working on mitosis, I never imagined ion channels were involved,” says Cheeseman.

He adds, “We’re really just scratching the surface of what can be unearthed from our data. We hope many others will not only benefit from — but also build upon — this resource.”

This work was supported by grants from the U.S. National Institutes of Health as well as support from the Gordon and Betty Moore Foundation, a National Defense Science and Engineering Graduate Fellowship, and a Natural Sciences and Engineering Research Council Fellowship.

Genome-wide screens could reveal the liver’s secrets

A new technique for studying liver cells within an organism could shed light on the genes required for regeneration.

Anne Trafton | MIT News Office
November 15, 2022

The liver’s ability to regenerate itself is legendary. Even if more than 70 percent of the organ is removed, the remaining tissue can regrow an entire new liver.

Kristin Knouse, an MIT assistant professor of biology, wants to find out how the liver is able to achieve this kind of regeneration, in hopes of learning how to induce other organs to do the same thing. To that end, her lab has developed a new way to perform genome-wide studies of the liver in mice, using the gene-editing system CRISPR.

With this new technique, researchers can study how each of the genes in the mouse genome affects a particular disease or behavior. In a paper describing the technique, the researchers uncovered several genes important for liver cell survival and proliferation that had not been seen before in studies of cells grown in a lab dish.

“If we really want to understand mammalian physiology and disease, we should study these processes in the living organism wherever possible, as that’s where we can investigate the biology in its most native context,” says Knouse, who is also a member of MIT’s Koch Institute for Integrative Cancer Research.

Knouse is the senior author of the new paper, which appears today in Cell Genomics. Heather Keys, director of the Functional Genomics Platform at the Whitehead Institute, is a co-author on the study.

Extracellular context

As a graduate student at MIT, Knouse used regenerating liver tissue as a model to study an aspect of cell division called chromosome segregation. During this study, she observed that cells dividing in the liver did not behave the same way as liver cells dividing in a lab dish.

“What I internalized from that research was the extent to which something as intrinsic to the cell as cell division, something we have long assumed to be independent of anything beyond the cell, is clearly influenced by the extracellular environment,” she says. “When we study cells in culture, we lose the impact of that extracellular context.”

However, many types of studies, including genome-wide screens that use technologies such as CRISPR, are more difficult to deploy at the scale of an entire organism. The CRISPR gene-editing system consists of an enzyme called Cas9 that cuts DNA in a given location, directed by a strand of RNA called a guide RNA. This allows researchers to knock out one gene per cell, in a huge population of cells.

While this approach can reveal genes and proteins involved in specific cellular processes, it has proven difficult to deliver CRISPR components efficiently to enough cells in the body to make it useful for animal studies. In some studies, researchers have used CRISPR to knock out about 100 genes of interest, which is useful if they know which genes they want to study, but this limited approach doesn’t reveal new genes linked to a particular function or disease.

A few research groups have used CRISPR to do genome-wide screens in the brain and in skin cells, but these studies required large numbers of mice to uncover significant hits.

“For us, and I think many other researchers, the limited experimental tractability of mouse models has long hindered our capacity to dive into questions of mammalian physiology and disease in an unbiased and comprehensive manner,” Knouse says. “That’s what I really wanted to change, to bring the experimental tractability that was once restricted to cell culture into the organism, so that we are no longer limited in our ability to explore fundamental principles of physiology and disease in their native context.”

To get guide RNA strands into hepatocytes, the predominant cell type in the liver, Knouse decided to use lentivirus, an engineered nonpathogenic virus that is commonly used to insert genetic material into the genome of cells. She injected the guide RNAs into newborn mice, such that once the guide RNA was integrated into the genome, it would be passed on to future generations of liver cells as the mice grew. After months of effort in the lab, she was able to get guide RNAs consistently expressed in tens of millions of hepatocytes, which is enough to do a genome-wide screen in just a single animal.

Cellular fitness

To test the system, the researchers decided to look for genes that influence hepatocyte fitness — the ability of hepatocytes to survive and proliferate. To do that, they delivered a library of more than 70,000 guide RNAs, targeting more than 13,000 genes, and then determined the effect of each knockout on cell fitness.

The mice used for the study were engineered so that Cas9 can be turned on at any point in their lifetime. Using a group of four mice — two male and two female — the researchers turned on expression of Cas9 when the mice were five days old. Three weeks later, the researchers screened their liver cells and measured how much of each guide RNA was present. If a particular guide RNA is abundant, that means the gene it targets can be knocked out without fatally damaging the cells. If a guide RNA doesn’t show up in the screen, it means that knocking out that gene was fatal to the cells.

This screen yielded hundreds of genes linked to hepatocyte fitness, and the results were very consistent across the four mice. The researchers also compared the genes they identified to genes that have been linked to human liver disease. They found that genes mutated in neonatal liver failure syndromes also caused hepatocyte death in their screen.

The screen also revealed critical fitness genes that had not been identified in studies of liver cells grown in a lab dish. Many of these genes are involved in interactions with immune cells or with molecules in the extracellular matrix that surrounds cells. These pathways likely did not turn up in screens done in cultured cells because they involve cellular interactions with their external environment, Knouse says.

By comparing the results from the male and female mice, the researchers also identified several genes that had sex-specific effects on fitness, which would not have been possible to pick up by studying cells alone.

Renew and regenerate

Knouse now plans to use this system to identify genes that are critical for liver regeneration.

“Many tissues such as the heart are unable to regenerate because they lack stem cells and the differentiated cells are unable to divide. However, the liver is also a highly differentiated tissue that lacks stem cells, yet it retains this amazing capacity to regenerate itself after injury,” she says. “Importantly, the genome of the liver cells is no different from the genome of the heart cells. All of these cells have the same instruction manual in their nucleus, but the liver cells are clearly reading different sentences in this manual in order to regenerate. What we don’t know is, what are those sentences? What are those genes? If we can identify those genes, perhaps someday we can instruct the heart to regenerate.”

This new screening technique could also be used to study conditions such as fatty liver disease and cirrhosis. Knouse’s lab is also working on expanding this approach to organs other than the liver.

“We need to find ways to get guide RNAs into other tissues at high efficiency,” she says. “In overcoming that technical barrier, then we can establish the same experimental tractability that we now have in the liver in the heart or other issues.”

The research was funded by the National Institutes of Health NIH Director’s Early Independence Award, the Koch Institute Support (core) Grant from the National Cancer Institute, and the Scott Cook and Signe Ostby Fund.

Introducing the Amon Award Winners
MIT Koch Institute
October 25, 2022

Cheers to the inaugural winners of the Koch Institute’s Angelika Amon Young Scientist Award, Alejandro Aguilera and Melanie de Almeida. The new award recognizes graduate students in the life sciences or biomedical research from institutions outside the U.S. who embody Dr. Amon’s infectious enthusiasm for discovery science.

Aguilera, a student at the Weizmann Institute of Science in Israel, has developed a platform for studying mammalian embryogenesis. De Almeida, who recently completed her doctoral work at the Research Institute of Molecular Pathology in Austria, develops CRISPR screens to explore cancer vulnerabilities and gene regulatory networks.

Aguilera and de Almeida will visit the Koch Institute in November to deliver scientific presentations to the MIT community and Amon Lab alumni.

A “door” into the mitochondrial membrane

Study finds the protein MTCH2 is responsible for shuttling various other proteins into the membrane of mitochondria. The finding could have implications for cancer treatments and MTCH2-linked conditions.

Eva Frederick | Whitehead Institute
October 25, 2022

Mitochondria — the organelles responsible for energy production in human cells — were once free-living organisms that found their way into early eukaryotic cells over a billion years ago. Since then, they have merged seamlessly with their hosts in a classic example of symbiotic evolution, and now rely on many proteins made in their host cell’s nucleus to function properly.

Proteins on the outer membrane of mitochondria are especially important; they allow the mitochondria to communicate with the rest of the cell, and play a role in immune functions and a type of programmed cell death called apoptosis. Over the course of evolution, cells evolved a specific mechanism by which to insert these proteins — which are made in the cell’s cytoplasm — into the mitochondrial membrane. But what that mechanism was, and what cellular players were involved, has long been a mystery.

A new paper from the labs of MIT Professor Jonathan Weissman and Caltech Professor Rebecca Voorhees provides a solution to that mystery. The work, published Oct. 21 in the journal Science, reveals that a protein called mitochondrial carrier homolog 2, or MTCH2 for short, which has been linked to many cellular processes and even diseases such as cancer and Alzheimer’s, is responsible for acting as a “door” for a variety of proteins to access the mitochondrial membrane.

“Until now, no one knew what MTCH2 was really doing — they just knew that when you lose it, all these different things happen to the cell,” says Weissman, who is also member of the Whitehead Institute for Biomedical Research and an investigator of the Howard Hughes Medical Institute. “It was sort of a mystery why this one protein affects so many different processes. This study gives a molecular basis for understanding why MTCH2 was implicated in Alzheimer’s and lipid biosynthesis and mitochondrial fission and fusion: because it was responsible for inserting all these different types of proteins in the membrane.”

“The collaboration between our labs was essential in understanding the biochemistry of this interaction, and has led to a really exciting new understanding of a fundamental question in cell biology,” Voorhees says.

The search for a door 

In order to find out how proteins from the cytoplasm — specifically a class called tail-anchored proteins — were being inserted into the outer membranes of mitochondria, Weismann Lab postdoc and first author of the study Alina Guna, alongside Voorhees Lab graduate student Taylor Stevens and postdoc Alison Inglis, decided to use a technique called used the CRISPR interference (or CRISPRi) screening approach, which was invented by Weissman and collaborators.

“The CRISPR screen let us systematically get rid of every gene, and then look and see what happened [to one specific tail-anchored protein],” says Guna. “We found one gene, MTCH2, where when we got rid of it there was a huge decrease in how much of our protein got to the mitochondrial membrane. So we thought, maybe this is the doorway to get in.”

To confirm that MTCH2 was acting as a doorway into the mitochondrial membrane, the researchers performed additional experiments to observe what happened when MTCH2 was not present in the cell. They found that MTCH2 was both necessary and sufficient to allow tail-anchored membrane proteins to move from the cytoplasm into the mitochondrial membrane.

MTCH2’s ability to shuttle proteins from the cytoplasm into the mitochondrial membrane is likely due to its specialized shape. The researchers ran the protein’s sequence through Alpha Fold, an artificial intelligence system that predicts a protein’s structure through its amino acid sequence, which revealed that it is a hydrophobic protein — perfect for inserting into the oily membrane — but with a single hydrophilic groove where other proteins could enter.

“It’s basically like a funnel,” Guna says. “Proteins come from the cytosol, they slip into that hydrophilic groove and then move from the protein into the membrane.”

To confirm that this groove was important in the protein’s function, Guna and her colleagues designed another experiment. “We wanted to play around with the structure to see if we could change its behavior, and we were able to do that,” Guna says. “We went in and made a single point mutation, and that point mutation was enough to really change how the protein behaved and how it interacted with substrates. And then we went on and found mutations that made it less active and mutations that made it super active.”

The new study has applications beyond answering a fundamental question of mitochondria research. “There’s a whole lot of things that come out of this,” Guna says.

For one thing, MTCH2 inserts proteins key to a type of programmed cell death called apoptosis, which researchers could potentially harness for cancer treatments. “We can make leukemia cells more sensitive to a cancer treatment by giving them a mutation that changes the activity of MTCH2,” Guna says. “The mutation makes MTCH2 act more ‘greedy’ and insert more things into the membrane, and some of those things that have inserts are like pro-apoptotic factors, so then those cells are more likely to die, which is fantastic in the context of a cancer treatment.”

The work also raises questions about how MTCH2 developed its function over time. MTCH2 evolved from a family of proteins called the solute carriers, which shuttle a variety of substances across cellular membranes. “We’re really interested in this evolution question of, how do you evolve a new function from an old, ubiquitous class of proteins?” Weissman says.

And researchers still have much to learn about how mitochondria interact with the rest of the cell, including how they react to stress and changes within the cell, and how proteins find their way to mitochondria in the first place. “I think that [this paper] is just the first step,” Weissman says. “This only applies to one class of membrane proteins — and it doesn’t tell you all of the steps that happen after the proteins are made in the cytoplasm. For example, how are they ferried to mitochondria? So stay tuned — I think we’ll be learning that we now have a very nice system for opening up this fundamental piece of cell biology.”