Drug overdose, mostly from opioid use, is the leading cause of accidental death in the United States. Prior studies of twins have revealed that genetics play a key role in opioid use disorder. Researchers know that a mixture of genetic and environmental risk factors contribute to heritability of the disorder, but identifying the specific risk factors is challenging. Opioid use disorder is complex, so instead of one or a few genes causing the disorder, there may be many contributing factors that can combine in different ways. Researchers want to understand which genes contribute to opioid use disorder because this will lead to a better understanding of its underlying biology and could help identify people who will be most at risk if exposed to opioids, enabling researchers, health care providers, and social services to develop strategies for prevention, treatment, and support.
The usual approach for finding genes associated with disease risk is to do a genome wide association study, which compares the genetics of many people to identify patterns in different gene versions occurring in association with a disease. This approach is being used to look at opioid use disorder, but requires many more patient samples than are currently available to reach clear conclusions. Researchers from multiple research universities and institutes, including Whitehead Institute Member Olivia Corradin and her former PhD advisor, Case Western Reserve University Professor Peter Scacheri; as well as Icahn School of Medicine Professor Schahram Akbarian; Eric O. Johnson, a distinguished fellow at RTI International; Dr. Kiran C. Patel College of Allopathic Medicine at Nova South Eastern University Professor Deborah C. Mash; and Richard Sallari of Axiotl, Inc., developed a shortcut for identifying genes that are associated with opioid use disorder and may contribute to it using only a small number of patient samples. Genome wide studies may require hundreds of thousands of samples, but this new method, described in their research published in the journal Molecular Psychiatry on March 17, uses only around 100 samples—51 cases and 51 controls—to narrow in on five candidate genes.
“With this work, we think we’re only seeing the tip of the iceberg of the complex, diverse factors contributing to opioid overdose,” says Corradin, who is also an assistant professor of biology at the Massachusetts Institute of Technology. “However, we hope our findings can help prioritize genes for further study, to speed up the identification of risk markers and possible therapeutic targets.”
In order to learn more about the underlying biology of opioid use disorder, the researchers analyzed brain tissue samples from people who had died of opioid overdoses and compared them with samples from people with no known opioid use history who died of other accidental causes. They specifically looked at neurons from the dorsolateral prefrontal cortex, an area of the brain known to play important roles in addiction. Instead of analyzing the genes in these cells directly, the researchers instead looked at the regulators of the genes’ activity, and searched for changes in these regulators that could point them to genes of interest.
To identify a gene, first map its community
Genes have DNA regions, often close to the gene, that can ratchet up and down the gene’s expression, or the strength of its activity in certain cells. Researchers have only recently been able to map the three-dimensional organization of DNA in a cell well enough to identify all of the regulators that are close to and acting upon target genes. Corradin and her collaborators call a gene’s collection of close regulatory elements its “plexus.” Their approach finds genes of interest by searching for patterns of variation across each gene’s entire plexus, which can be easier to spot with a small sample size.
The patterns that the researchers look for in a plexus are epigenetic changes: differences in the chemical tags that affect regulatory DNA and in turn, modify the expression of the regulators’ target gene. In this case, the researchers looked at a type of epigenetic tag called H3K27 acetylation, which is linked to increases in the activity of regulatory regions. They found nearly 400 locations in the DNA that consistently had less H3K27 acetylation in the brains of people who died of opioid overdose, which would lower activity of target genes. They also identified under-acetylated DNA locations that were often specific to individuals rather than uniform across all opioid overdose cases. The researchers then looked at how many of those locations belonged to regulatory elements in the same plexus. Surprisingly, these individual-specific changes often occurred within the same gene’s plexus. A gene whose plexus had been heavily affected as a collective was flagged as a possible contributor to opioid use disorder.
“We know that the factors that contribute to opioid use disorder are numerous, and that it’s an extremely complex disease that by definition is going to be extremely heterogeneous,” Scacheri says. “The idea was to figure out an approach that embraces that heterogeneity, and then try to spot the themes within it.”
Using this approach, the researchers identified five candidate genes, ASTN2, KCNMA1, DUSP4, GABBR2, and ENOX1. One of the genes, ASTN2, is related to pain tolerance, while KCNMA1, DUSP4, and GABBR2 are active in signaling pathways that have been linked more broadly to addiction. Follow up experiments can confirm whether these genes contribute to opioid use disorder.
The five genes and their plexi are also involved in the heritability of generalized anxiety disorder, metrics of tolerance for risk-taking, and educational attainment. Heritability of these traits and opioid use disorder have previously been found to coincide, and people with opioid use disorder often also have generalized anxiety. Furthermore, heritability of these traits and opioid use disorder all have been associated with early childhood adversity. These connections suggest the possibility that early childhood adversity could be contributing to the epigenetic changes observed by the researchers in the brains of people who died of opioid overdose—a useful hypothesis for further research.
The researchers hope that these results will provide some insights into the genetics and neurobiology of opioid use disorder. They are interested in moving their research forward in several ways: they would like to see if they can identify more candidate genes by increasing their sample number, examine different parts of the brain and different cell types, and further analyze the genes already identified. They also hope that their results demonstrate the potency of their approach, which was able to discern useful patterns and identify candidate genes from the neurons of only 51 cases.
“We’re trying a different approach here that relies on this idea of convergence and leverages our understanding of the three-dimensional architecture of DNA, and I hope this approach will be applied to further our understanding of all sorts of complex diseases,” Scacheri says.
Eva Frederick | Whitehead Institute
April 20, 2022
The Australian stinging tree (Dendrocnide moroides) is a plant that many peopleavoid at all costs. The tree, which is a member of the nettle family, is covered in thin silicon needles laced with one of nature’s most excruciating toxins, a compound called moroidin. “It’s notorious for causing extreme pain, which lingers for a very long time,” said Whitehead Institute Member Jing-Ke Weng.
There’s another side to moroidin, though; in addition to causing pain, the compound binds to cells’ cytoskeletons, preventing them from dividing, which makes moroidin a promising candidate for chemotherapy drugs.
Harvesting enough of the chemical to study has proven difficult, for obvious reasons. Now, in a paper published April 19 in the Journal of the American Chemical Society, Weng, who is also an associate professor of biology at the Massachusetts Institute of Technology (MIT) and former postdoc Roland Kersten, now an assistant professor at the University of Michigan College of Pharmacy, present the first published method to biosynthesize moroidin within the tissues of harmless plants such as tobacco, facilitating research on the compound’s utility for cancer treatments.
Taking a leaf out of plants’ book to create peptides
Moroidin is a bicyclic peptide — a type of molecule made up of building blocks called amino acids and circularized to contain two connected rings. For synthetic chemists, moroidin has proved nearly impossible to synthesize due to its complex chemical structure. Weng and Kersten wanted to dig deeper into what methods the plants were using to create this molecule.
In plant cells, cyclic peptides are made from specific precursor proteins synthesized by the ribosome, the macromolecular machine that produces proteins by translating messenger RNAs. After leaving the ribosome, these precursor proteins are further processed by other enzymes in the cell to give rise to the final cyclic peptides. In 2018, Weng and Kersten had elucidated the biosynthetic mechanism of another type of plant peptides called lyciumins, first found in the goji berry plant, which gave them some insight into how post-translational modifications might play a role in creating different types of plant peptide chemistry. “We learned a lot about the principal elements of this system by studying lyciumins,” said Weng.
When they began to look into how moroidin was synthesized, the researchers found a few other plants, such as Kerria japonica and Celosia argentea, also produce peptides with similar chemistry to moroidin. “That really gave us the very critical insight that this is a new class of peptides,” Weng said.
Weng and Kersten previously learned that the BURP domain, which is part of the precursor proteins for lyciumins and several other plant cyclic peptides, catalyzes key reactions involved in the peptide ring formation. They found that the BURP domain was present in the precursor proteins for moroidins in Kerria japonica, and seemed to be essential for creating the two-ring structure of the molecules. The BURP domain creates ring chemistry when in the presence of copper, and when the researchers incubated the moroidin precursor protein with copper chloride in the lab together with other downstream proteolytic enzymes, they were able to create moroidin-like peptides.
With this information, they were able to produce a variety of moroidin analogs in tobacco plants by transgenically expressing the moroidin precursor gene of Kerria japonica and varying the core motif sequence corresponding to moroidin peptides. “We show that you can produce the same moroidin chemistry in a different host plant,” Weng said. “Tobacco itself is easier to be farmed on a large scale, and we also think in the future we can derive a plant cell line from the existing tobacco cell lines that we put in the moroidin precursor peptide, then we can use the cell line to produce the molecule, which really enables us to scale up for medicine production.”
Future use of moroidin
Moroidin’s anti-cancer property is due, at least in part, to the compound’s unique structure that allows it to bind to a protein called tubulin. Tubulin forms a skeletal system for living cells, and provides the means by which cells separate their chromosomes as they prepare to divide. Currently, two existing anti-cancer drugs, vincristine and paclitaxel, work by binding tubulin. These two compounds are derived from plants as well (the Madagascar periwinkle and Pacific yew tree, respectively).
In their new work, Weng and Kersten synthesized a moroidin analog called celogentin C. They tested its anti-cancer activity against a human lung cancer cell line, and found that the compound was toxic to the cancer cells. Their new study also suggests potentially new anti-cancer mechanisms specific to this lung cancer cell line in addition to tubulin inhibition.
In the past, researchers have run into issues when trying to create effective drugs from peptides. “There are two major challenges for peptides as medicine,” Weng said. “For one thing they are not very stable in vivo, and for another they are not very bioavailable and don’t readily pass the membrane of a cell.”
But cyclic peptides like moroidin and its analogs are a bit different. “These peptides essentially evolve to be drug-like,” Weng said. “In the case of the Australian stinging tree, the peptides are present because the plants want to deter any animals that want to eat the leaves. So over millions of years of evolution these plants eventually figured out a way to construct these specific cyclic peptides that are stable, bioavailable and can get to the animal that is trying to eat the plants.”
It’s likely that the painful reaction that occurs when moroidin enters the body through a sting from the tree would not be an issue in traditional methods of administering chemotherapy. “The pain is really caused if you get injections of the compound into the skin,” Weng said. “If you take it orally or intravenously, your body will most likely not sense the pain.”
Somewhat counterintuitively, the compound could also be used as a pain reliever. “If something causes pain, you can sometimes use that as an anti-pain medicine,” Weng said. “You could essentially exhaust the pain receptors, or if you alter the structure a little bit, you could turn an agonist into an antagonist and potentially block the pain.”
On a more fundamental level, moroidin could help researchers study pain receptors. “We don’t know exactly why being stung by the stinging tree produces that enormous amount of pain, and there may be additional pain receptors people haven’t identified,” Weng said. “Being able to synthesize moroidin provides a chemical probe that allows us to study this unknown pain perception in humans.”
In the future, the researchers hope to create analogs of moroidin to study, and hopefully create an optimal version for use in cancer therapy. “We want to generate a library of moroidin-like peptides,” Weng said. “We’ve done this for lyciumins, and since the initial moroidins are anti-tubulin molecules, we can use this system to find an improved version that binds to tubulin even tighter and contains other pharmacological properties making it suitable to be used as a therapeutic.
Greta Friar | Whitehead Institute
April 20, 2022
Stem cells are the versatile building blocks from which every cell type in the body, from neurons, to skin cells, to blood cells, is ultimately descended. Researchers have also figured out how to turn stem cells into different cell types in the lab, which has been helpful for studying health and disease in their normal cellular contexts, and could be used to generate cells for medical transplants. Whitehead Institute Founding Member Rudolf Jaenisch not only uses these cells in his research, but has spent much of his career discovering and improving the methods for making accurate laboratory models out of stem cell-derived cells.
One challenge that Jaenisch’s lab is focusing on is how to eliminate the differences between cell types as they are found in the body and their stem cell-derived equivalents. In particular, they have found that stem-cell derived cells are often immature, more closely resembling the cells found in fetuses rather than in adults. These differences can make the cells less accurate research models and prevent them from being medically useful as functional transplant cells. Stem cells in the body receive complex cocktails of molecular signals as they transform into different cell types. The challenge for researchers lies in figuring out which of the many molecular signals in the body are relevant and then get the recipe exactly right in their recreations.
Postdoc Haiting Ma in the Jaenisch lab decided to tackle this problem for hepatocytes, the main type of cell in the liver. In work published in Cell Stem Cell on April 21, Jaenisch and Ma share their findings on why stem cell-derived liver cells resemble fetal liver cells, and what’s needed to make them mature—including an important role for a thyroid hormone.
The liver filters everything that enters the body through the digestive system. It helps to store and modify nutrients, safely break down toxins and waste, process medications, and more. There is still a lot to learn about how the liver functions, and what goes wrong in a number of liver-associated diseases, and accurate stem cell-derived models will help with that research. Liver cells are also needed to treat end-stage liver disease, and if researchers could mass produce stem cell-derived liver cells that can function safely in an adult liver, this could help to meet the demand for liver cell transfusions.
For this study, Jaenisch and Ma grew liver cells from stem cells in two setups: a typical 2D culture, in which the cells were grown in a dish, and a 3D spheroid, in which cells that started out in the normal culture were then allowed to grow into three-dimensional balls of cells. The spheroids can be designed to mimic some aspects of the cells’ natural environment in ways that a 2D culture cannot. In each case, the researchers exposed the cells to a carefully timed mixture of signals to prompt them to develop into liver cells. The researchers then analyzed cells from both the 2D and 3D cultures and compared them to primary liver cells, or cells from a body, using a variety of techniques to look for differences related to DNA and gene expression. They found that the cells cultivated in the 3D system were closer to cells from the adult body than those in the 2D system.
“The 3D culture not only contributes to maturation of the liver cells, but it can also be used to scale up production of the cells, which could be very useful for cell therapies in the future,” Ma says.
However, both sets of lab-derived cells lacked important features of adult liver cells. The analyses pointed to one important missing factor in particular: in the adult liver cells, a hormone receptor called Thyroid Hormone Receptor Beta (THRB) binds to a number of places in the DNA. THRB then senses the presence or absence of thyroid hormones, and regulates a variety of gene expression processes accordingly. However, the researchers found that while the stem cell-derived liver cells made the right amount of THRB, something was preventing it from binding where it should and performing its function.
Normally, THRB has a partner that helps it bind to DNA, the thyroid hormone T3. When the researchers added T3 to their 2D and 3D cultures, this led to more typical binding of THRB, which in turn made the cells—especially the cells from the 3D culture—more closely resemble adult liver cells in a number of ways. Improved THRB binding increased the expression of key liver genes, restored the activity of regulatory elements in the DNA that modify gene expression, and reduced the expression of a fetal liver gene. The researchers also gained insights into the molecules that THRB interacts with and the mechanisms by which it affects liver maturation, painting a more complete picture of its key roles in liver cells.
Altogether, this work led to a better recipe for making adult liver cells from stem cells in the lab–using the 3D spheroid culture and adding T3. When cells developed with this approach were incorporated into the livers of mice, the cells integrated successfully and the liver maintained normal function long term.
The new and improved stem cell-derived liver cells are still not a perfect match for adult liver cells—the researchers have ideas about which missing characteristics they could tackle next—but the current cells’ ability to seamlessly integrate into the liver, as well as indicators from the analyses that they would be good models for liver-associated diseases, suggest that they will be useful in a variety of projects.
“As we improve the authenticity of our stem cell-derived cell types, we open up new opportunities for research,” Jaenisch says. “We can build more accurate models in which to study high-impact diseases, such as liver diseases, diabetes, and chronic viral infections, and using those models we can develop strategies for treatment and prevention.”
Merrill Meadow | Whitehead Institute
April 20, 2022
This year’s Vanderbilt Prize in Biomedical Science will be awarded to Whitehead Institute director Ruth Lehmann. It recognizes women scientists with a stellar record of research accomplishments who also have made significant contributions to mentoring other women in science.
“Dr. Lehmann’s determination to solve the deepest mysteries of life while encouraging others at the beginning of their careers exemplifies the spirit of the Vanderbilt Prize in Biomedical Science,” says Jeff Balser, president and chief executive officer of Vanderbilt University Medical Center – which bestows the Prize – and dean of Vanderbilt University School of Medicine. “I’m honored to congratulate her as the 2022 Vanderbilt Prize recipient.”
“I’m thrilled to be receiving this honor, recognizing the importance of mentoring and empowering the next generation of scientists,” says Lehmann, who has mentored scores of students and research fellows during her career, and has developed a mentorship program specifically designed to encourage and empower junior faculty in science. Read more here.
Graduate student En Ze Linda Zhong-Johnson is creating new methods to measure and enhance enzyme activity — which she hopes will help restore a plastic-choked world.
Grace van Deelen
April 21, 2022
After graduating with her undergraduate degree in molecular genetics from the University of Toronto in 2016, En Ze Linda Zhong-Johnson celebrated with a trip to Alaska. There, she saw a pristine landscape unlike the plastic-littered shores of the Toronto waterfront. “What I saw up there was so different from what I saw in the city,” Zhong-Johnson says. “I realized there shouldn’t be all this waste floating everywhere, in our water, in our environment. It’s not natural.”
As a trained biologist, Zhong-Johnson began to think about the problem of plastic pollution from a biological perspective. One solution, she thought, could be biological recycling: a process by which living organisms break down materials, using digestion or other metabolic processes to turn these materials into smaller pieces or new compounds. Composting, for example, is a type of biological recycling — microbes in the soil break down discarded food, speeding up the decomposition process. Zhong-Johnson wondered if there were any organisms on Earth that could use the carbon in polyethylene terephthalate (PET), a common plastic used in water bottle and food packaging, as an energy source.
Earlier that same year, Japanese scientists discovered that a bacterium, Ideonella sakaiensis, could do just that by producing enzymes that could break down PET. The two main PET-degrading enzymes, referred to as IsPETase and IsMHETase, are able to turn PET into two chemical compounds, terephthalic acid and ethylene glycol, which I. sakaiensis can use for food.
The discovery of these enzymes opened up many new questions and possible applications that scientists have continued to work on since. However, because there was — and still is — much to learn about PET-degrading enzymes, they are still not widely used to recycle consumer products. Zhong-Johnson figured that, in graduate school, she could build on the existing IsPETase research and help to accelerate their use at recycling facilities. Specifically, she wanted to engineer the enzyme to work faster at lower temperatures, and study how, fundamentally, the enzymes worked on the surface of PET plastic to degrade it.
“I hopped on the excitement train, along with the rest of the world,” she says.
A better enzyme
After receiving her acceptance to MIT to complete her PhD, Zhong-Johnson approached various professors, pitching her idea to speed up IsPETase activity. Christopher Voigt, the Daniel I. C. Wang Professor of Biological Engineering, and Anthony Sinskey, professor of biology, were interested, and formed a co-advisorship to support Zhong-Johnson’s project. Sinskey, in particular, was impressed by her idea to help solve the world’s plastic problem with PET-degrading enzymes.
Zhong-Johnson screens for enzyme variants with improved activity. Credit: Grace van Deelen
“Plastic pollution is a big problem,” he says, “and that’s the kind of problem my lab likes to tackle.” Plus, he says, he feels “committed to helping graduate students who want to apply their science and technology learnings to the environment.”
While the idea of a plastic-degrading enzyme seems like a panacea, the enzyme’s practical applications have been limited by its biology. The wild-type IsPETase is a mesophilic enzyme, meaning the structure of the enzyme is only stable around ambient temperatures, and the enzyme loses its activity above that threshold. This restriction on temperature limits the number and types of facilities that can use IsPETase, as well as the rate of the enzyme reaction, and drives up the cost of their use.
However, Zhong-Johnson thinks that, with combined approaches of biological and chemical engineering, it’s possible to scale up the use of the enzymes by increasing their stability and activity. For example, an enzyme that’s highly active at lower temperatures could work in unheated facilities, or even be sprinkled directly into landfills or oceans to degrade plastic waste — a process called bioremediation. Increasing the activity of the enzyme at ambient temperatures could also expand the possible applications.
“Most of the environments where plastic is present are not above 50 degrees Celsius,” said Zhong-Johnson. “If we can increase enzyme activity at lower temperatures, that’s really interesting for bioremediation purposes.”
Now a fifth-year graduate student, Zhong-Johnson has honed her project, and is focusing on increasing the activity of IsPETase. To do so, she’s using directed evolution — creating random mutations in the IsPETase gene, and selecting for IsPETase variants that digest PET faster. When they do, she combines the beneficial mutations and uses that as template for the next round of library generation, to improve the enzyme even further. The evolution is “directed” because Zhong-Johnson herself, rather than nature, is picking out which gene sequences of enzyme proceed through to the next round of random mutagenesis, and which don’t. Her ultimate goal is to create a more efficient and hardier enzyme that will, hopefully, work faster at ambient temperatures.
A better protocol
Just as Zhong-Johnson was beginning her project, she ran into an obstacle: There wasn’t a standard way to measure whether her experiments were successful. In particular, no immediately applicable method existed to measure enzyme kinetics for IsPETases on solid substrates like plastic bottles and other plasticware. That was a problem for Zhong-Johnson because understanding enzyme activity was a crucial part of how she selected her enzymes in the directed evolution process.
Usually, enzyme activity is measured via product accumulation: When enzymes metabolize a substance, they create a new substance in return, called a product. Measuring the amount of product created by an enzyme after a certain amount of time gives the researcher a snapshot of that enzyme’s activity.
There are two problems with the product accumulation method, though. First, it is usually done using liquid or soluble substrates. In other words, the material that the enzyme is targeting is dissolved, like sugar dissolved in water. Then, the enzyme is added to that liquid concoction and mixed evenly throughout. However, the substrate Zhong-Johnson wanted to use — PET — was not soluble but solid, meaning it could not be evenly distributed like a soluble substrate. Second, the product accumulation measurement methods available were only practical for measuring less than a handful of timepoints for a few enzyme or substrate concentrations. As a result, many in the field opted to measure a single time point, late in the enzyme reaction, which doesn’t provide an indication of how an enzyme’s rate of digestion actually changes over the course of time — something that can be measured through kinetic measurements.
Taking kinetic measurements would help researchers like Zhong-Johnson illustrate the full pattern of enzyme activity and answer questions like: When is the enzyme most active? Does most product accumulation happen at the beginning of the reaction or the end? How does temperature impact the rate of these reactions over time? To answer these questions, she realized she would have to develop the method herself.
Through a serendipitous discussion with a group of chemical engineering undergraduate students that Zhong-Johnson was mentoring, she came up with a solution, which she published in a 2021 paper in Scientific Reports. The undergraduates brought to her attention many factors that she had overlooked about the enzyme, and she says she would not have realized the importance of kinetic measurements if it weren’t for the fact that she was trying to design an experiment that the undergraduates could perform over the course of three hours.
The paper outlined a new way to measure enzyme activity, which Zhong-Johnson calls “the bulk absorbance method.” Instead of measuring the final product accumulation at very late time points, the bulk absorbance method involves taking multiple kinetic measurements at early time intervals during the experiment. This technique informs Zhong-Johnson’s directed evolution approach: If she can find which enzymes are most active at low temperatures, she can select the best possible enzyme for the next round of analyses. She hasn’t yet engineered an enzyme she’s completely happy with, but she’s gotten much closer to her ultimate goal.
Solving big problems together
Zhong-Johnson’s discoveries have been made possible by the collaboration between her and her two co-advisors, Voigt and Sinskey, who have supported her independence throughout her five years at MIT.
Zhong-Johnson and her advisor, professor Anthony Sinskey, in his office. Credit: Grace van Deelen
When she first started her graduate work, neither Voigt nor Sinskey had expertise in enzyme biochemistry involving solid substrates: Sinkey’s lab focuses on bacterial metabolism, while Voigt’s lab focuses on genetic engineering (though Voigt did have experience with directed evolution research). Additionally, Zhong-Johnson’s path to her project was rather unconventional. Most grad students do not come to potential advisors proposing entire dissertations, which posed a unique challenge for Zhong-Johnson.
Despite not having specific expertise in enzyme biochemistry involving solid substrates, Voigt and Sinskey have supported Zhong-Johnson in other ways: by helping her to develop critical thinking skills and connecting her to other people in her field, such as potential collaborators, who can help her project thrive in the future. Zhong-Johnson has supplemented her MIT experience by having enzyme experts as part of her dissertation committee as well.
Sinskey says that, in the future — once Zhong-Johnson has engineered the ideal enzyme — they would like to partner with industry, and work on making the enzyme into a product that waste companies might use to recycle plastic. Additionally, Sinskey says, the plastic problem and the IsPETase solution raise so many interesting questions that Zhong-Johnson’s project will probably live on in the Voigt and Sinskey labs even after she graduates. He’d like to see other graduate students working to understand the enzyme’s activity and progressing the directed evolution that Zhong-Johnson started.
Zhong-Johnson is already working on understanding the specifics of how IsPETase act on PET. “How does it eat a hole in a plastic bottle? How does it move along and make the hole bigger as it moves through the process? Does it jump around? Or does it keep degrading a single polymer chain until its completely broken down? We just don’t know the answers yet,” says Sinskey.
But Zhong-Johnson is up to the task. “My graduate students have to have three skills, in my opinion,” Sinskey says. “One, they have to be intelligent. Two, they have to be energetic, and three, they have to be of high integrity, in research and behavior.” Zhong-Johnson, he says, has all three qualities.
A Climate Grand Challenges flagship project aims to reduce agriculture-driven emissions while making food crop plants heartier and more nutritious.
Merrill Meadow | Whitehead Institute
April 20, 2022
On April 11, MIT announced five multiyear flagship projects in the first-ever Climate Grand Challenges, a new initiative to tackle complex climate problems and deliver breakthrough solutions to the world as quickly as possible. This article is the fourth in a five-part series highlighting the most promising concepts to emerge from the competition and the interdisciplinary research teams behind them.
The impact of our changing climate on agriculture and food security — and how contemporary agriculture contributes to climate change — is at the forefront of MIT’s multidisciplinary project “Revolutionizing agriculture with low-emissions, resilient crops.” The project The project is one of five flagship winners in the Climate Grand Challenges competition, and brings together researchers from the departments of Biology, Biological Engineering, Chemical Engineering, and Civil and Environmental Engineering.
“Our team’s research seeks to address two connected challenges: first, the need to reduce the greenhouse gas emissions produced by agricultural fertilizer; second, the fact that the yields of many current agricultural crops will decrease, due to the effects of climate change on plant metabolism,” says the project’s faculty lead, Christopher Voigt, the Daniel I.C. Wang Professor in MIT’s Department of Biological Engineering. “We are pursuing six interdisciplinary projects that are each key to our overall goal of developing low-emissions methods for fertilizing plants that are bioengineered to be more resilient and productive in a changing climate.”
Whitehead Institute members Mary Gehring and Jing-Ke Weng, plant biologists who are also associate professors in MIT’s Department of Biology, will lead two of those projects.
Promoting crop resilience
For most of human history, climate change occurred gradually, over hundreds or thousands of years. That pace allowed plants to adapt to variations in temperature, precipitation, and atmospheric composition. However, human-driven climate change has occurred much more quickly, and crop plants have suffered: Crop yields are down in many regions, as is seed protein content in cereal crops.
“If we want to ensure an abundant supply of nutritious food for the world, we need to develop fundamental mechanisms for bioengineering a wide variety of crop plants that will be both hearty and nutritious in the face of our changing climate,” says Gehring. In her previous work, she has shown that many aspects of plant reproduction and seed development are controlled by epigenetics — that is, by information outside of the DNA sequence. She has been using that knowledge and the research methods she has developed to identify ways to create varieties of seed-producing plants that are more productive and resilient than current food crops.
But plant biology is complex, and while it is possible to develop plants that integrate robustness-enhancing traits by combining dissimilar parental strains, scientists are still learning how to ensure that the new traits are carried forward from one generation to the next. “Plants that carry the robustness-enhancing traits have ‘hybrid vigor,’ and we believe that the perpetuation of those traits is controlled by epigenetics,” Gehring explains. “Right now, some food crops, like corn, can be engineered to benefit from hybrid vigor, but those traits are not inherited. That’s why farmers growing many of today’s most productive varieties of corn must purchase and plant new batches of seeds each year. Moreover, many important food crops have not yet realized the benefits of hybrid vigor.”
The project Gehring leads, “Developing Clonal Seed Production to Fix Hybrid Vigor,” aims to enable food crop plants to create seeds that are both more robust and genetically identical to the parent — and thereby able to pass beneficial traits from generation to generation.
The process of clonal (or asexual) production of seeds that are genetically identical to the maternal parent is called apomixis. Gehring says, “Because apomixis is present in 400 flowering plant species — about 1 percent of flowering plant species — it is probable that genes and signaling pathways necessary for apomixis are already present within crop plants. Our challenge is to tweak those genes and pathways so that the plant switches reproduction from sexual to asexual.”
The project will leverage the fact that genes and pathways related to autonomous asexual development of the endosperm — a seed’s nutritive tissue — exist in the model plant Arabidopsis thaliana. In previous work on Arabidopsis, Gehring’s lab researched a specific gene that, when misregulated, drives development of an asexual endosperm-like material. “Normally, that seed would not be viable,” she notes. “But we believe that by epigenetic tuning of the expression of additional relevant genes, we will enable the plant to retain that material — and help achieve apomixis.”
If Gehring and her colleagues succeed in creating a gene-expression “formula” for introducing endosperm apomixis into a wide range of crop plants, they will have made a fundamental and important achievement. Such a method could be applied throughout agriculture to create and perpetuate new crop breeds able to withstand their changing environments while requiring less fertilizer and fewer pesticides.
Creating “self-fertilizing” crops
Roughly a quarter of greenhouse gas (GHG) emissions in the United States are a product of agriculture. Fertilizer production and use accounts for one third of those emissions and includes nitrous oxide, which has heat-trapping capacity 298-fold stronger than carbon dioxide, according to a 2018 Frontiers in Plant Science study. Most artificial fertilizer production also consumes huge quantities of natural gas and uses minerals mined from nonrenewable resources. After all that, much of the nitrogen fertilizer becomes runoff that pollutes local waterways. For those reasons, this Climate Grand Challenges flagship project aims to greatly reduce use of human-made fertilizers.
One tantalizing approach is to cultivate cereal crop plants — which account for about 75 percent of global food production — capable of drawing nitrogen from metabolic interactions with bacteria in the soil. Whitehead Institute’s Weng leads an effort to do just that: genetically bioengineer crops such as corn, rice, and wheat to, essentially, create their own fertilizer through a symbiotic relationship with nitrogen-fixing microbes.
“Legumes such as bean and pea plants can form root nodules through which they receive nitrogen from rhizobia bacteria in exchange for carbon,” Weng explains. “This metabolic exchange means that legumes release far less greenhouse gas — and require far less investment of fossil energy — than do cereal crops, which use a huge portion of the artificially produced nitrogen fertilizers employed today.
“Our goal is to develop methods for transferring legumes’ ‘self-fertilizing’ capacity to cereal crops,” Weng says. “If we can, we will revolutionize the sustainability of food production.”
The project — formally entitled “Mimicking legume-rhizobia symbiosis for fertilizer production in cereals” — will be a multistage, five-year effort. It draws on Weng’s extensive studies of metabolic evolution in plants and his identification of molecules involved in formation of the root nodules that permit exchanges between legumes and nitrogen-fixing bacteria. It also leverages his expertise in reconstituting specific signaling and metabolic pathways in plants.
Weng and his colleagues will begin by deciphering the full spectrum of small-molecule signaling processes that occur between legumes and rhizobium bacteria. Then they will genetically engineer an analogous system in nonlegume crop plants. Next, using state-of-the-art metabolomic methods, they will identify which small molecules excreted from legume roots prompt a nitrogen/carbon exchange from rhizobium bacteria. Finally, the researchers will genetically engineer the biosynthesis of those molecules in the roots of nonlegume plants and observe their effect on the rhizobium bacteria surrounding the roots.
While the project is complex and technically challenging, its potential is staggering. “Focusing on corn alone, this could reduce the production and use of nitrogen fertilizer by 160,000 tons,” Weng notes. “And it could halve the related emissions of nitrous oxide gas.”
Seven staff members are recognized for their dedication to the School of Science and to MIT.
School of Science
April 15, 2022
The MIT School of Science has announced the winners of the 2022 Infinite Mile Award. The selected staff members were nominated by their colleagues for going above and beyond in their roles at the Institute. Their outstanding contributions have made MIT a better place.
The following are the 2022 Infinite Mile Award winners in the School of Science:
• Christina Andujar, senior administrative assistant in the Department of Physics, was nominated by Peter Fisher, Edmund Bertschinger, and Matt Cubstead because Andujar “has gone far beyond her assigned role and duties to improve the lives of a great many students at MIT.”
• Monika Avello, an instructor in the Department of Biology, was nominated by Barbara Imperiali, Cathy Drennan, Graham Walker, Adam Martin, Lenny Guarente, David Des Marais, Seychelle Vos, and Jing-Ke Weng because Avello “was always meticulous in attention to detail and never hesitated when we threw out crazy ideas that might make the students gain something unique from the class — even if it gave her ever more things to do.”
• David Orenstein, director of communications in The Picower Institute for Learning and Memory, was nominated by Li-Huei Tsai, Mriganka Sur, Earl Miller, Gloria Choi, William Lawson, Asha Bhakar, Julie Pryor, Raleigh McElvery, and Julia Keller because Orenstein is “always willing to help out in whatever way is needed, whether as a part of a brainstorming session about any given topic, or lending a helping hand for an event or something else going on with the Institute. His dedication to the mission of the Picower Institute is unquestionable and it is evident in everything he does.”
• Dennis Porche, assistant to the department head in the Department of Mathematics, was nominated by Michel Goemans, Gigliola Staffilani, Michael Sipser, and Amanda Kuhl because Porche “has been amazingly dedicated to the well-being of the mathematics department at MIT, and cares tremendously about everything that goes on in the department. He will spend many hours making sure everything is perfect, nothing or no one is omitted, everyone is properly acknowledged, and everything goes smoothly.”
• Joshua Stone, administrative assistant in the Department of Biology, was nominated by Michael Laub, Hallie Dowling-Huppert, Alex Pike, Rebecca Chamberlain, and Janice Chang because Stone “has driven a movement to create an inclusive environment for staff within the biology department, implementing programs for welcoming new staff and establishing peer mentoring to increase the sense of inclusion within the department. These efforts are essential to shifting the culture and integrating pillars of DEI into the everyday operations of the biology department.”
• Sierra Vallin, academic administrator in the Department of Brain and Cognitive Sciences, was nominated by Michale Fee, Laura Schulz, Rebecca Saxe, Joshua McDermott, Mehrdad Jazayeri, Mark Harnett, Kate White, Laura Frawley, Kian Caplan, Di Kang, Halie Olson, Tobias Kaiser, and Julianne Ormerod because Vallin is “truly incredible” and “goes way above and beyond the call of duty to help students and other staff,” and for her “willingness to stand up for staff throughout our building, and to support our ongoing diversity efforts.”
• Shannon Wagner, senior administrative assistant in the Department of Chemistry, was nominated by Troy Van Voorhis, Stephen Buchwald, Jeremiah Johnson, Rick Danheiser, Richard Wilk, and Jennifer Weisman because Wagner “is someone who goes far above and beyond her usual call of duty. Her work has positively impacted many in the department including our students. She demonstrates an exceptional commitment to every aspect of her work and the staff with whom she works. Our department is a far better place with her in it.”
Innovative brain-wide mapping study shows that “engrams,” the ensembles of neurons encoding a memory, are widely distributed, including among regions not previously realize
Picower Institute
April 12, 2022
A new study by scientists at The Picower Institute for Learning and Memory at MIT provides the most comprehensive and rigorous evidence yet that the mammalian brain stores a single memory across a widely distributed, functionally connected complex spanning many brain regions, rather than in just one or even a few places.
Memory pioneer Richard Semon had predicted such a “unified engram complex” more than a century ago, but achieving the new study’s affirmation of his hypothesis required the application of several technologies developed only recently. In the study, the team identified and ranked dozens of areas that were not previously known to be involved in memory and showed that memory recall becomes more behaviorally powerful when multiple memory-storing regions are reactivated, rather than just one.
“When talking about memory storage we all usually talk about the hippocampus or the cortex,” said co-lead and co-corresponding author Dheeraj Roy. He began the research while a graduate student in the RIKEN-MIT Laboratory for Neural Circuit Genetics at The Picower Institute led by senior author Susumu Tonegawa, Picower Professor in the Departments of Biology and Brain and Cognitive Sciences. “This study reflects the most comprehensive description of memory encoding cells, or memory ‘engrams,’ distributed across the brain, not just in the well-known memory regions. It basically provides the first rank-ordered list for high-probability engram regions. This list should lead to many future studies, which we are excited about, both in our labs and by other groups.”
In addition to Roy, who is now a McGovern Fellow in the Broad Institute of MIT and Harvard and the lab of MIT neuroscience Professor Guoping Feng, the study’s other lead authors are Young-Gyun Park, Minyoung Kim, Ying Zhang and Sachie Ogawa.
Mapping Memory
The team was able to map regions participating in an engram complex by conducting an unbiased analysis of more than 247 brain regions in mice who were taken from their home cage to another cage where they felt a small but memorable electrical zap. In one group of mice their neurons were engineered to become fluorescent when they expressed a gene required for memory encoding. In another group, cells activated by naturally recalling the zap memory (e.g. when the mice returned to the scene of the zap) were fluorescently labeled instead. Cells that were activated by memory encoding or by recall could therefore readily be seen under a microscope after the brains were preserved and optically cleared using a technology called SHIELD, developed by co-corresponding author Kwanghun Chung, Associate Professor in The Picower Institute, the Institute for Medical Engineering & Science and the Department of Chemical Engineering. By using a computer to count fluorescing cells in each sample, the team produced brain-wide maps of regions with apparently significant memory encoding or recall activity.
The maps highlighted many regions expected to participate in memory but also many that were not. To help factor out regions that might have been activated by activity unrelated to the zap memory, the team compared what they saw in zap-encoding or zap-recalling mice to what they saw in the brains of controls who were simply left in their home cage. This allowed them to calculate an “engram index” to rank order 117 brain regions with a significant likelihood of being involved in the memory engram complex. They deepened the analysis by engineering new mice in which neurons involved in both memory encoding and in recall could be doubly labeled, thereby revealing which cells exhibited overlap of those activities.
To really be an engram cell, the authors noted, a neuron should be activated both in encoding and recall.
“These experiments not only revealed significant engram reactivation in known hippocampal and amygdala regions, but also showed reactivation in many thalamic, cortical, midbrain and brainstem structures,” the authors wrote. “Importantly when we compared the brain regions identified by the engram index analysis with these reactivated regions, we observed that ~60 percent of the regions were consistent between analyses.”
Memory manipulations
Having ranked regions significantly likely to be involved in the engram complex, the team engaged in several manipulations to directly test their predictions and to determine how engram complex regions might work together.
For instance, they engineered mice such that cells activated by memory encoding would also become controllable with flashes of light (a technique called “optogenetics”). The researchers then applied light flashes to select brain regions from their engram index list to see if stimulating those would artificially reproduce the fear memory behavior of freezing in place, even when mice were placed in a “neutral” cage where the zap had not occurred.
“Strikingly, all these brain regions induced robust memory recall when they were optogenetically stimulated,” the researchers observed. Moreover, stimulating areas that their analysis suggested were insignificant to zap memory indeed produced no freezing behavior.
The team then demonstrated how different regions within an engram complex connect. They chose two well-known memory regions, CA1 of the hippocampus and the basolateral amygdala (BLA), and optogenetically activated engram cells there to induce memory recall behavior in a neutral cage. They found that stimulating those regions produced memory recall activity in specific “downstream” areas identified as being probable members of the engram complex. Meanwhile, optogenetically inhibiting natural zap memory recall in CA1 or the BLA (i.e. when mice were placed back in the cage where they experienced the zap) led to reduced activity in downstream engram complex areas compared to what they measured in mice with unhindered natural recall.
Further experiments showed that optogenetic reactivations of engram complex neurons followed similar patterns as those observed in natural memory recall. So having established that natural memory encoding and recall appears to occur across a wide engram complex, the team decided to test whether reactivating multiple regions would improve memory recall compared to reactivating just one. After all, prior experiments have shown that activating just one engram area does not produce recall as vividly as natural recall. This time the team used a chemical means to stimulate different engram complex regions and when they did, they found that indeed stimulating up to three involved regions simultaneously produced more robust freezing behavior than stimulating just one or two.
Meaning of distributed storage
Roy said that by storing a single memory across such a widespread complex the brain might be making memory more efficient and resilient.
“Different memory engrams may allow us to recreate memories more efficiently when we are trying to remember a previous event (and similarly for the initial encoding where different engrams may contribute different information from the original experience),” he said. “Secondly, in disease states, if a few regions are impaired, distributed memories would allow us to remember previous events and in some ways be more robust against regional damages.”
In the long term that second idea might suggest a clinical strategy for dealing with memory impairment: “If some memory impairments are because of hippocampal or cortical dysfunction, could we target understudied engram cells in other regions and could such a manipulation restore some memory functions?”
That’s just one of many new questions researchers can ask now that the study has revealed a listing of where to look for at least one kind of memory in the mammalian brain.
The paper’s other authors are Nicholas DiNapoli, Xinyi Gu, Jae Cho, Heejin Choi, Lee Kamentsky, Jared Martin, Olivia Mosto and Tomomi Aida.
Funding sources included the JPB Foundation, the RIKEN Center for Brain Science, the Howard Hughes Medical Institute, a Warren Alpert Distinguished Scholar Award, the National Institutes of Health, the Burroughs Wellcome Fund, the Searle Scholars Program, a Packard Award in Science and Engineering, a NARSAD Young Investigator Award, the McKnight Foundation Technology Award, the NCSOFT Cultural Foundation, and the Institute for Basic Science.
Greta Friar | Whitehead Institute
April 11, 2022
Cancer is at its most deadly when it spreads and forms tumors in new tissues. This process, called metastasis, is responsible for the vast majority of cancer deaths, and yet there is still a lot that researchers do not know about how and when it happens. Whitehead Institute Founding Member Robert Weinberg, also the Daniel K. Ludwig Professor for Cancer Research at the Massachusetts Institute of Technology, studies the mechanisms behind metastasis. One such mechanism is a process called the epithelial-mesenchymal transition (EMT), which causes epithelial cells, which normally stick tightly together, to lose their cohesion, enabling them to move around and even invade nearby tissue. This EMT program also operates during embryonic development. Cancer cells can co-opt this process and use it travel from their original tumor site to distant tissues throughout the body. Some of the cancer cells that spread are able, on rare occasions, to form new tumors in these tissues—metastases—while the great majority of these cells remain dormant after entering the distant tissues.
New research from Weinberg and postdoc Yun Zhang shows that cells change in diverse ways through the actions of the EMT, which can influence whether cells are able to form new tumors after they spread. The work, published in Nature Cell Biology on April 11, 2022, also identifies two regulators of the EMT and shows that loss of each regulator leads to a different metastatic risk profile.
“Using triple negative breast cancer as a model, we are trying to go a bit deeper into understanding the molecular mechanisms that regulate the EMT, how cells enter into different EMT intermediate states, and which of these states contribute to metastasis,” Zhang says.
The EMT was originally imagined as a sort of binary switch, in which cells start out epithelial and become mesenchymal, much like a light switch being flicked from off to on. However, researchers are learning that the EMT works more like a dimmer switch that can be shifted along a spectrum of brightness. Cells that undergo the EMT usually end up in hybrid states between the epithelial and mesenchymal extremes. These cells in the middle of the spectrum, which have some characteristics of each extreme, are called “quasi-mesenchymal” cells, and it turns out that they–rather than cells that become fully mesenchymal–are the most capable of metastasizing and forming new tumors throughout the body.
Protected versus plastic cells
Weinberg and Zhang set out to better understand the EMT spectrum and what controls cells’ movement along it. First, they compared epithelial cells to each other and found that some were more plastic or prone to transitioning along the EMT spectrum than others. They also used the CRISPR gene editing tool to screen for genes that might be regulating the cells’ plasticity. If researchers can learn what makes a cell become quasi-mesenchymal—posing a high risk for metastasis—they might be able use this information, at some time in the future, to develop strategies to prevent cells from entering this high-risk state.
The CRISPR gene screen turned up a number of molecules that seemed to influence cells’ epithelial-mesenchymal plasticity. Two groups of these molecules had especially strong effects: PRC2, a complex that operates in chromosomes to silence or inactivate genes, and KMT2D-COMPASS, a complex that helps activate genes. Both complexes help to keep cells in a stable epithelial state. Loss of either complex makes cells more prone to moving along the EMT spectrum.
The researchers then determined how the loss of either complex enables the EMT. PRC2 normally silences several key EMT-related genes. When PRC2 is lost, those genes activate, which in turn sensitizes the cell to a signal that can trigger the EMT. The loss of KMT2D-COMPASS affects how well PRC2 can bind its targets, leading to the same signal sensitivity. In spite of the similar mechanisms at play, the loss of PRC2 versus KMT2D-COMPASS leads cells to transition to end up in different EMT states, an exciting finding for the researchers. Cells without KMT2D-COMPASS became fully mesenchymal, while cells without PRC2 became hybrid or quasi-mesenchymal. Consequently, cells without PRC2 were much more capable of metastasis than cells without KMT2D-COMPASS (or cells in which both complexes were active) in mouse models. When the researchers looked at historical data from breast cancer patients, they observed the same pattern: people with faulty PRC2 component genes had worse outcomes. These findings provide further evidence that cells in the middle of the EMT spectrum are most likely to metastasize.
This work supports the understanding of the EMT as a spectrum rather than a simple switch, and shows that different EMT regulators can program cells to transition to different parts of the EMT spectrum. Additionally, the finding that loss of PRC2 is linked to metastasis has implications for cancer drugs currently in development that work by inactivating PRC2. Benefits of the drugs may outweigh risks for patients with certain types of cancer for which PRC2 is an effective target. However, Weinberg and Zhang caution that researchers leading clinical trials of PRC2-targeting drugs should be careful about selecting patients and monitoring outcomes. In the types of cancer cells that the researchers looked at, even temporary PRC2 inactivation, such as from a therapy trial, was sufficient to trigger cells to become EMT hybrids with increased metastatic capacity.
Weinberg and Zhang intend to continue exploring the genes identified in their CRISPR screen to see if they can identify other hybrid states along the EMT spectrum, in which cells have different combinations of epithelial and mesenchymal features. They hope that by deepening their understanding of the gene expression profiles of cancer cells associated with different EMT trajectories, they can contribute to the development of therapies for people with potentially metastatic cancers.
“Understanding when and how cancer cells become able to form life-threatening metastases is crucial in order to help the many patients for whom this is a risk,” Weinberg says. “This work provides new insights into the mechanisms that enable cells to metastasize and the roles that different EMT programs can play.”
Institute Professor Sallie “Penny” Chisholm is best known for her role in discovering the tiny bacteria called Prochlorococcus — the world’s most abundant photosynthetic organism. But she has also played a pivotal role in pioneering and advocating for women’s rights at MIT and beyond.
Celina Zhao
March 31, 2022
Without the ancestors of the ocean microbe that Sallie “Penny” Watson Chisholm discovered in 1986, humans may not have ever evolved on Earth. The tiny microbe is called Prochlorococcus, and it’s full of superlatives. One hundred of them can fit on the width of a single strand of human hair, making them the smallest photosynthetic organisms on the planet. At the same time, they’re also the most abundant, comprising an integral piece of the oceans’ “invisible forest.” In fact, you can thank them for the oxygen you breathe in every twentieth breath you take.
With their population, which numbers in the billion billion billions and weighs a collective 220 million Volkswagen Beetles, you might expect Prochlorococcus to be easy to find. But they were not uncovered until the 1980s — and by accident, at that. Since that fateful discovery, Chisholm has dedicated her life and career to studying these intriguing little cells. They have earned her a National Medal of Science from former President Barack Obama, led her to a debate with the Dalai Lama, and even sparked a meeting with the Wu-Tang Clan’s GZA, a rapper interested in featuring the bacterium in his album.
This extraordinary journey, however, was not a straight path — in part because being a pioneering female researcher was no easy feat.
Early life and education
Chisholm was born on November 5, 1947 in Marquette, Michigan, a small town located on the shores of Lake Superior. While growing up, her passion was not science, but skiing, and when it came time to apply for college, she had little ambition and few dreams in higher education.
Her parents, however, intervened. Chisholm’s mother, a traditional 1950s housewife, hated not having her own job or income. In particular, she stressed to her daughter the importance of being able to get an education and career. So, Chisholm traveled east to Saratoga Springs, New York, enrolling at Skidmore College — then a private liberal arts women’s college.
Chisholm on the RV Ellen B. Scripps during her postdoctoral years at the Scripps Institute of Oceanography. Credit: David Karl, University of Hawaii
Living at Skidmore, where she studied biology and chemistry, was a formative experience. “It unconsciously builds a confidence,” Chisholm says of the absence of men competing for attention and resources. In her senior year, she participated in her first fieldwork expedition, studying the prevalence of the mineral manganese in a local lake. Noticing her interest and potential in research, her advisor suggested she attend graduate school. Though the idea had never occurred to her before, it sounded much more interesting than entering the workforce, and so she agreed.
After a year as a graduate student at Cornell University, Chisholm transferred to the State University of New York (SUNY) to study freshwater plankton. With her PhD in hand — the first PhD in her entire extended family — she moved to the sunny beaches of La Jolla, California for her postdoctoral studies. At the University of California San Diego’s Scripps Institute of Oceanography, she began studying the massive system to which she’d dedicate the rest of her career: the ocean.
Just a few months after arriving at Scripps, Chisholm sailed around the Gulf of California on her first research cruise, the R/V Alpha Helix. She stood out from most of the members of the ship in a prominent way — she was one of only a few women aboard. Women were only just starting to be allowed on research vessels in the 1970s, an imbalance that would persist on Chisholm’s other cruises at Scripps.
And, though she loved her time at Scripps, by 1976 it was time to move on. She had three options: a small marine lab in Maine, an oceanography department in Canada, or the Civil and Environmental Engineering Department at MIT. In the case of the latter, she would not simply be the only biologist; she would be the only woman. Should she choose a comfortable route, or should she choose the challenge? Then, one of her mentors at Scripps told her, “Penny, you don’t turn down MIT.” It was a once-in-a-lifetime opportunity, so she decided to give it a shot.
Settling into MIT
In 1976, Chisholm arrived at MIT to continue studying the physiology of various phytoplankton species. One of the instruments she used was a flow cytometer. Though traditionally only used in medical settings, she’d discovered that flow cytometers — with their ability to move individual cells in single file past a laser for a convenient close-up view — were also excellent for her research. In addition, she’d noticed that the unique pigments of phytoplankton fluoresced distinctive colors in the laser’s presence. For example, the green chlorophyll found in phytoplankton would emit red light when struck by a blue laser. Accessory pigments in some would emit orange light.
One day, she and her team came to a game-changing idea: “Wouldn’t it be cool if we could take an instrument like this out on a ship and just squirt sea water through it to see what the diversity of phytoplankton look like?” she mused. This idea immediately opened a whole new set of doors for possible research.
At the start of Chisholm’s career, scientists had a highly restricted picture of microbes in the ocean. This was largely due to limitations in microscope technology: phytoplankton smaller than 5 microns were extremely difficult to see using standard microscopy of the day. In 1979, however, John Waterbury of the Woods Hole Oceanographic Institution, using a technique known as epifluorescence microscopy, discovered tiny photosynthetic cyanobacteria only about 2 microns in diameter, which glowed orange. He named the bacterium Synechococcus. Chisholm was intrigued, and her team set off to the Caribbean in 1985 with their flow cytometer to study them.
While studying images of Synechococcus, they started seeing extremely tiny red signals on their instrument. They didn’t think much of it at first — assuming it was probably just electronic noise. But, as these signals kept showing up, they began to wonder: Could these signals be coming from something that was alive?
The Department of Civil and Environmental Engineering faculty picture from 1978 shows that Chisholm (middle, second row from the back) was the only female faculty member.
It was an intriguing possibility, with evidence in its favor. She and her team noticed that the signals varied depending on the depth and temperature of the water sample being analyzed. There was no reason for electronic noise to increase with depth. But there was good reason for cells to have more chlorophyll (hence the red color under the blue laser of the flow cytometer) the deeper they were under the surface of the water, in order to help with photosynthesis.
Over the next few years, collaborators discovered the same little cells in other seas across the globe. In 1988, Chisholm and her team published their findings in the journal Nature. In the paper, the researchers referred to them as a “new group of pico-plankters,” but members of the lab affectionally called them “little greens” because they contained chlorophyll b, which is a characteristic of green plant chloroplasts. For its formal name, they eventually chose “Prochlorococcus,” meaning “little round progenitors of chloroplasts,” or more colloquially “primitive green berries.”
Chisholm ultimately refocused her entire lab on Prochlorococcus, which revealed itself to be a fascinating subject. She had been looking for a phytoplankton species that could be both easily studied in the lab and easily found in the oceans. Prochlorococcus seemed like a promising model system through which she and her lab could begin understanding how the ocean works.
It turns out that Prochlorococcus wasn’t just one uniform organism, but a collective, composed of more than 30 clusters of strains called “ecotypes” with their own unique survival tactics. This collective has adapted to various environments, dividing up vast swaths of the oceans with various light, temperature, and nutrient combinations. Each ecotype is the “most efficient photosynthetic machine” at its particular conditions — and added together produce 10% of the oxygen in the atmosphere.
To Chisholm, this was an unmistakable sign that there was something truly remarkable about Prochlorococcus. But it would take a few more years for technology to develop far enough to uncover more.
The intriguing new cells glowed red under the flow cytometer (left), but appeared like mere specks of dust or electronic noise under the commonly-used light microscopes (right). Credit: Rob Olson, Woods Hole Oceanographic Institute
Taking a stance on women’s rights at MIT
Deep in the thralls of Procholorococcus research, Chisholm worked early mornings and stayed late into the night. She was in her late forties, and her life revolved around her job. In 1994, during one of those typical long days in her office, she received a phone call that dramatically changed the way she viewed her career.
It was from Nancy Hopkins, a fellow MIT professor whom she casually knew. Hopkins was rallying the support of the 17 other senior female faculty at MIT for a purpose: She believed MIT was discriminating against all of them in multiple areas, including lab space, pay, and support. Hopkins asked Chisholm if she would be willing to come to a meeting and discuss these issues.
Chisholm didn’t know what would come of the meeting, nor did she expect much change to happen. In fact, she had never really thought about what it meant for her to be among the few female faculty at MIT. “I was just doing my work,” she says, “and I liked being one of the guys. I wasn’t plugged into feminist issues or anything.”
But she agreed to attend, and in a few days, found herself in a discreet location on campus. The room was small. Some women sat on chairs, while others sprawled on the floor.
It was awkward at first. “We didn’t know each other that well, so nobody wanted to speak out. We didn’t want to be that one woman faculty member to complain because that meant you couldn’t cut it,” she recalls. But soon everyone started shedding their protective shells. Hours went by as they all found common ground.
Chisholm came to a realization, which she later articulated in her Killian Award acceptance speech: “There’s always a general sense of not being part of the club. It’s like there’s a playbook for this whole enterprise we’re involved in, and it was written by men. As a woman, you’re just constantly trying to figure out what the game is and what the playbook is.”
In her office in 1988, Chisholm holds an image of Prochlorococcus. Credit: Donna Coveney
Armed with a letter calling for investigation, they marched into Dean Robert J. Birgeneau’s office. One of the women even put on a skirt for the first time. They had no idea how he’d react, but preliminary evidence seemed to be on their side. Not only were there 194 tenured male professors compared to only 15 tenured women faculty in the School of Science, the percentage of female faculty hadn’t budged from 8% in 20 years.
Seeing their determination and the evidence, Birgeneau agreed to help. Together with MIT President Charles Vest, he established the MIT Committee on Women Faculty in the MIT School of Science. Birgeneau and Vest charged the committee “to study the status of women in science at MIT and, among other things, to determine the reasons for [MIT’s] failure in the School of Science to hire and promote significant numbers of women faculty.”
The committee’s findings over a two year period were published in a report in 1999. The report made headlines in nearly every major newspaper when President Vest admitted to and apologized for the gender discrimination.
“It was a shot heard around the world,” Chisholm says. And it was true — the MIT administration acted quickly and decisively to right its wrongs. Salaries were adjusted, space and equipment allocation were corrected, retirement packages were increased, among many other changes. Importantly, several other higher education institutions followed MIT’s lead.
As a result of their shared experience, the group of women faculty grew close. Chisholm says, “In my mind, that was one of the best outcomes — on top of all the positive changes by the administration — in terms of my quality of life at MIT: having a community of women that I could talk to.”
Increasing recognition for Prochlorococcus and legacy at MIT
Parallel to the push for equity for women faculty at MIT was the development of one of the most ambitious science expeditions to date — sequencing the human genome. After the Human Genome Project released the sequence of all 20,000 human genes in 2003, there was an abundance of DNA sequencing machines available for other projects. Chisholm leapt at the chance to learn more about her “little greens.” A strain of Prochlorococcus called MED4 became the second microbial genome to be sequenced.
The sequencing “completely opened up the black box,” Chisholm says. With only 1,700 genes, that strain of cyanobacteria is one of the simplest self-sustaining organisms known. Chisholm wrote in a chapter of “Microbes and Evolution: The World that Darwin Never Saw”, “This cell is truly the ‘essence’ of life. As a photosynthesizer, it can do what humans cannot, even with all of our technology: It can split water using sunlight and make hydrogen and oxygen — all with only 1,700 genes.”
The cyanobacterium Prochlorococcus was Chisholm’s muse throughout the majority of her career. Credit: Luke Thompson and Nicki Watson
Together, Chisholm and her lab eventually learned that though each Prochlorococcus strain contains fewer than 2,000 genes, the collective as a whole contains more than 80,000 — four times the size of the human genome. There is a core set of genes (about 1,200) that all Prochlorococcus share, and a few hundred more that are shared only by a subset of strains. In addition, each individual strain has an additional 80-200 genes that are completely unique to it. That incredible diversity allows Prochlorococcus to thrive in all sorts of environmental conditions, from 40 degrees north to 40 degrees south latitudes to almost 200 meters under the ocean where there’s less than 1% light penetration. As the environment shifts, so does the ecotype composition, stabilizing the total population of the species.
Additionally, it turns out that Prochlorococcus is also integral to its local environment. The 5 billion tons of living biomass it produces though photosynthesis each year is eaten by small microorganisms, then zooplankton, then fish. Ultimately, Prochlorococcus feeds 10% of all the creatures in the sea.
Over the years, Chisholm has led the charge in uncovering more about these intriguing cyanobacteria, including how they interact with other things in their environment like viruses or different microorganisms, as well as the unusual molecules Prochlorococcus produce. This persistent dedication has earned her many awards and honors. In 2011, former President Barack Obama presented her with the National Medal of Science, the White House’s highest honor for American scientists. In 2014, she won the MIT James R. Killian Faculty Award, and in 2015 she was named an MIT Institute Professor — the highest title that MIT bestows, and one that only 13 faculty presently hold. In 2019, she was awarded the Crafoord Prize in Biological Sciences by the Swedish Academy of Sciences, the equivalent of a Nobel Prize for biosciences.
As she was gaining worldwide recognition for her research, Chisholm also served as teacher and mentor to many classes of students at MIT. In 1993, she joined the Department of Biology to teach 7.014 (Introductory Biology). Her class was the only introductory biology class to feature ecology. In her lab, she advises undergraduate and graduate students with varying backgrounds, from biology, to chemistry, to oceanography, to civil and environmental engineering.
Her advocacy for women faculty’s rights at MIT has also paid off over the years, with statistics slowly but surely improving to more equal footing. As of June 2019, 250 of the approximately 1,050 faculty members are women, and within the Schools of Science and Engineering, women comprise 22% of the faculty.
And, nearly four decades later, Prochlorococcus still remains an irresistible siren to her. “I should probably be thinking about retiring, but I’m not because Prochlorococcus is too darn interesting,” Chisholm says. “I’m really very grateful to have this organism in my life.”
