Category: News Briefs

Professor Anthony Sinskey, biologist, inventor, entrepreneur, and Center for Biomedical Innovation co-founder, dies at 84

Colleagues remember the longtime MIT professor as a supportive, energetic collaborator who seemed to know everyone at the Institute.

Zach Winn | MIT News
February 20, 2025

Longtime MIT Professor Anthony “Tony” Sinskey ScD ’67, who was also the co-founder and faculty director of the Center for Biomedical Innovation (CBI), passed away on Feb. 12 at his home in New Hampshire. He was 84.

Deeply engaged with MIT, Sinskey left his mark on the Institute as much through the relationships he built as the research he conducted. Colleagues say that throughout his decades on the faculty, Sinskey’s door was always open.

“He was incredibly generous in so many ways,” says Graham Walker, an American Cancer Society Professor at MIT. “He was so willing to support people, and he did it out of sheer love and commitment. If you could just watch Tony in action, there was so much that was charming about the way he lived. I’ve said for years that after they made Tony, they broke the mold. He was truly one of a kind.”

Sinskey’s lab at MIT explored methods for metabolic engineering and the production of biomolecules. Over the course of his research career, he published more than 350 papers in leading peer-reviewed journals for biology, metabolic engineering, and biopolymer engineering, and filed more than 50 patents. Well-known in the biopharmaceutical industry, Sinskey contributed to the founding of multiple companies, including Metabolix, Tepha, Merrimack Pharmaceuticals, and Genzyme Corporation. Sinskey’s work with CBI also led to impactful research papers, manufacturing initiatives, and educational content since its founding in 2005.

Across all of his work, Sinskey built a reputation as a supportive, collaborative, and highly entertaining friend who seemed to have a story for everything.

“Tony would always ask for my opinions — what did I think?” says Barbara Imperiali, MIT’s Class of 1922 Professor of Biology and Chemistry, who first met Sinskey as a graduate student. “Even though I was younger, he viewed me as an equal. It was exciting to be able to share my academic journey with him. Even later, he was continually opening doors for me, mentoring, connecting. He felt it was his job to get people into a room together to make new connections.”

Sinskey grew up in the small town of Collinsville, Illinois, and spent nights after school working on a farm. For his undergraduate degree, he attended the University of Illinois, where he got a job washing dishes at the dining hall. One day, as he recalled in a 2020 conversation, he complained to his advisor about the dishwashing job, so the advisor offered him a job washing equipment in his microbiology lab.

In a development that would repeat itself throughout Sinskey’s career, he befriended the researchers in the lab and started learning about their work. Soon he was showing up on weekends and helping out. The experience inspired Sinskey to go to graduate school, and he only applied to one place.

Sinskey earned his ScD from MIT in nutrition and food science in 1967. He joined MIT’s faculty a few years later and never left.

“He loved MIT and its excellence in research and education, which were incredibly important to him,” Walker says. “I don’t know of another institution this interdisciplinary — there’s barely a speed bump between departments — so you can collaborate with anybody. He loved that. He also loved the spirit of entrepreneurship, which he thrived on. If you heard somebody wanted to get a project done, you could run around, get 10 people, and put it together. He just loved doing stuff like that.”

Working across departments would become a signature of Sinskey’s research. His original office was on the first floor of MIT’s Building 56, right next to the parking lot, so he’d leave his door open in the mornings and afternoons and colleagues would stop in and chat.

“One of my favorite things to do was to drop in on Tony when I saw that his office door was open,” says Chris Kaiser, MIT’s Amgen Professor of Biology. “We had a whole range of things we liked to catch up on, but they always included his perspectives looking back on his long history at MIT. It also always included hopes for the future, including tracking trajectories of MIT students, whom he doted on.”

Long before the internet, colleagues describe Sinskey as a kind of internet unto himself, constantly leveraging his vast web of relationships to make connections and stay on top of the latest science news.

“He was an incredibly gracious person — and he knew everyone,” Imperiali says. “It was as if his Rolodex had no end. You would sit there and he would say, ‘Call this person.’ or ‘Call that person.’ And ‘Did you read this new article?’ He had a wonderful view of science and collaboration, and he always made that a cornerstone of what he did. Whenever I’d see his door open, I’d grab a cup of tea and just sit there and talk to him.”

When the first recombinant DNA molecules were produced in the 1970s, it became a hot area of research. Sinskey wanted to learn more about recombinant DNA, so he hosted a large symposium on the topic at MIT that brought in experts from around the world.

“He got his name associated with recombinant DNA for years because of that,” Walker recalls. “People started seeing him as Mr. Recombinant DNA. That kind of thing happened all the time with Tony.”

Sinskey’s research contributions extended beyond recombinant DNA into other microbial techniques to produce amino acids and biodegradable plastics. He co-founded CBI in 2005 to improve global health through the development and dispersion of biomedical innovations. The center adopted Sinskey’s collaborative approach in order to accelerate innovation in biotechnology and biomedical research, bringing together experts from across MIT’s schools.

“Tony was at the forefront of advancing cell culture engineering principles so that making biomedicines could become a reality. He knew early on that biomanufacturing was an important step on the critical path from discovering a drug to delivering it to a patient,” says Stacy Springs, the executive director of CBI. “Tony was not only my boss and mentor, but one of my closest friends. He was always working to help everyone reach their potential, whether that was a colleague, a former or current researcher, or a student. He had a gentle way of encouraging you to do your best.”

“MIT is one of the greatest places to be because you can do anything you want here as long as it’s not a crime,” Sinskey joked in 2020. “You can do science, you can teach, you can interact with people — and the faculty at MIT are spectacular to interact with.”

Sinskey shared his affection for MIT with his family. His wife, the late ChoKyun Rha ’62, SM ’64, SM ’66, ScD ’67, was a professor at MIT for more than four decades and the first woman of Asian descent to receive tenure at MIT. His two sons also attended MIT — Tong-ik Lee Sinskey ’79, SM ’80 and Taeminn Song MBA ’95, who is the director of strategy and strategic initiatives for MIT Information Systems and Technology (IS&T).

Song recalls: “He was driven by same goal my mother had: to advance knowledge in science and technology by exploring new ideas and pushing everyone around them to be better.”

Around 10 years ago, Sinskey began teaching a class with Walker, Course 7.21/7.62 (Microbial Physiology). Walker says their approach was to treat the students as equals and learn as much from them as they taught. The lessons extended beyond the inner workings of microbes to what it takes to be a good scientist and how to be creative. Sinskey and Rha even started inviting the class over to their home for Thanksgiving dinner each year.

“At some point, we realized the class was turning into a close community,” Walker says. “Tony had this endless supply of stories. It didn’t seem like there was a topic in biology that Tony didn’t have a story about either starting a company or working with somebody who started a company.”

Over the last few years, Walker wasn’t sure they were going to continue teaching the class, but Sinskey remarked it was one of the things that gave his life meaning after his wife’s passing in 2021. That decided it.

After finishing up this past semester with a class-wide lunch at Legal Sea Foods, Sinskey and Walker agreed it was one of the best semesters they’d ever taught.

In addition to his two sons, Sinskey is survived by his daughter-in-law Hyunmee Elaine Song, five grandchildren, and two great grandsons. He has two brothers, Terry Sinskey (deceased in 1975) and Timothy Sinskey, and a sister, Christine Sinskey Braudis.

Gifts in Sinskey’s memory can be made to the ChoKyun Rha (1962) and Anthony J Sinskey (1967) Fund.

MIT biologists discover a new type of control over RNA splicing

They identified proteins that influence splicing of about half of all human introns, allowing for more complex types of gene regulation.

Anne Trafton | MIT News
February 20, 2025

RNA splicing is a cellular process that is critical for gene expression. After genes are copied from DNA into messenger RNA, portions of the RNA that don’t code for proteins, called introns, are cut out and the coding portions are spliced back together.

This process is controlled by a large protein-RNA complex called the spliceosome. MIT biologists have now discovered a new layer of regulation that helps to determine which sites on the messenger RNA molecule the spliceosome will target.

The research team discovered that this type of regulation, which appears to influence the expression of about half of all human genes, is found throughout the animal kingdom, as well as in plants. The findings suggest that the control of RNA splicing, a process that is fundamental to gene expression, is more complex than previously known.

“Splicing in more complex organisms, like humans, is more complicated than it is in some model organisms like yeast, even though it’s a very conserved molecular process. There are bells and whistles on the human spliceosome that allow it to process specific introns more efficiently. One of the advantages of a system like this may be that it allows more complex types of gene regulation,” says Connor Kenny, an MIT graduate student and the lead author of the study.

Christopher Burge, the Uncas and Helen Whitaker Professor of Biology at MIT, is the senior author of the study, which appears today in Nature Communications.

Building proteins

RNA splicing, a process discovered in the late 1970s, allows cells to precisely control the content of the mRNA transcripts that carry the instructions for building proteins.

Each mRNA transcript contains coding regions, known as exons, and noncoding regions, known as introns. They also include sites that act as signals for where splicing should occur, allowing the cell to assemble the correct sequence for a desired protein. This process enables a single gene to produce multiple proteins; over evolutionary timescales, splicing can also change the size and content of genes and proteins, when different exons become included or excluded.

The spliceosome, which forms on introns, is composed of proteins and noncoding RNAs called small nuclear RNAs (snRNAs). In the first step of spliceosome assembly, an snRNA molecule known as U1 snRNA binds to the 5’ splice site at the beginning of the intron. Until now, it had been thought that the binding strength between the 5’ splice site and the U1 snRNA was the most important determinant of whether an intron would be spliced out of the mRNA transcript.

In the new study, the MIT team discovered that a family of proteins called LUC7 also helps to determine whether splicing will occur, but only for a subset of introns — in human cells, up to 50 percent.

Before this study, it was known that LUC7 proteins associate with U1 snRNA, but the exact function wasn’t clear. There are three different LUC7 proteins in human cells, and Kenny’s experiments revealed that two of these proteins interact specifically with one type of 5’ splice site, which the researchers called “right-handed.” A third human LUC7 protein interacts with a different type, which the researchers call “left-handed.”

The researchers found that about half of human introns contain a right- or left-handed site, while the other half do not appear to be controlled by interaction with LUC7 proteins. This type of control appears to add another layer of regulation that helps remove specific introns more efficiently, the researchers say.

“The paper shows that these two different 5’ splice site subclasses exist and can be regulated independently of one another,” Kenny says. “Some of these core splicing processes are actually more complex than we previously appreciated, which warrants more careful examination of what we believe to be true about these highly conserved molecular processes.”

“Complex splicing machinery”

Previous work has shown that mutation or deletion of one of the LUC7 proteins that bind to right-handed splice sites is linked to blood cancers, including about 10 percent of acute myeloid leukemias (AMLs). In this study, the researchers found that AMLs that lost a copy of the LUC7L2 gene have inefficient splicing of right-handed splice sites. These cancers also developed the same type of altered metabolism seen in earlier work.

“Understanding how the loss of this LUC7 protein in some AMLs alters splicing could help in the design of therapies that exploit these splicing differences to treat AML,” Burge says. “There are also small molecule drugs for other diseases such as spinal muscular atrophy that stabilize the interaction between U1 snRNA and specific 5’ splice sites. So the knowledge that particular LUC7 proteins influence these interactions at specific splice sites could aid in improving the specificity of this class of small molecules.”

Working with a lab led by Sascha Laubinger, a professor at Martin Luther University Halle-Wittenberg, the researchers found that introns in plants also have right- and left-handed 5’ splice sites that are regulated by Luc7 proteins.

The researchers’ analysis suggests that this type of splicing arose in a common ancestor of plants, animals, and fungi, but it was lost from fungi soon after they diverged from plants and animals.

“A lot what we know about how splicing works and what are the core components actually comes from relatively old yeast genetics work,” Kenny says. “What we see is that humans and plants tend to have more complex splicing machinery, with additional components that can regulate different introns independently.”

The researchers now plan to further analyze the structures formed by the interactions of Luc7 proteins with mRNA and the rest of the spliceosome, which could help them figure out in more detail how different forms of Luc7 bind to different 5’ splice sites.

The research was funded by the U.S. National Institutes of Health and the German Research Foundation.

A planarian’s guide to growing a new head

Researchers at the Whitehead Institute have described a pathyway by which planarians, freshwater flatworms with spectacular regenerative capabilities, can restore large portions of their nervous system, even regenerating a new head with a fully functional brain.

Shafaq Zia | Whitehead Institute
February 6, 2025

Cut off any part of this worm’s body and it will regrow. This is the spectacular yet mysterious regenerative ability of freshwater flatworms known as planarians. The lab of Whitehead Institute Member Peter Reddien investigates the principles underlying this remarkable feat. In their latest study, published in PLOS Genetics on February 6, first author staff scientist M. Lucila Scimone, Reddien, and colleagues describe how planarians restore large portions of their nervous system—even regenerating a new head with a fully functional brain—by manipulating a signaling pathway.

This pathway, called the Delta-Notch signaling pathway, enables neurons to guide the differentiation of a class of progenitors—immature cells that will differentiate into specialized types—into glia, the non-neuronal cells that support and protect neurons. The mechanism ensures that the spatial pattern and relative numbers of neurons and glia at a given location are precisely restored following injury.

“This process allows planarians to regenerate neural circuits more efficiently because glial cells form only where needed, rather than being produced broadly within the body and later eliminated,” said Reddien, who is also a professor of biology at Massachusetts Institute of Technology and an Investigator with the Howard Hughes Medical Institute.

Coordinating regeneration

Multiple cell types work together to form a functional human brain. These include neurons and a more abundant group of cells called glial cells—astrocytes, microglia, and oligodendrocytes. Although glial cells are not the fundamental units of the nervous system, they perform critical functions in maintaining the connections between neurons, called synapses, clearing away dead cells and other debris, and regulating neurotransmitter levels, effectively holding the nervous system together like glue. A few years ago, Reddien and colleagues discovered cells in planarians that looked like glial cells and performed similar neuro-supportive functions. This led to the first characterization of glial cells in planarians in 2016.

Unlike in mammals where the same set of neural progenitors give rise to both neurons and glia, glial cells in planarians originate from a separate, specialized group of progenitors. These progenitors, called phagocytic progenitors, can not only give rise to glial cells but also pigment cells that determine the worm’s coloration, as well as other, lesser understood cell types.

Why neurons and glia in planarians originate from distinct progenitors—and what factors ultimately determine the differentiation of phagocytic progenitors into glia—are questions that still puzzled Reddien and team members. Then, a study showing that planarian neurons regenerate before glia formation led the researchers to wonder whether a signaling mechanism between neurons and phagocytic progenitors guides the specification of glia in planarians.

The first step to unravel this mystery was to look at the Notch signaling pathway, which is known to play a crucial role in the development of neurons and glia in other organisms, and determine its role in planarian glia regeneration. To do this, the researchers used RNA interference (RNAi)—a technique that decreases or completely silences the expression of genes—to turn off key genes involved in the Notch pathway and amputated the planarian’s head. It turned out Notch signaling is essential for glia regeneration and maintenance in planarians—no glial cells were found in the animal following RNAi, while the differentiation of other types of phagocytic cells was unaffected.

Of the different Notch signaling pathway components the researchers tested, turning of the genes notch-1, delta-2, and suppressor of hairless produced this phenotype. Interestingly, the signaling molecules Delta-2 was found on the surface of neurons, whereas Notch-1 was expressed in phagocytic progenitors.

With these findings in hand, the researchers hypothesized that interaction between Delta-2 on neurons and Notch-1 on phagocytic progenitors could be governing the final fate determination of glial cells in planarians.

To test the hypothesis, the researchers transplanted eyes either from planarians lacking the notch-1 gene or from planarians lacking the delta-2 gene into wild-type animals and assessed the formation of glial cells around the transplant site. They observed that glial cells still formed around the notch-1 deficient eyes, as notch-1 was still active in the glial progenitors of the host wild-type animal. However, no glial cells formed around the delta-2 deficient eyes, even with the Notch signaling pathway intact in phagocytic progenitors, confirming that delta-2 in the photoreceptor neurons is required for the differentiation of phagocytic progenitors into glia near the eye.

“This experiment really showed us that you have two faces of the same coin—one is the phagocytic progenitors expressing Notch-1, and one is the neurons expressing Delta-2—working together to guide the specification of glia in the organism,”said Scimone.

The researchers have named this phenomenon coordinated regeneration, as it allows neurons to influence the pattern and number of glia at specific locations without the need for a separate mechanism to adjust the relative numbers of neurons and glia.

The group is now interested in investigating whether the same phenomenon might also be involved in the regeneration of other tissue types.

AI model deciphers the code in proteins that tells them where to go

Whitehead Institute and CSAIL researchers created a machine-learning model to predict and generate protein localization, with implications for understanding and remedying disease.

Greta Friar | Whitehead Institute
February 13, 2025

Proteins are the workhorses that keep our cells running, and there are many thousands of types of proteins in our cells, each performing a specialized function. Researchers have long known that the structure of a protein determines what it can do. More recently, researchers are coming to appreciate that a protein’s localization is also critical for its function. Cells are full of compartments that help to organize their many denizens. Along with the well-known organelles that adorn the pages of biology textbooks, these spaces also include a variety of dynamic, membrane-less compartments that concentrate certain molecules together to perform shared functions. Knowing where a given protein localizes, and who it co-localizes with, can therefore be useful for better understanding that protein and its role in the healthy or diseased cell, but researchers have lacked a systematic way to predict this information.

Meanwhile, protein structure has been studied for over half-a-century, culminating in the artificial intelligence tool AlphaFold, which can predict protein structure from a protein’s amino acid code, the linear string of building blocks within it that folds to create its structure. AlphaFold and models like it have become widely used tools in research.

Proteins also contain regions of amino acids that do not fold into a fixed structure, but are instead important for helping proteins join dynamic compartments in the cell. MIT Professor Richard Young and colleagues wondered whether the code in those regions could be used to predict protein localization in the same way that other regions are used to predict structure. Other researchers have discovered some protein sequences that code for protein localization, and some have begun developing predictive models for protein localization. However, researchers did not know whether a protein’s localization to any dynamic compartment could be predicted based on its sequence, nor did they have a comparable tool to AlphaFold for predicting localization.

Now, Young, also member of the Whitehead Institute for Biological Research; Young lab postdoc Henry Kilgore; Regina Barzilay, the School of Engineering Distinguished Professor for AI and Health at MIT’s Computer Science and Artificial Intelligence Laboratory (CSAIL); and colleagues have built such a model, which they call ProtGPS. In a paper published on Feb. 6 in the journal Science, with first authors Kilgore and Barzilay lab graduate students Itamar Chinn, Peter Mikhael, and Ilan Mitnikov, the cross-disciplinary team debuts their model. The researchers show that ProtGPS can predict to which of 12 known types of compartments a protein will localize, as well as whether a disease-associated mutation will change that localization. Additionally, the research team developed a generative algorithm that can design novel proteins to localize to specific compartments.

“My hope is that this is a first step towards a powerful platform that enables people studying proteins to do their research,” Young says, “and that it helps us understand how humans develop into the complex organisms that they are, how mutations disrupt those natural processes, and how to generate therapeutic hypotheses and design drugs to treat dysfunction in a cell.”

The researchers also validated many of the model’s predictions with experimental tests in cells.

“It really excited me to be able to go from computational design all the way to trying these things in the lab,” Barzilay says. “There are a lot of exciting papers in this area of AI, but 99.9 percent of those never get tested in real systems. Thanks to our collaboration with the Young lab, we were able to test, and really learn how well our algorithm is doing.”

Developing the model

The researchers trained and tested ProtGPS on two batches of proteins with known localizations. They found that it could correctly predict where proteins end up with high accuracy. The researchers also tested how well ProtGPS could predict changes in protein localization based on disease-associated mutations within a protein. Many mutations — changes to the sequence for a gene and its corresponding protein — have been found to contribute to or cause disease based on association studies, but the ways in which the mutations lead to disease symptoms remain unknown.

Figuring out the mechanism for how a mutation contributes to disease is important because then researchers can develop therapies to fix that mechanism, preventing or treating the disease. Young and colleagues suspected that many disease-associated mutations might contribute to disease by changing protein localization. For example, a mutation could make a protein unable to join a compartment containing essential partners.

They tested this hypothesis by feeding ProtGOS more than 200,000 proteins with disease-associated mutations, and then asking it to both predict where those mutated proteins would localize and measure how much its prediction changed for a given protein from the normal to the mutated version. A large shift in the prediction indicates a likely change in localization.

The researchers found many cases in which a disease-associated mutation appeared to change a protein’s localization. They tested 20 examples in cells, using fluorescence to compare where in the cell a normal protein and the mutated version of it ended up. The experiments confirmed ProtGPS’s predictions. Altogether, the findings support the researchers’ suspicion that mis-localization may be an underappreciated mechanism of disease, and demonstrate the value of ProtGPS as a tool for understanding disease and identifying new therapeutic avenues.

“The cell is such a complicated system, with so many components and complex networks of interactions,” Mitnikov says. “It’s super interesting to think that with this approach, we can perturb the system, see the outcome of that, and so drive discovery of mechanisms in the cell, or even develop therapeutics based on that.”

The researchers hope that others begin using ProtGPS in the same way that they use predictive structural models like AlphaFold, advancing various projects on protein function, dysfunction, and disease.

Moving beyond prediction to novel generation

The researchers were excited about the possible uses of their prediction model, but they also wanted their model to go beyond predicting localizations of existing proteins, and allow them to design completely new proteins. The goal was for the model to make up entirely new amino acid sequences that, when formed in a cell, would localize to a desired location. Generating a novel protein that can actually accomplish a function — in this case, the function of localizing to a specific cellular compartment — is incredibly difficult. In order to improve their model’s chances of success, the researchers constrained their algorithm to only design proteins like those found in nature. This is an approach commonly used in drug design, for logical reasons; nature has had billions of years to figure out which protein sequences work well and which do not.

Because of the collaboration with the Young lab, the machine learning team was able to test whether their protein generator worked. The model had good results. In one round, it generated 10 proteins intended to localize to the nucleolus. When the researchers tested these proteins in the cell, they found that four of them strongly localized to the nucleolus, and others may have had slight biases toward that location as well.

“The collaboration between our labs has been so generative for all of us,” Mikhael says. “We’ve learned how to speak each other’s languages, in our case learned a lot about how cells work, and by having the chance to experimentally test our model, we’ve been able to figure out what we need to do to actually make the model work, and then make it work better.”

Being able to generate functional proteins in this way could improve researchers’ ability to develop therapies. For example, if a drug must interact with a target that localizes within a certain compartment, then researchers could use this model to design a drug to also localize there. This should make the drug more effective and decrease side effects, since the drug will spend more time engaging with its target and less time interacting with other molecules, causing off-target effects.

The machine learning team members are enthused about the prospect of using what they have learned from this collaboration to design novel proteins with other functions beyond localization, which would expand the possibilities for therapeutic design and other applications.

“A lot of papers show they can design a protein that can be expressed in a cell, but not that the protein has a particular function,” Chinn says. “We actually had functional protein design, and a relatively huge success rate compared to other generative models. That’s really exciting to us, and something we would like to build on.”

All of the researchers involved see ProtGPS as an exciting beginning. They anticipate that their tool will be used to learn more about the roles of localization in protein function and mis-localization in disease. In addition, they are interested in expanding the model’s localization predictions to include more types of compartments, testing more therapeutic hypotheses, and designing increasingly functional proteins for therapies or other applications.

“Now that we know that this protein code for localization exists, and that machine learning models can make sense of that code and even create functional proteins using its logic, that opens up the door for so many potential studies and applications,” Kilgore says.

A sum of their parts

Researchers in the Department of Biology at MIT use an AI-driven approach to computationally predict short amino acid sequences that can bind to or inhibit a target, with a potential for great impact on fundamental biological research and therapeutic applications.

Lillian Eden | Department of Biology
February 6, 2025

All biological function is dependent on how different proteins interact with each other. Protein-protein interactions facilitate everything from transcribing DNA and controlling cell division to higher-level functions in complex organisms.

Much remains unclear about how these functions are orchestrated on the molecular level, however, and how proteins interact with each other — either with other proteins or with copies of themselves. 

Recent findings have revealed that small protein fragments have a lot of functional potential. Even though they are incomplete pieces, short stretches of amino acids can still bind to interfaces of a target protein, recapitulating native interactions. Through this process, they can alter that protein’s function or disrupt its interactions with other proteins. 

Protein fragments could therefore empower both basic research on protein interactions and cellular processes and could potentially have therapeutic applications. 

Recently published in Proceedings of the National Academy of Sciences, a new computational method developed in the Department of Biology at MIT builds on existing AI models to computationally predict protein fragments that can bind to and inhibit full-length proteins in E. coli. Theoretically, this tool could lead to genetically encodable inhibitors against any protein. 

The work was done in the lab of Associate Professor of Biology and HHMI Investigator Gene-Wei Li in collaboration with the lab of Jay A. Stein (1968) Professor of Biology, Professor of Biological Engineering and Department Head Amy Keating.

Leveraging Machine Learning

The program, called FragFold, leverages AlphaFold, an AI model that has led to phenomenal advancements in biology in recent years due to its ability to predict protein folding and protein interactions. 

The goal of the project was to predict fragment inhibitors, which is a novel application of AlphaFold. The researchers on this project confirmed experimentally that more than half of FragFold’s predictions for binding or inhibition were accurate, even when researchers had no previous structural data on the mechanisms of those interactions. 

“Our results suggest that this is a generalizable approach to find binding modes that are likely to inhibit protein function, including for novel protein targets, and you can use these predictions as a starting point for further experiments,” says co-first and corresponding author Andrew Savinov, a postdoc in the Li Lab. “We can really apply this to proteins without known functions, without known interactions, without even known structures, and we can put some credence in these models we’re developing.”

One example is FtsZ, a protein that is key for cell division. It is well-studied but contains a region that is intrinsically disordered and, therefore, especially challenging to study. Disordered proteins are dynamic, and their functional interactions are very likely fleeting — occurring so briefly that current structural biology tools can’t capture a single structure or interaction. 

The researchers leveraged FragFold to explore the activity of fragments of FtsZ, including fragments of the intrinsically disordered region, to identify several new binding interactions with various proteins. This leap in understanding confirms and expands upon previous experiments measuring FtsZ’s biological activity. 

This progress is significant in part because it was made without solving the disordered region’s structure, and because it exhibits the potential power of FragFold.

“This is one example of how AlphaFold is fundamentally changing how we can study molecular and cell biology,” Keating says. “Creative applications of AI methods, such as our work on FragFold, open up unexpected capabilities and new research directions.”

Inhibition, and beyond

The researchers accomplished these predictions by computationally fragmenting each protein and then modeling how those fragments would bind to interaction partners they thought were relevant.

They compared the maps of predicted binding across the entire sequence to the effects of those same fragments in living cells, determined using high-throughput experimental measurements in which millions of cells each produce one type of protein fragment. 

AlphaFold uses co-evolutionary information to predict folding, and typically evaluates the evolutionary history of proteins using something called multiple sequence alignments for every single prediction run. The MSAs are critical, but are a bottleneck for large-scale predictions — they can take a prohibitive amount of time and computational power. 

For FragFold, the researchers instead pre-calculated the MSA for a full-length protein once and used that result to guide the predictions for each fragment of that full-length protein. 

Savinov, together with Keating Lab alum Sebastian Swanson, PhD ‘23, predicted inhibitory fragments of a diverse set of proteins in addition to FtsZ. Among the interactions they explored was a complex between lipopolysaccharide transport proteins LptF and LptG. A protein fragment of LptG inhibited this interaction, presumably disrupting the delivery of lipopolysaccharide, which is a crucial component of the E. coli outer cell membrane essential for cellular fitness.

“The big surprise was that we can predict binding with such high accuracy and, in fact, often predict binding that corresponds to inhibition,” Savinov says. “For every protein we’ve looked at, we’ve been able to find inhibitors.”

The researchers initially focused on protein fragments as inhibitors because whether a fragment could block an essential function in cells is a relatively simple outcome to measure systematically. Looking forward, Savinov is also interested in exploring fragment function outside inhibition, such as fragments that can stabilize the protein they bind to, enhance or alter its function, or trigger protein degradation. 

Design, in principle 

This research is a starting point for developing a systemic understanding of cellular design principles, and what elements deep-learning models may be drawing on to make accurate predictions. 

“There’s a broader, further-reaching goal that we’re building towards,” Savinov says. “Now that we can predict them, can we use the data we have from predictions and experiments to pull out the salient features to figure out what AlphaFold has actually learned about what makes a good inhibitor?” 

Savinov and collaborators also delved further into how protein fragments bind, exploring other protein interactions and mutating specific residues to see how those interactions change how the fragment interacts with its target. 

Experimentally examining the behavior of thousands of mutated fragments within cells, an approach known as deep mutational scanning, revealed key amino acids that are responsible for inhibition. In some cases, the mutated fragments were even more potent inhibitors than their natural, full-length sequences. 

“Unlike previous methods, we are not limited to identifying fragments in experimental structural data,” says Swanson. “The core strength of this work is the interplay between high-throughput experimental inhibition data and the predicted structural models: the experimental data guides us towards the fragments that are particularly interesting, while the structural models predicted by FragFold provide a specific, testable hypothesis for how the fragments function on a molecular level.”

Savinov is excited about the future of this approach and its myriad applications.

“By creating compact, genetically encodable binders, FragFold opens a wide range of possibilities to manipulate protein function,” Li agrees. “We can imagine delivering functionalized fragments that can modify native proteins, change their subcellular localization, and even reprogram them to create new tools for studying cell biology and treating diseases.” 

Kingdoms collide as bacteria and cells form captivating connections

Studying the pathogen R. parkeri, researchers discovered the first evidence of extensive and stable interkingdom contacts between a pathogen and a eukaryotic organelle.

Lillian Eden | Department of Biology
January 24, 2025

In biology textbooks, the endoplasmic reticulum is often portrayed as a distinct, compact organelle near the nucleus, and is commonly known to be responsible for protein trafficking and secretion. In reality, the ER is vast and dynamic, spread throughout the cell and able to establish contact and communication with and between other organelles. These membrane contacts regulate processes as diverse as fat metabolism, sugar metabolism, and immune responses.

Exploring how pathogens manipulate and hijack essential processes to promote their own life cycles can reveal much about fundamental cellular functions and provide insight into viable treatment options for understudied pathogens.

New research from the Lamason Lab in the Department of Biology at MIT recently published in the Journal of Cell Biology has shown that Rickettsia parkeri, a bacterial pathogen that lives freely in the cytosol, can interact in an extensive and stable way with the rough endoplasmic reticulum, forming previously unseen contacts with the organelle.

It’s the first known example of a direct interkingdom contact site between an intracellular bacterial pathogen and a eukaryotic membrane.

The Lamason Lab studies R. parkeri as a model for infection of the more virulent Rickettsia rickettsii. R. rickettsii, carried and transmitted by ticks, causes Rocky Mountain Spotted Fever. Left untreated, the infection can cause symptoms as severe as organ failure and death.

Rickettsia is difficult to study because it is an obligate pathogen, meaning it can only live and reproduce inside living cells, much like a virus. Researchers must get creative to parse out fundamental questions and molecular players in the R. parkeri life cycle, and much remains unclear about how R. parkeri spreads.

Detour to the junction

First author Yamilex Acevedo-Sánchez, a BSG-MSRP-Bio program alum and a graduate student at the time, stumbled across the ER and R. parkeri interactions while trying to observe Rickettsia reaching a cell junction.

The current model for Rickettsia infection involves R. parkeri spreading cell to cell by traveling to the specialized contact sites between cells and being engulfed by the neighboring cell in order to spread. Listeria monocytogenes, which the Lamason Lab also studies, uses actin tails to forcefully propel itself into a neighboring cell. By contrast, R. parkeri can form an actin tail, but loses it before reaching the cell junction. Somehow, R. parkeri is still able to spread to neighboring cells.

After an MIT seminar about the ER’s lesser-known functions, Acevedo-Sánchez developed a cell line to observe whether Rickettsia might be spreading to neighboring cells by hitching a ride on the ER to reach the cell junction.

Instead, she saw an unexpectedly high percentage of R. parkeri surrounded and enveloped by the ER, at a distance of about 55 nanometers. This distance is significant because membrane contacts for interorganelle communication in eukaryotic cells form connections from 10-80 nanometers wide. The researchers ruled out that what they saw was not an immune response, and the sections of the ER interacting with the R. parkeri were still connected to the wider network of the ER.

“I’m of the mind that if you want to learn new biology, just look at cells,” Acevedo-Sánchez says. “Manipulating the organelle that establishes contact with other organelles could be a great way for a pathogen to gain control during infection.”

The stable connections were unexpected because the ER is constantly breaking and reforming connections, lasting seconds or minutes. It was surprising to see the ER stably associating around the bacteria. As a cytosolic pathogen that exists freely in the cytosol of the cells it infects, it was also unexpected to see R. parkeri surrounded by a membrane at all.

Small margins

Acevedo-Sánchez collaborated with the Center for Nanoscale Systems at Harvard University to view her initial observations at higher resolution using focused ion beam scanning electron microscopy. FIB-SEM involves taking a sample of cells and blasting them with a focused ion beam in order to shave off a section of the block of cells. With each layer, a high-resolution image is taken. The result of this process is a stack of images.

From there, Acevedo-Sánchez marked what different areas of the images were — such as the mitochondria, Rickettsia, or the ER — and a program called ORS Dragonfly, a machine learning program, sorted through the thousand or so images to identify those categories. That information was then used to create 3D models of the samples.

Acevedo-Sánchez noted that less than 5 percent of R. parkeri formed connections with the ER — but small quantities of certain characteristics are known to be critical for R. parkeri infection. R. parkeri can exist in two states: motile, with an actin tail, and nonmotile, without it. In mutants unable to form actin tails, R. parkeri are unable to progress to adjacent cells — but in nonmutants, the percentage of R. parkeri that have tails starts at about 2 percent in early infection and never exceeds 15 percent at the height of it.

The ER only interacts with nonmotile R. parkeri, and those interactions increased 25-fold in mutants that couldn’t form tails.

Creating connections

Co-authors Acevedo-Sánchez, Patrick Woida, and Caroline Anderson also investigated possible ways the connections with the ER are mediated. VAP proteins, which mediate ER interactions with other organelles, are known to be co-opted by other pathogens during infection.

During infection by R. parkeri, VAP proteins were recruited to the bacteria; when VAP proteins were knocked out, the frequency of interactions between R. parkeri and the ER decreased, indicating R. parkeri may be taking advantage of these cellular mechanisms for its own purposes during infection.

Although Acevedo-Sánchez now works as a senior scientist at AbbVie, the Lamason Lab is continuing the work of exploring the molecular players that may be involved, how these interactions are mediated, and whether the contacts affect the host or bacteria’s life cycle.

Senior author and associate professor of biology Rebecca Lamason noted that these potential interactions are particularly interesting because bacteria and mitochondria are thought to have evolved from a common ancestor. The Lamason Lab has been exploring whether R. parkeri could form the same membrane contacts that mitochondria do, although they haven’t proven that yet. So far, R. parkeri is the only cytosolic pathogen that has been observed behaving this way.

“It’s not just bacteria accidentally bumping into the ER. These interactions are extremely stable. The ER is clearly extensively wrapping around the bacterium, and is still connected to the ER network,” Lamason says. “It seems like it has a purpose — what that purpose is remains a mystery.”

Cellular interactions help explain vascular complications due to COVID-19 virus infection

Whitehead Institute Founding Member Rudolf Jaenisch and colleagues have found that cellular interactions help explain how SARS-CoV-2, the virus that causes COVID-19, could have such significant vascular complications, including blood clots, heart attacks, and strokes.

Greta Friar | Whitehead Institute
December 31, 2024

COVID-19 is a respiratory disease primarily affecting the lungs. However, the SARS-CoV-2 virus that causes COVID-19 surprised doctors and scientists by triggering an unusually large percentage of patients to experience vascular complications – issues related to blood flow, such as blood clots, heart attacks, and strokes.

Whitehead Institute Founding Member Rudolf Jaenisch and colleagues wanted to understand how this respiratory virus could have such significant vascular effects. They used pluripotent stem cells to generate three relevant vascular and perivascular cell types—cells that surround and help maintain blood vessels—so they could closely observe the effects of SARS-CoV-2 on the cells. Instead of using existing methods to generate the cells, the researchers developed a new approach, providing them with fresh insights into the mechanisms by which the virus causes vascular problems. The researchers found that SARS-CoV-2 primarily infects perivascular cells and that signals from these infected cells are sufficient to cause dysfunction in neighboring vascular cells, even when the vascular cells are not themselves infected. In a paper published in the journal Nature Communications on December 30, Jaenisch, postdoc in his lab Alexsia Richards, Harvard University Professor and Wyss Institute for Biologically Inspired Engineering Member David Mooney, and then-postdoc in the Jaenisch and Mooney labs Andrew Khalil share their findings and present a scalable stem cell-derived model system with which to study vascular cell biology and test medical therapies.

A new problem requires a new approach

When the COVID-19 pandemic began, Richards, a virologist, quickly pivoted her focus to SARS-CoV-2. Khalil, a bioengineer, had already been working on a new approach to generate vascular cells. The researchers realized that a collaboration could provide Richards with the research tool she needed and Khalil with an important research question to which his tool could be applied.

The three cell types that Khalil’s approach generated were endothelial cells, the vascular cells that form the lining of blood vessels; and smooth muscle cells and pericytes, perivascular cells that surround blood vessels and provide them with structure and maintenance, among other functions. Khalil’s biggest innovation was to generate all three cell types in the same media—the mixture of nutrients and signaling molecules in which stem cell-derived cells are grown.

The combination of signals in the media determines the final cell type into which a stem cell will mature, so it is much easier to grow each cell type separately in specially tailored media than to find a mixture that works for all three. Typically, Richards explains, virologists will generate a desired cell type using the easiest method, which means growing each cell type and then observing the effects of viral infection on it in isolation. However, this approach can limit results in several ways. Firstly, it can make it challenging to distinguish the differences in how cell types react to a virus from the differences caused by the cells being grown in different media.

“By making these cells under identical conditions, we could see in much higher resolution the effects of the virus on these different cell populations, and that was essential in order to form a strong hypothesis of the mechanisms of vascular symptom risk and progression,” Khalil says.

Secondly, infecting isolated cell types with a virus does not accurately represent what happens in the body, where cells are in constant communication as they react to viral exposure. Indeed, Richards’ and Khalil’s work ultimately revealed that the communication between infected and uninfected cell types plays a critical role in the vascular effects of COVID-19.

“The field of virology often overlooks the importance of considering how cells influence other cells and designing models to reflect that,” Richards says. “Cells do not get infected in isolation, and the value of our model is that it allows us to observe what’s happening between cells during infection.”

Viral infection of smooth muscle cells has broader, indirect effects

When the researchers exposed their cells to SARS-CoV-2, the smooth muscle cells and pericytes became infected—the former at especially high levels, and this infection resulted in strong inflammatory gene expression—but the endothelial cells resisted infection. Endothelial cells did show some response to viral exposure, likely due to interactions with proteins on the virus’ surface. Typically, endothelial cells press tightly together to form a firm barrier that keeps blood inside of blood vessels and prevents viruses from getting out. When exposed to SARS-CoV-2, the junctions between endothelial cells appeared to weaken slightly. The cells also had increased levels of reactive oxygen species, which are damaging byproducts of certain cellular processes.

However, big changes in endothelial cells only occurred after the cells were exposed to infected smooth muscle cells. This triggered high levels of inflammatory signaling within the endothelial cells. It led to changes in the expression of many genes relevant to immune response. Some of the genes affected were involved in coagulation pathways, which thicken blood and so can cause blood clots and related vascular events. The junctions between endothelial cells experienced much more significant weakening after exposure to infected smooth muscle cells, which would lead to blood leakage and viral spread. All of these changes occurred without SARS-CoV-2 ever infecting the endothelial cells.

This work shows that viral infection of smooth muscle cells, and their resultant signaling to endothelial cells, is the lynchpin in the vascular damage caused by SARS-CoV-2. This would not have been apparent if the researchers had not been able to observe the cells interacting with each other.

Clinical relevance of stem cell results

The effects that the researchers observed were consistent with patient data. Some of the genes whose expression changed in their stem cell-derived model had been identified as markers of high risk for vascular complications in COVID-19 patients with severe infections. Additionally, the researchers found that a later strain of SARS-CoV-2, an Omicron variant, had much weaker effects on the vascular and perivascular cells than did the original viral strain. This is consistent with the reduced levels of vascular complications seen in COVID-19 patients infected with recent strains.

Having identified smooth muscle cells as the main site of SARS-Cov-2 infection in the vascular system, the researchers next used their model system to test one drug’s ability to prevent infection of smooth muscle cells. They found that the drug, N, N-Dimethyl-D-erythro-sphingosine, could reduce infection of the cell type without harming smooth muscle or endothelial cells. Although preventing vascular complications of COVID-19 is not as pressing a need with current viral strains, the researchers see this experiment as proof that their stem cell model could be used for future drug development. New coronaviruses and other pathogens are frequently evolving, and when a future virus causes vascular complications, this model could be used to quickly test drugs to find potential therapies while the need is still high. The model system could also be used to answer other questions about vascular cells, how these cells interact, and how they respond to viruses.

“By integrating bioengineering strategies into the analysis of a fundamental question in viral pathology, we addressed important practical challenges in modeling human disease in culture and gained new insights into SARS-CoV-2 infection,” Mooney says.

“Our interdisciplinary approach allowed us to develop an improved stem cell model for infection of the vasculature,” says Jaenisch, who is also a professor of biology at the Massachusetts Institute of Technology. “Our lab is already applying this model to other questions of interest, and we hope that it can be a valuable tool for other researchers.”

An abundant phytoplankton feeds a global network of marine microbes

New findings illuminate how Prochlorococcus’ nightly “cross-feeding” plays a role in regulating the ocean’s capacity to cycle and store carbon.

Jennifer Chu | MIT News
January 3, 2025

One of the hardest-working organisms in the ocean is the tiny, emerald-tinged Prochlorococcus marinus. These single-celled “picoplankton,” which are smaller than a human red blood cell, can be found in staggering numbers throughout the ocean’s surface waters, making Prochlorococcus the most abundant photosynthesizing organism on the planet. (Collectively, Prochlorococcus fix as much carbon as all the crops on land.) Scientists continue to find new ways that the little green microbe is involved in the ocean’s cycling and storage of carbon.

Now, MIT scientists have discovered a new ocean-regulating ability in the small but mighty microbes: cross-feeding of DNA building blocks. In a study appearing today in Science Advances, the team reports that Prochlorococcus shed these extra compounds into their surroundings, where they are then “cross-fed,” or taken up by other ocean organisms, either as nutrients, energy, or for regulating metabolism. Prochlorococcus’ rejects, then, are other microbes’ resources.

What’s more, this cross-feeding occurs on a regular cycle: Prochlorococcus tend to shed their molecular baggage at night, when enterprising microbes quickly consume the cast-offs. For a microbe called SAR11, the most abundant bacteria in the ocean, the researchers found that the nighttime snack acts as a relaxant of sorts, forcing the bacteria to slow down their metabolism and effectively recharge for the next day.

Through this cross-feeding interaction, Prochlorococcus could be helping many microbial communities to grow sustainably, simply by giving away what it doesn’t need. And they’re doing so in a way that could set the daily rhythms of microbes around the world.

“The relationship between the two most abundant groups of microbes in ocean ecosystems has intrigued oceanographers for years,” says co-author and MIT Institute Professor Sallie “Penny” Chisholm, who played a role in the discovery of Prochlorococcus in 1986. “Now we have a glimpse of the finely tuned choreography that contributes to their growth and stability across vast regions of the oceans.”

Given that Prochlorococcus and SAR11 suffuse the surface oceans, the team suspects that the exchange of molecules from one to the other could amount to one of the major cross-feeding relationships in the ocean, making it an important regulator of the ocean carbon cycle.

“By looking at the details and diversity of cross-feeding processes, we can start to unearth important forces that are shaping the carbon cycle,” says the study’s lead author, Rogier Braakman, a research scientist in MIT’s Department of Earth, Atmospheric and Planetary Sciences (EAPS).

Other MIT co-authors include Brandon Satinsky, Tyler O’Keefe, Shane Hogle, Jamie Becker, Robert Li, Keven Dooley, and Aldo Arellano, along with Krista Longnecker, Melissa Soule, and Elizabeth Kujawinski of Woods Hole Oceanographic Institution (WHOI).

Spotting castaways

Cross-feeding occurs throughout the microbial world, though the process has mainly been studied in close-knit communities. In the human gut, for instance, microbes are in close proximity and can easily exchange and benefit from shared resources.

By comparison, Prochlorococcus are free-floating microbes that are regularly tossed and mixed through the ocean’s surface layers. While scientists assume that the plankton are involved in some amount of cross-feeding, exactly how this occurs, and who would benefit, have historically been challenging to probe; any stuff that Prochlorococcus cast away would have vanishingly low concentrations,and be exceedingly difficult to measure.

But in work published in 2023, Braakman teamed up with scientists at WHOI, who pioneered ways to measure small organic compounds in seawater. In the lab, they grew various strains of Prochlorococcus under different conditions and characterized what the microbes released. They found that among the major “exudants,” or released molecules, were purines and pyridines, which are molecular building blocks of DNA. The molecules also happen to be nitrogen-rich — a fact that puzzled the team. Prochlorococcus are mainly found in ocean regions that are low in nitrogen, so it was assumed they’d want to retain any and all nitrogen-containing compounds they can. Why, then, were they instead throwing such compounds away?

Global symphony

In their new study, the researchers took a deep dive into the details of Prochlorococcus’ cross-feeding and how it influences various types of ocean microbes.

They set out to study how Prochlorococcus use purine and pyridine in the first place, before expelling the compounds into their surroundings. They compared published genomes of the microbes, looking for genes that encode purine and pyridine metabolism. Tracing the genes forward through the genomes, the team found that once the compounds are produced, they are used to make DNA and replicate the microbes’ genome. Any leftover purine and pyridine is recycled and used again, though a fraction of the stuff is ultimately released into the environment. Prochlorococcus appear to make the most of the compounds, then cast off what they can’t.

The team also looked to gene expression data and found that genes involved in recycling purine and pyrimidine peak several hours after the recognized peak in genome replication that occurs at dusk. The question then was: What could be benefiting from this nightly shedding?

For this, the team looked at the genomes of more than 300 heterotrophic microbes — organisms that consume organic carbon rather than making it themselves through photosynthesis. They suspected that such carbon-feeders could be likely consumers of Prochlorococcus’ organic rejects. They found most of the heterotrophs contained genes that take up either purine or pyridine, or in some cases, both, suggesting microbes have evolved along different paths in terms of how they cross-feed.

The group zeroed in on one purine-preferring microbe, SAR11, as it is the most abundant heterotrophic microbe in the ocean. When they then compared the genes across different strains of SAR11, they found that various types use purines for different purposes, from simply taking them up and using them intact to breaking them down for their energy, carbon, or nitrogen. What could explain the diversity in how the microbes were using Prochlorococcus’ cast-offs?

It turns out the local environment plays a big role. Braakman and his collaborators performed a metagenome analysis in which they compared the collectively sequenced genomes of all microbes in over 600 seawater samples from around the world, focusing on SAR11 bacteria. Metagenome sequences were collected alongside measurements of various environmental conditions and geographic locations in which they are found. This analysis showed that the bacteria gobble up purine for its nitrogen when the nitrogen in seawater is low, and for its carbon or energy when nitrogen is in surplus — revealing the selective pressures shaping these communities in different ocean regimes.

“The work here suggests that microbes in the ocean have developed relationships that advance their growth potential in ways we don’t expect,” says co-author Kujawinski.

Finally, the team carried out a simple experiment in the lab, to see if they could directly observe a mechanism by which purine acts on SAR11. They grew the bacteria in cultures, exposed them to various concentrations of purine, and unexpectedly found it causes them to slow down their normal metabolic activities and even growth. However, when the researchers put these same cells under environmentally stressful conditions, they continued growing strong and healthy cells, as if the metabolic pausing by purines helped prime them for growth, thereby avoiding the effects of the stress.

“When you think about the ocean, where you see this daily pulse of purines being released by Prochlorococcus, this provides a daily inhibition signal that could be causing a pause in SAR11 metabolism, so that the next day when the sun comes out, they are primed and ready,” Braakman says. “So we think Prochlorococcus is acting as a conductor in the daily symphony of ocean metabolism, and cross-feeding is creating a global synchronization among all these microbial cells.”

This work was supported, in part, by the Simons Foundation and the National Science Foundation.

Imperiali Lab News Brief: combining bioinformatics and biochemistry

Parsing endless possibilities

Lillian Eden | Department of Biology
December 11, 2024

New research from the Imperiali Lab in the Department of Biology at MIT combines bioinformatics and biochemistry to reveal critical players in assembling glycans, the large sugar molecules on bacterial cell surfaces responsible for behaviors such as evading immune responses and causing infections.

In most cases, single-celled organisms such as bacteria interact with their environment through complex chains of sugars known as glycans bound to lipids on their outer membranes. Glycans orchestrate biological responses and interactions, such as evading immune responses and causing infections. 

The first step in assembling most bacterial glycans is the addition of a sugar-phosphate group onto a lipid, which is catalyzed by phosphoglycosyl transferases (PGTs) on the inner membrane. This first sugar is then further built upon by other enzymes in subsequent steps in an assembly-line-like pathway. These critical biochemical processes are challenging to explore because the proteins involved in these processes are embedded in membranes, which makes them difficult to isolate and study. 

Although glycans are found in all living organisms, the sugar molecules that compose glycans are especially diverse in bacteria. There are over 30,000 known bacterial PGTs, and hundreds of sugars for them to act upon. 

Research recently published in PNAS from the Imperiali Lab in the Department of Biology at MIT uses a combination of bioinformatics and biochemistry to predict clusters of “like-minded” PGTs and verify which sugars they will use in the first step of glycan assembly. 

Defining the biochemical machinery for these assembly pathways could reveal new strategies for tackling antibiotic-resistant strains of bacteria. This comprehensive approach could also be used to develop and test inhibitors, halting the assembly pathway at this critical first step. 

Exploring Sequence Similarity

First author Theo Durand, an undergraduate student from Imperial College London who studied at MIT for a year, worked in the Imperiali Lab as part of a research placement. Durand was first tasked with determining which sugars some PGTs would use in the first step of glycan assembly, known as the sugar substrates of the PGTs. When initially those substrate-testing experiments didn’t work, Durand turned to the power of bioinformatics to develop predictive tools. 

Strategically exploring the sugar substrates for PGTs is challenging due to the sheer number of PGTs and the diversity of bacteria, each with its own assorted set of glycans and glycoconjugates. To tackle this problem, Durand deployed a tool called a Sequence Similarity Network (SSN), part of a computational toolkit developed by the Enzyme Function Initiative. 

According to senior author Barbara Imperiali, Class of 1922 Professor of Biology and Chemistry, an SSN provides a powerful way to analyze protein sequences through comparisons of the sequences of tens of thousands of proteins. In an optimized SSN, similar proteins cluster together, and, in the case of PGTs, proteins in the same cluster are likely to share the same sugar substrate. 

For example, a previously uncharacterized PGT that appears in a cluster of PGTs whose first sugar substrate is FucNAc4N would also be predicted to use FucNAc4N. The researchers could then test that prediction to verify the accuracy of the SSN. 

FucNAc4N is the sugar substrate for the PGT of Fusobacterium nucleatum (F. nucleatum), a bacterium that is normally only present in the oral cavity but is correlated with certain cancers and endometriosis, and Streptococcus pneumoniae, a bacterium that causes pneumonia. 

Adjusting the assay

The critical biochemical process of assembling glycans has historically been challenging to define, mainly because assembly is anchored to the interior side of the inner membrane of the bacterium. The purification process itself can be difficult, and the purified proteins don’t necessarily behave in the same manner once outside their native membrane environment.

To address this, the researchers modified a commercially available test to work with proteins still embedded in the membrane of the bacterium, thus saving them weeks of work to purify the proteins. They could then determine the substrate for the PGT by measuring whether there was activity. This first step in glycan assembly is chemically unique, and the test measures one of the reaction products. 

For PGTs whose substrate was unknown, Durand did a deep dive into the literature to find new substrates to test. FucNAc4N, the first sugar substrate for F. nucleatum, was, in fact, Durand’s favorite sugar – he found it in the literature and reached out to a former Imperiali Lab postdoc for the instructions and materials to make it. 

“I ended up down a rabbit hole where I was excited every time I found a new, weird sugar,” Durand recalls with a laugh. “These bacteria are doing a bunch of really complicated things and any tools to help us understand what is actually happening is useful.” 

Exploring inhibitors

Imperiali noted that this research both represents a huge step forward in our understanding of bacterial PGTs and their substrates and presents a pipeline for further exploration. She’s hoping to create a searchable database where other researchers can seed their own sequences into the SSN for their organisms of interest. 

This pipeline could also reveal antibiotic targets in bacteria. For example, she says, the team is using this approach to explore inhibitor development. 

The Imperiali lab worked with Karen Allen, a professor of Chemistry at Boston University, and graduate student Roxanne Siuda to test inhibitors, including ones for F. nucleatum, the bacterium correlated with certain cancers and endometriosis whose first sugar substrate is FucNAc4N. They are also hoping to obtain structures of inhibitors bound to the PGT to enable structure-guided optimization.

“We were able to, using the network, discover the substrate for a PGT, verify the substrate, use it in a screen, and test an inhibitor,” Imperiali says. “This is bioinformatics, biochemistry, and probe development all bundled together, and represents the best of functional genomics.”

Study suggests how the brain, with sleep, learns meaningful maps of spaces

Place cells are well known to encode individual locations, but new experiments and analysis indicate that stitching together a “cognitive map” of a whole environment requires a broader ensemble of cells, aided by sleep, to build a richer network over several days, according to new research from the Wilson Lab.

David Orenstein | The Picower Institute for Learning and Memory
December 10, 2024

On the first day of your vacation in a new city your explorations expose you to innumerable individual places. While the memories of these spots (like a beautiful garden on a quiet side street) feel immediately indelible, it might be days before you have enough intuition about the neighborhood to direct a newer tourist to that same site and then maybe to the café you discovered nearby. A new study in mice by MIT neuroscientists at The Picower Insitute for Learning and Memory provides new evidence for how the brain forms cohesive cognitive maps of whole spaces and highlights the critical importance of sleep for the process.

Scientists have known for decades that the brain devotes neurons in a region called the hippocampus to remembering specific locations. So-called “place cells” reliably activate when an animal is at the location the neuron is tuned to remember. But more useful than having markers of specific spaces is having a mental model of how they all relate in a continuous overall geography. Though such “cognitive maps” were formally theorized in 1948, neuroscientists have remained unsure of how the brain constructs them. The new study in the December edition of Cell Reports finds that the capability may depend upon subtle but meaningful changes over days in the activity of cells that are only weakly attuned to individual locations, but that increase the robustness and refinement of the hippocampus’s encoding of the whole space. With sleep, the study’s analyses indicate, these “weakly spatial” cells increasingly enrich neural network activity in the hippocampus to link together these places into a cognitive map.

“On day 1, the brain doesn’t represent the space very well,” said lead author Wei Guo, a research scientist in the lab of senior author Matthew Wilson, Sherman Fairchild Professor in The Picower Institute and MIT’s Departments of Biology and Brain and Cognitive Sciences. “Neurons represent individual locations, but together they don’t form a map. But on day 5 they form a map. If you want a map, you need all these neurons to work together in a coordinated ensemble.”

Mice mapping mazes

To conduct the study, Guo and Wilson along with labmates Jie “Jack” Zhang and Jonathan Newman introduced mice to simple mazes of varying shapes and let them explore them freely for about half an hour a day for several days. Importantly, the mice were not directed to learn anything specific through the offer of any rewards. They just wandered. Previous studies have shown that mice naturally demonstrate “latent learning” of spaces from this kind of unrewarded experience after several days.

To understand how latent learning takes hold, Guo and his colleagues visually monitored hundreds of neurons in the CA1 area of the hippocampus by engineering cells to flash when a buildup of calcium ions made them electrically active. They not only recorded the neurons’ flashes when the mice were actively exploring, but also while they were sleeping. Wilson’s lab has shown that animals “replay” their previous journeys during sleep, essentially refining their memories by dreaming about their experiences.

Analysis of the recordings showed that the activity of the place cells developed immediately and remained strong and unchanged over several days of exploration.  But this activity alone wouldn’t explain how latent learning or a cognitive map evolves over several days. So unlike in many other studies where scientists focus solely on the strong and clear activity of place cells, Guo extended his analysis to the more subtle and mysterious activity of cells that were not so strongly spatially tuned. Using an emerging technique called “manifold learning” he was able to discern that many of the “weakly spatial” cells gradually correlated their activity not with locations, but with activity patterns among other neurons in the network. As this was happening, Guo’s analyses showed, the network encoded a cognitive map of the maze that increasingly resembled the literal, physical space.

“Although not responding to specific locations like strongly spatial cells, weakly spatial cells specialize in responding to ‘‘mental locations,’’ i.e., specific ensemble firing patterns of other cells,” the study authors wrote. “If a weakly spatial cell’s mental field encompasses two subsets of strongly spatial cells that encode distinct locations, this weakly spatial cell can serve as a bridge between these locations.”

In other words, the activity of the weakly spatial cells likely stitches together the individual locations represented by the place cells into a mental map.

The need for sleep

Studies by Wilson’s lab and many others have shown that memories are consolidated, refined and processed by neural activity, such as replay, that occurs during sleep and rest. Guo and Wilson’s team therefore sought to test whether sleep was necessary for the contribution of weakly spatial cells to latent learning of cognitive maps.

To do this they let some mice explore a new maze twice during the same day with a three-hour siesta in between. Some of the mice were allowed to sleep but some were not. The ones that did showed a significant refinement of their mental map, but the ones that weren’t allowed to sleep showed no such improvement. Not only did the network encoding of the map improve, but also measures of the tuning of individual cells during showed that sleep helped cells become better attuned both to places and to patterns of network activity, so called “mental places” or “fields.”

Mental map meaning

The “cognitive maps” the mice encoded over several days were not literal, precise maps of the mazes, Guo notes. Instead they were more like schematics. Their value is that they provide the brain with a topology that can be explored mentally, without having to be in the physical space. For instance, once you’ve formed your cognitive map of the neighborhood around your hotel, you can plan the next morning’s excursion (e.g. you could imagine grabbing a croissant at the bakery you observed a few blocks west and then picture eating it on one of those benches you noticed in the park along the river).

Indeed, Wilson hypothesized that the weakly spatial cells’ activity may be overlaying salient non-spatial information that brings additional meaning to the maps (i.e. the idea of a bakery is not spatial, even if it’s closely linked to a specific location). The study, however, included no landmarks within the mazes and did not test any specific behaviors among the mice. But now that the study has identified that weakly spatial cells contribute meaningfully to mapping, Wilson said future studies can investigate what kind of information they may be incorporating into the animals’ sense of their environments. We seem to intuitively regard the spaces we inhabit as more than just sets of discrete locations.

“In this study we focused on animals behaving naturally and demonstrated that during freely exploratory behavior and subsequent sleep, in the absence of reinforcement, substantial neural plastic changes at the ensemble level still occur,” the authors concluded. “This form of implicit and unsupervised learning constitutes a crucial facet of human learning and intelligence, warranting further in-depth investigations.”

The Freedom Together Foundation, The Picower Institute for Learning and Memory and the National Institutes of Health funded the study.