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Sallie “Penny” Chisholm awarded the 2019 Crafoord Prize

Institute Professor honored for discovering <i>Prochlorococcus,</i> the most abundant photosynthesizing organism on Earth.

Allison Dougherty | Department of Civil and Environmental Engineering
January 22, 2019

MIT Institute Professor Sallie “Penny” Chisholm of the departments of Civil and Environmental Engineering and Biology is the recipient of the 2019 Crafoord Prize.

Announced on Jan. 17, Chisholm was awarded the prize “for the discovery and pioneering studies of the most abundant photosynthesizing organism on Earth, Prochlorococcus.”

Prochlorococcus is a type of phytoplankton found in the ocean that is able to photosynthesize like plants on land.  The process of photosynthesis is responsible for the oxygen humans breathe, which makes it critical to life on Earth. Prochlorococcus accounts for approximately 10 percent of all ocean photosynthesis, which draws carbon dioxide out of the atmosphere, provides it with oxygen, and forms the base of the food chain.

While the organism is the most abundant photosynthesizer on the planet (the total amount of Prochlorococcus on Earth has been estimated to be 3*1027, or 3,000,000,000,000,000,000,000,000,000), it wasn’t until the mid-1980’s that Prochlorococcus was discovered by Chisholm and colleagues at the Woods Hole Oceanographic Institution. The reason the organism remained unknown for so long can be attributed to its small size. The tiny bacteria is half of a micrometer in size, 1/100 the width of a human hair, making it the smallest photosynthesizing organism.

Since its discovery, Chisholm and her team have found that although each cell has only 2,000 genes, the species as a whole has more than 80,000 different genes in its gene pool, which is four times more than the genetic makeup of humans. This vast diversity of genes distributed among the global population contributes to why Prochlorococcus is able to exist prominently in various environments containing different levels of light, heat, and nutrients.

Chisholm, who has been at MIT since 1976, now studies how Prochlorococcus interacts with various components of seawater and other microorganisms found in the ocean; its role in shaping the ocean ecosystem over evolutionary time; and how its populations may shift in response to climate change.

In April, Chisholm delivered a TED Talk that dove deeper into the properties of Prochlorococcus, comparing the organism’s genetic diversity to iPhone apps, and expanded on the the beauty of this microorganism as the smallest living thing that can convert solar energy and carbon dioxide into fuel through photosynthesis. Understanding its simple design could aid in efforts to engineer artificial photosynthesis machines — reducing our dependency on fossil fuels.

Prochlorococcus has even inspired Chisholm to educate future generations of scientists through a series of children’s books called the “Sunlight Series,” with co-author and illustrator Molly Bang. The series describes the Earth’s natural processes in layman’s terms and through imagery. While none of Chisholm’s books mention Prochlorococcus by name, Chisholm says the simplicity of Prochlorococcus compelled her to create the series.

Chisholm will present her prize lecture in Sweden at Lund University on May 13, and will receive her prize at the Royal Swedish Academy of Sciences prize award ceremony on May 15, in the presence of H. M. King Carl XVI Gustaf and H. M. Queen Silvia of Sweden.

The Crafoord Prize is awarded in partnership between the Royal Swedish Academy of Sciences and the Crafoord Foundation, with the academy responsible for selecting the Crafoord Laureates. Awards are presented in one of four disciplines each year: mathematics and astronomy, geosciences, biosciences, or polyarthritis (such as rheumatoid arthritis).

From microfluidics to metastasis

New platform enables longitudinal studies of circulating tumor cells in mouse models of cancer.

Bendta Schroeder | Koch Institute
January 23, 2019

Circulating tumor cells (CTCs) — an intermediate form of cancer cell between a primary and metastatic tumor cell — carry a treasure trove of information that is critical to treating cancer. Numerous engineering advancements over the years have made it possible to extract cells via liquid biopsy and analyze them to monitor an individual patient’s response to treatment and predict relapse.

Thanks to significant progress toward creating genetically engineered mouse models, liquid biopsies hold great promise for the lab as well. These mouse models mimic many aspects of human tumor development and have enabled informative studies that cannot be performed in patients. For example, these models can be used to trace the evolution of cells from initial mutation to eventual metastasis, a process in which CTCs play a critical role. But since it has not been possible to monitor CTCs over time in mice, scientists’ ability to study important features of metastasis has been limited.

The challenge lies in capturing enough cells to conduct such longitudinal studies. Although primary tumors shed CTCs constantly, the density of CTCs in blood is very low — fewer than 100 CTCs per milliliter. For human patients undergoing liquid biopsy, this does not present a problem. Clinicians can withdraw enough blood to guarantee a sufficient sample of CTCs, just a few milliliters out of five or so liters on average, with minimal impact to the patient.

A mouse, on the other hand, only has about 1.5 milliliters of blood in total. If researchers want to study CTCs over time, they may safely withdraw no more than a few microliters of blood from a mouse each day — nowhere near enough to ensure that many (or any) CTCs are collected.

But with a new approach developed by researchers at the Koch Institute for Integrative Cancer Research, it is now possible to collect CTCs from mice over days and even weeks, and analyze them as the disease progresses. The system, described in the Proceedings of the National Academy of Sciences the week of Jan. 21, diverts blood to a microfluidic cell-sorting chip that extracts individual CTCs before returning the blood back to an awake mouse.

A menu of sorts

The inspiration for the project was cooked up, not in the lab, but during a chance encounter in the Koch Café between Tyler Jacks, director of the Koch Institute and the David H. Koch Professor of Biology, and Scott Manalis, the Andrew and Erna Viterbi Professor in the departments of Biological Engineering and Mechanical Engineering and a member of the Koch Institute.

As luck and lunch lines would have it, the pair would discuss thesis work being done by then-graduate student Shawn Davidson, who was using a dialysis-like system to track metabolites in the bloodstream of mice in the laboratory of Matthew Vander Heiden, an associate professor of biology. Jacks and Manalis were inspired: Could a similar approach could be used to study rare CTCs in real time?

Along with their Koch Institute colleague Alex K. Shalek, the Pfizer-Laubach Career Development Assistant Professor of Chemistry and a core member of the Institute for Medical Engineering and Science (IMES) at MIT, it would take Jacks and Manalis more than five years to put all the pieces of the system together, drawing from different areas of expertise around the Koch Institute. The Jacks lab supplied its fluorescent small cell lung cancer model, the Manalis lab developed the real-time CTC isolation platform, and the Shalek lab provided genomic profiles of the collected CTCs using single-cell RNA sequencing.

“This is a project that could not have succeeded without a sustained effort from several labs with very different sets of expertise. For my lab, which primarily consists of engineers, the opportunity to participate in this type of research has been incredibly exciting and is the reason why we are in the Koch Institute,” Manalis says.

The CTC sorter uses laser excitation to identify tumor cells expressing a fluorescent marker that is incorporated in the mouse model. The system draws blood from the mouse and passes it through a microfluidic chip to detect and extract the fluorescing CTCs before returning the blood back to the mouse. A minute amount of blood — approximately 100 nanoliters — is diverted with every detected CTC into a collection tube, which then is purified further to extract individual CTCs from the thousands of other blood cells.

“The real-time detection of CTCs happens at a flow rate of approximately 2 milliliters per hour which allows us to scan nearly the entire blood volume of an awake and moving mouse within an hour,” says Bashar Hamza, a graduate student in the Manalis lab and one of the lead authors on the paper.

Biology in their blood

With the development of a real-time cell sorter, the researchers could now, for the first time, longitudinally collect CTCs from the same mouse.

Previously, the low blood volume of mice and the rarity of CTCs meant that groups of mice had to be sacrificed at successive times so that their CTCs could be pooled. However, CTCs from different mice often have significantly different gene expression profiles that can obscure subtle changes that occur from the evolution of the tumor or a perturbation such as a drug.

To demonstrate that their cell-sorter could capture these differences, the researchers treated mice with a compound called JQ1, which is known to inhibit the proliferation of cancer cells and perturb gene expression. CTCs were collected and profiled with single-cell RNA sequencing for two hours prior to the treatment, and then every 24 hours after the initial treatment for four days.

When the researchers pooled data for all mice that had been treated with JQ1, they found that the data clustered based on individual mice, offering no confirmation that the drug affects CTC gene expression over time. However, when the researchers analyzed single-mouse data, they observed gene expression shift with time.

“What’s so exciting about this platform and our approach is that we finally have the opportunity to comprehensively study longitudinal CTC responses without worrying about the potentially confounding effects of mouse-to-mouse variability. I, for one, can’t wait to see what we will be able to learn as we profile more CTCs, and their matched primary and metastatic tumors,” says Shalek.

Researchers believe their approach, which they intend to use in additional cancer types including non-small cell lung, pancreatic, and breast cancer, could open new avenues of inquiry in the study of CTCs, such as studying long-term drug responses, characterizing their relationship to metastatic tumors, and measuring their rate of production in short timeframes — and the entire metastatic cascade. In future work, researchers also plan to use their approach for profiling rare immune cells and monitoring cells in dynamic contexts such as wound healing and tumor formation.

“The ability to study CTCs as well other rare cells in the blood longitudinally gives us a powerful view into cancer development. This sorter represents a real breakthrough for the field and it is a great example of the Koch Institute in action,” says Jacks.

The paper’s other co-lead authors are graduate students Sheng Rong Ng from the Jacks lab and Sanjay Prakadan from the Shalek lab. The research is supported, in part, by the Ludwig Center at MIT, the National Cancer Institute, the National Institutes of Health and the Searle Scholars Program.

Study shows how specific gene variants may raise bipolar disorder risk

Findings could help inform new therapies, improve diagnosis.

David Orenstein | Picower Institute for Learning and Memory
January 18, 2019

A new study by researchers at the Picower Institute for Learning and Memory at MIT finds that the protein CPG2 is significantly less abundant in the brains of people with bipolar disorder (BD) and shows how specific mutations in the SYNE1 gene that encodes the protein undermine its expression and its function in neurons.

Led by Elly Nedivi, professor in MIT’s departments of Biology and Brain and Cognitive Sciences, and former postdoc Mette Rathje, the study goes beyond merely reporting associations between genetic variations and psychiatric disease. Instead, the team’s analysis and experiments show how a set of genetic differences in patients with bipolar disorder can lead to specific physiological dysfunction for neural circuit connections, or synapses, in the brain.

The mechanistic detail and specificity of the findings provide new and potentially important information for developing novel treatment strategies and for improving diagnostics, Nedivi says.

“It’s a rare situation where people have been able to link mutations genetically associated with increased risk of a mental health disorder to the underlying cellular dysfunction,” says Nedivi, senior author of the study online in Molecular Psychiatry. “For bipolar disorder this might be the one and only.”

The researchers are not suggesting that the CPG2-related variations in SYNE1 are “the cause” of bipolar disorder, but rather that they likely contribute significantly to susceptibility to the disease. Notably, they found that sometimes combinations of the variants, rather than single genetic differences, were required for significant dysfunction to become apparent in laboratory models.

“Our data fit a genetic architecture of BD, likely involving clusters of both regulatory and protein-coding variants, whose combined contribution to phenotype is an important piece of a puzzle containing other risk and protective factors influencing BD susceptibility,” the authors wrote.

CPG2 in the bipolar brain

During years of fundamental studies of synapses, Nedivi discovered CPG2, a protein expressed in response to neural activity, that helps regulate the number of receptors for the neurotransmitter glutamate at excitatory synapses. Regulation of glutamate receptor numbers is a key mechanism for modulating the strength of connections in brain circuits. When genetic studies identified SYNE1 as a risk gene specific to bipolar disorder, Nedivi’s team recognized the opportunity to shed light into the cellular mechanisms of this devastating neuropsychiatric disorder typified by recurring episodes of mania and depression.

For the new study, Rathje led the charge to investigate how CPG2 may be different in people with the disease. To do that, she collected samples of postmortem brain tissue from six brain banks. The samples included tissue from people who had been diagnosed with bipolar disorder, people who had neuropsychiatric disorders with comorbid symptoms such as depression or schizophrenia, and people who did not have any of those illnesses. Only in samples from people with bipolar disorder was CPG2 significantly lower. Other key synaptic proteins were not uniquely lower in bipolar patients.

“Our findings show a specific correlation between low CPG2 levels and incidence of BD that is not shared with schizophrenia or major depression patients,” the authors wrote.

From there they used deep-sequencing techniques on the same brain samples to look for genetic variations in the SYNE1 regions of BD patients with reduced CPG2 levels. They specifically looked at ones located in regions of the gene that could regulate expression of CPG2 and therefore its abundance.

Meanwhile, they also combed through genomic databases to identify genetic variants in regions of the gene that code CPG2. Those mutations could adversely affect how the protein is built and functions.

Examining effects

The researchers then conducted a series of experiments to test the physiological consequences of both the regulatory and protein coding variants found in BD patients.

To test effects of non-coding variants on CPG2 expression, they cloned the CPG2 promoter regions from the human SYNE1 gene and attached them to a “reporter” that would measure how effective they were in directing protein expression in cultured neurons. They then compared these to the same regions cloned from BD patients that contained specific variants individually or in combination. Some did not affect the neurons’ ability to express CPG2 but some did profoundly. In two cases, pairs of variants (but neither of them individually), also reduced CPG2 expression.

Previously Nedivi’s lab showed that human CPG2 can be used to replace rat CPG2 in culture neurons, and that it works the same way to regulate glutamate receptor levels. Using this assay they tested which of the coding variants might cause problems with CPG2’s cellular function. They found specific culprits that either reduced the ability of CPG2 to locate in the “spines” that house excitatory synapses or that decreased the proper cycling of glutamate receptors within synapses.

The findings show how genetic variations associated with BD disrupt the levels and function of a protein crucial to synaptic activity and therefore the health of neural connections. It remains to be shown how these cellular deficits manifest as biopolar disorder.

Nedivi’s lab plans further studies including assessing behavioral implications of difference-making variants in lab animals. Another is to take a deeper look at how variants affect glutamate receptor cycling and whether there are ways to fix it. Finally, she said, she wants to continue investigating human samples to gain a more comprehensive view of how specific combinations of CPG2-affecting variants relate to disease risk and manifestation.

In addition to Rathje and Nedivi, the paper’s other authors are Hannah Waxman, Marc Benoit, Prasad Tammineni, Costin Leu, and Sven Loebrich.

The JPB Foundation, the Gail Steel Fund, the Carlsberg Foundation, the Lundbeck Foundation and the Danish Council for Independent Research funded the study.

Revising the textbook on introns

Whitehead Institute researchers uncover a group of introns in yeast that possess surprising stability and function.

Nicole Davis | Whitehead Institute
January 16, 2019

A research team from Whitehead Institute has uncovered a surprising and previously unrecognized role for introns, the parts of genes that lack the instructions for making proteins and are typically cut away and rapidly destroyed. Through studies of baker’s yeast, the researchers identified a highly unusual group of introns that linger and accumulate, in their fully intact form, long after they have been freed from their neighboring sequences, which are called exons. Importantly, these persistent introns play a role in regulating yeast growth, particularly under stressful conditions.

The researchers, whose work appears online in the journal Nature, suggest that some introns also might accumulate and carry out functions in other organisms.

“This is the first time anyone has found a biological role for full-length, excised introns,” says senior author David Bartel, a member of the Whitehead Institute. “Our findings challenge the view of these introns as simply byproducts of gene expression, destined for rapid degradation.”

Imagine the DNA that makes up your genes as the raw footage of a movie. The exons are the scenes used in the final cut, whereas the introns are the outtakes — shots that are removed, or spliced out, and therefore not represented in the finished product.

Despite their second-class status, introns are known to play a variety of important roles. Yet these activities are primarily confined to the period prior to splicing — that is, before introns are separated from their nearby exons. After splicing, some introns can be whittled down and retained for other uses — part of a group of so-called “non-coding RNAs.” But by and large, introns have been thought to be relegated to the genome’s cutting room floor.

Bartel and his Whitehead Institute colleagues, including world-renowned yeast expert Gerald Fink, now add an astonishing new dimension to this view: Full-length introns — that is, those that have been cut out but remain otherwise intact — can persist and carry out useful biological functions. As reported in their Nature paper, the team discovered that these extraordinary introns are regulated by and function within the essential TORC1 growth signaling network, forming a previously unknown branch of this network that controls cell growth during periods of stress.

“Our initial reaction was: ‘This is really weird,’” recalls first author Jeffrey Morgan, a former graduate student in Bartel’s lab who is now a postdoc in Jared Rutter’s lab at the University of Utah. “We came across genes where the introns were much more abundant than the exons, which is the exact opposite of what you’d expect.”

The researchers identified a total of 34 of these unusually stable introns, representing 11 percent of all introns in the yeast, also known as Saccharomyces cerevisiae. Surprisingly, there are very few criteria that determine which introns will become stable introns. For example, the genetic sequences of the introns or the regions that surround them are of no significance. The only defining — and necessary — feature, the team found, is a structural one, and involves the precise shape the introns adopt as they are being excised from their neighboring exons. Excised introns typically form a lasso-shaped structure, known as a lariat. The length of the lasso’s handle appears to dictate whether an intron will be stabilized or not.

Remarkably, both yeast and introns have been studied for several decades. Yet until now, these unique introns went undetected. One reason, Bartel and his colleagues believe, is the conditions under which yeast are typically grown. Often, researchers study yeast that are growing very rapidly — so-called log-phase growth. That is because abnormalities are often easiest to detect when cells are multiplying quickly.

“Biologists have focused heavily on log-phase for very good reasons, but in the wild, yeast are very rarely in that condition, whether it’s because of limited nutrients or other stresses,” says Bartel, who is also professor of biology at MIT and a Howard Hughes Medical Institute investigator.

He and his colleagues decided to grow yeast under more stressful circumstances, and that is what ultimately led them to their discovery. Although their experiments were confined to yeast, the researchers believe it is possible other organisms may harbor this long-overlooked class of introns — and that similar approaches using less-often-studied conditions could help illuminate them.

“Right now, we can say it is happening in yeast, but we’d be surprised if this is the only organism in which it is happening,” Bartel says.

The research was supported by the National Institutes of Health and the Howard Hughes Medical Institute.

School of Science honors postdocs and research staff with 2018 Infinite Kilometer Awards

Five winners are recognized for their outstanding contributions to colleagues, the school, and the Institute.

School of Science
December 25, 2018

The MIT School of Science has announced the 2018 winners of the Infinite Kilometer Award. The Infinite Kilometer Award was established in 2012 to highlight and reward the extraordinary work of the school’s postdocs and research staff.

Recipients of the award are exceptional contributors to their research programs. In many cases, they are also deeply committed to their local or global MIT community, and are frequently involved in mentoring and advising their junior colleagues, participating in the school’s educational programs, making contributions to the MIT Postdoctoral Association, or contributing to some other facet of the MIT community.

In addition to a monetary award, the honorees and their colleagues, friends, and family are invited to a celebratory reception in the spring semester.

The 2018 Infinite Kilometer winners are:

Matthew Golder, a National Institutes of Health Postdoctoral Fellow in the Department of Chemistry, nominated by Jeremiah Johnson, an associate professor of chemistry;

Robert Grant, manager of the crystallography lab in the Department of Biology, nominated by Michael Laub, a professor of biology;

Slawomir Gras, a research scientist on the LIGO project at the MIT Kavli Institute for Astrophysics and Space Research, nominated by Nergis Mavalvala, the Curtis and Kathleen Marble Professor of Astrophysics, and Matthew Evans, an associate professor of physics;

Yeong Shin Yim, a postdoc at the McGovern Institute for Brain Research, nominated by Gloria Choi, an assistant professor of brain and cognitive sciences; and

Yong Zhao, a postdoc in the Laboratory for Nuclear Science, nominated by Iain Stewart, a professor of physics.

The School of Science is also currently accepting nominations for its Infinite Mile Awards. All School of Science employees are eligible, and nominations are due by Feb. 15, 2019. The Infinite Mile Awards will be presented with the Infinite Kilometer Awards this spring.

Real-time readouts of thinking in rats

New open-source system provides fast, accurate neural decoding and real-time readouts of where rats think they are.

David Orenstein | Picower Institute for Learning and Memory
December 19, 2018

The rat in a maze may be one of the most classic research motifs in brain science, but a new innovation described in Cell Reports by an international collaboration of scientists shows just how far such experiments are still pushing the cutting edge of technology and neuroscience alike.

In recent years, scientists have shown that by recording the electrical activity of groups of neurons in key areas of the brain they could read a rat’s thoughts of where it was, both after it actually ran the maze and also later when it would dream of running the maze in its sleep — a key process in consolidating its memory. In the new study, several of the scientists involved in pioneering such mind-reading methods now report they can read out those signals in real-time as the rat runs the maze, with a high degree of accuracy and the ability to account for the statistical relevance of the readings almost instantly after they are made.

The ability to so robustly track the rat’s spatial representations in real-time opens the door to a whole new class of experiments, the researchers said. They predict these experiments will produce new insights into learning, memory, navigation and cognition by allowing them to not only decode rat thinking as it happens, but also to instantaneously intervene and study the effects of those perturbations.

“The use of real-time decoding and closed-loop control of neural activity will fundamentally transform our studies of the brain,” says study co-author Matthew Wilson, the Sherman Fairchild Professor in Neurobiology at MIT’s Picower Institute for Learning and Memory.

The collaboration behind the new paper began in Wilson’s lab at MIT almost 10 years ago. At that time, corresponding authors Zhe (Sage) Chen, now an associate professor of psychiatry and neuroscience and physiology at New York University, and Fabian Kloosterman, now a principal investigator at Neuro-Electronics Research Flanders and a professor at KU Leuven in Belgium, were both postdocs at MIT.

After demonstrating how neural decoding can be used to read out what places are covertly replayed in the brain, the team began a series of technical innovations that progressively improved the field’s ability to accurately decode how the brain represents place both during navigation and in sleep or rest. They reached a first milestone in 2013 when the team published their novel decoding approach in a paper in the Journal of Neurophysiology. The new approach allows researchers to directly decipher hippocampal spatiotemporal patterns detected from tetrode recordings without the need for spike sorting, a computational process that is time-consuming and error prone.

In the new study, the team shows that by implementing their neural decoding software on a graphical processing unit (GPU) chip, the same kind of highly parallel processing hardware favored by video gamers, they were able to achieve unprecedented increases in decoding and analysis speed. In the study, the team shows that the GPU-based system was 20-50 times faster than ones using conventional multi-core CPU chips.

They also show that the system remains rapid and accurate even when handling more than 1,000 input channels. This is important because it extends the real-time decoding approach to new high-density brain recording devices, such as the Neuropixels probe co-developed by imec, HHMI and other institutions — think of a many electrodes recording from many hundreds of cells — that promise to measure cellular brain activity at larger scales and in more detail.

In addition, the new study reports the ability for the software to provide a rapid statistical assessment of whether a set of reactivated neural spatiotemporal activity patterns truly pertains to the task, or is perhaps unrelated.

“We are proposing an elegant solution using GPU computing to not only decode information on the fly but also to evaluate the significance of the information on the fly,” says Chen, whose graduate student, Sile Hu, is the new paper’s lead author.

Hu tested a wide range of neural recordings in brain areas such as the hippocampus, the thalamus and cortex in multiple rats as they ran a variety of mazes ranging from simple tracks to a wide-open space. In a video accompanying the paper, the system’s readout from 36 electrode channels in the hippocampus tracks the rat’s actual measured position in open space and provides real-time estimates of the decoded position from brain activity. Only occasionally and briefly do the trajectories diverge by much.

The software of the system is open source and available for fellow neuroscientists to download and use freely, Chen and Wilson say.

Prior experiments recording neural representations of place have helped to show that animals replay their spatial experiences during sleep and have allowed researchers to understand more about how animals rely on memory when making decisions about how to navigate — for instance to maximize the rewards they can find along the way. Traditionally, though, the brain readings have been analyzed offline (after the fact. More recently, scientists have begun to perform real-time analyses but these have been limited both in the detail of the content and also in the ability to understand whether the readings are statistically significant and therefore relevant.

In a recent major step forward, Kloosterman and two other co-authors of the new study, graduate students Davide Ciliberti and Frédéric Michon, published a paper in eLife on a real-time, closed-loop read-out of hippocampal memory replay as rats navigated a three-arm maze. That system used multi-core CPUs.

“The new GPU system will bring the field even closer to having a detailed, real-time and highly scalable read-out of the brain’s internal deliberations,” says Kloosterman, “That will be necessary to increase our understanding of how these replay events drive memory formation and behavior.”

By combining these capabilities with optogenetics — a technology that makes neurons controllable with flashes of light — the researchers could conduct what they call “closed-loop” studies in which they could use their instantaneous readout of spatial thinking to trigger experimental manipulations. For example, they could see what happens to navigational performance the day after they interfered with replay during sleep, or they could determine what temporarily disrupting communication between the cortex and hippocampus might do when a rat faces a key decision about which direction to go.

Hu is also affiliated with Zhejiang University in China. In addition to Hu, Wilson, Chen, Kloosterman, Ciliberti, and Michon, the paper’s other authors are Andres Grosmark of Columbia University, Daoyun Ji of Baylor College of Medicine, Hector Penagos of MIT’s Picower Institute, and György Buzsáki of NYU.

Funding for the study came from the U.S. National Institutes of Health, the National Science Foundation, MIT’s NSF-funded Center for Brains Minds and Machines, Research Foundation – Flanders (FWO), the National Science Foundation of China, and the Simons Foundation.

A tough case cracked
Greta Friar | Whitehead Institute
December 17, 2018

CAMBRIDGE, Mass. — For hundreds of millions of years, plants thrived in the Earth’s oceans, safe from harsh conditions found on land, such as drought and UV radiation. Then, roughly 450 million years ago, plants found a way to make the move to land: They evolved spores—small reproductive cells—and eventually pollen grains with tough, protective outer walls that could withstand the harsh conditions in the terrestrial environment until they could germinate and grow into a plant or fertilize an ovule. A key component of the walls is sporopollenin, a durable polymer — a large molecule made up of many small subunits — that is absent in algae but remains ubiquitous in all land plants to this day.

Understanding the molecular composition of polymers found in nature is a fundamental pursuit of biology, with a long history tracing back to the early days of elucidating DNA and protein structures. However, the very toughness that makes sporopollenin so important for all land plants also makes it tough for researchers to study. It is extremely inert, resistant to reacting with other chemicals, including the ones researchers typically use to determine the structures of other plant biopolymers, such as polysaccharides, lignin, and natural rubber. Consequently, scientists have struggled for decades to figure out exactly what the sporopollenin polymer is made of. Now, in an article published in the journal Nature Plants on December 17, Whitehead Institute Member Jing-Ke Weng and first author and Weng lab postdoc Fu-Shuang Li, together with collaborators Professor Mei Hong and graduate student Pyae Phyo from the Massachusetts Institute of Technology (MIT) Department of Chemistry, have used innovative chemical degradation methods and state-of-the-art nuclear magnetic resonance (NMR) spectroscopy to determine the chemical structure of sporopollenin.

“Plants could not have colonized the land if they had not developed a way to withstand harsh environments,” says Weng, who is also an assistant professor of biology at MIT. “Sporopollenin helped make the terrestrial ecosystem as we know it possible.”

In addition to solving a longstanding puzzle in plant chemistry, identifying the structure of sporopollenin opens the door for its potential use in a host of other applications. Sporopollenin’s inertness is a desirable attribute to replicate in the development of, for example, medical implants such as stents, which prop open clogged arteries, to prevent negative interactions between the device and the body. It could also be a good model for durable paints and coatings, such as those used on boats, where its inertness would prevent reactions with compounds in the water and so protect the ship’s hull from environmental degradation.

Finding the shape and composition of sporopollenin was not a simple task. The first challenge was getting enough of the material to study, as pollen amounts that can be collected from most plants are minute. However, pollen from the pitch pine, Pinus rigida, is sold in bulk in China as a topping for rice cakes, so Weng used an unconventional sample collection method: He asked his parents in China to ship him copious quantities of pitch pine pollen.

A common approach to determine a complex plant polymer’s structure is to dissolve it in solutions with specific chemical compounds that will break it apart into smaller and smaller pieces from which the complete structure can be deduced. But since sporopollenin is inert and does not react with the researchers’ usual cadre of chemicals, figuring out how to break down the molecule was a key challenge.

In order to crack this problem — and make the sporopollenin dissolve more easily — Li used a specially designed grinder known as the high-energy ball mill to physically shear the tiny pollen coat into even finer pieces. Then he began testing different chemical mixtures to find ones that could break apart the sporopollenin polymer into more accessible fragments. The big breakthrough came when he tried a chemical degradation process called thioacidolysis, an acid catalyzed reaction with a pinch of a special sulfur-containing compound. This allowed Li to consistently break down 50% of the total sporopollenin polymer into small pieces, with the structure of each of these pieces resolved one by one.

To help complete the puzzle, the researchers collaborated with Mei Hong’s group in MIT’s Department of Chemistry and used magic-angle-spinning solid-state NMR spectroscopy, which can determine the chemical structures of insoluble compounds by having them interact with magnetic fields. This investigation narrowed the possible structures for sporopollenin. Combined with more chemical degradation tests to verify certain possibilities and eliminate others, it ultimately led to the complete structure.

With the structure of sporopollenin in hand, the researchers were then able to identify aspects of this unique polymer that make it such a good protective wall for spores and pollen.

A key finding was that sporopollenin molecules contain two types of cross-linkages, esters and acetals, that act like chemical clips, binding the chains of the molecule together. Other known plant polymers have only one main type of cross-link, and this unique characteristic likely provides the extreme chemical inertness of sporopollenin. Ester bonds are resistant to mildly acidic conditions, while acetals are resistant to basic conditions, meaning the molecule won’t break down in either type of environment in the wild or in the lab.

Other components of sporopollenin that the researchers found include multiple molecules known to provide UV protection, as well as fatty acids, which are water resistant and may protect spores and pollen from drought or other changes in water availability.

The researchers are now looking for differences in sporopollenin between species. Pine is not a flowering plant, but the majority of plants of interest to agriculture and medicine are, so Weng and Li are investigating how sporopollenin may have changed with the evolution of the flowering plants.

“Since I was a student, inspired by the magnificent discovery of the structure of DNA, I have been driven to discover the fundamental forms of things in nature,” Weng says. “It has been so rewarding to illuminate the structure of this crucial biopolymer in plants.”

This work was supported by the Pew Scholar Program in the Biomedical Sciences and the Searle Scholars Program, and the U.S. Department of Energy (# DE-SC0001090).

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Jing-Ke Weng’s primary affiliation is with Whitehead Institute for Biomedical Research, where his laboratory is located and all his research is conducted. He is also an assistant professor of biology at Massachusetts Institute of Technology.

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Full citation:
“The molecular structure of plant sporopollenin”
Nature Plants, December 17, 2018, DOI: 10.1038/s41477-018-0330-7
Fu-Shuang Li (1), Pyae Phyo (2), Joseph Jacobowitz (1,3), Mei Hong (2), Jing-Ke Weng (1,3)
1. Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, United States.
2. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.
3. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.
Engineering “capture compounds” to probe cell growth

Researchers develop a method to investigate how bacteria respond to starvation and to identify which proteins bind to what they call the “magic spot” — ppGpp.

Raleigh McElvery | Department of Biology
December 17, 2018

In 1969, scientist Michael Cashel was analyzing the compounds produced by starved bacteria when he noticed two spots appearing on his chromatogram as if by magic. Today, we know one of these “magic spots,” as researchers call them, as guanosine tetraphosphate, or ppGpp for short. We also understand that it is a signaling molecule present in virtually all bacteria, helping tune cell growth and size based on nutrient availability.

And yet, despite decades of study, precisely how ppGpp regulates bacterial growth has remained rather mysterious. Delving further requires a more comprehensive list of the molecules that ppGpp binds to exert its effects.

Now, collaborators from MIT’s departments of Biology and Chemistry have developed a method to do just that, and used their new approach to pinpoint over 50 ppGpp targets in Escherichia coli — roughly half which had not been identified previously. Many of these targets are enzymes required to produce nucleotides, the building blocks of DNA and RNA. During times when the bacteria do not have enough nutrients to grow and divide normally, the researchers propose that ppGpp prevents these enzymes from creating new nucleotides from scratch, helping cells enter a dormant state.

“With small molecules or metabolites like ppGpp, it’s been difficult historically to determine which proteins they bind,” says Michael Laub, a professor of biology, a Howard Hughes Medical Institute investigator, and the senior author of the study. “This has been an intractable problem that’s held the field back for some time, but our new approach allows you to nail down the likely targets in a matter of weeks.”

Postdoc Boyuan Wang is the first author of the study, which appeares in Nature Chemical Biology on Dec. 17.

Since ppGpp was discovered nearly 50 years ago, it has been shown to suppress DNA replication, transcription, translation, and various metabolic pathways. It puts the brakes on cell growth and allows bacteria to persist in the face of starvation, stress, and antibiotics. Its influence over numerous regulatory processes has remained somewhat of a mystery, however — after all, it doesn’t just modulate a single pathway but coordinates multiple operations simultaneously to orchestrate a mass shutdown of the cell.

In order to discern which proteins ppGpp binds to effect such widespread change, the researchers built what they call “capture compounds” that contain ppGpp, allowing them to fish out its targets from bacterial extracts. These compounds included a photoreactive crosslinker that latched tightly onto the proteins of interest in the presence of light, and a biotin handle that helped the scientists pull out the proteins to identify them. Most importantly, they were joined to ppGpp in such a way that they wouldn’t interfere with its ability to bind to its targets. This method is more efficient and accurate compared to more traditional means of distinguishing ppGpp targets, which are far more arduous and lack sensitivity.

“Our approach solves these problems because you’re no longer required to do such labor-intensive protocols in order to identify ppGpp targets — and it works even in bacteria beyond E. coli,” says Wang. “Although ppGpp is common among many bacterial species, it seems to exert its effects through different mechanisms, which complicates things. Our capture compounds provide a way to unravel this diversity, and in short order.”

Although the 56 ppGpp targets Wang identified in his screen control a myriad of cellular processes, he homed in on the enzyme PurF — which initiates the biosynthesis of purine nucleotides bearing adenine and guanine bases, also known as A and G.

When bacteria are stressed or starved, they enter a dormant state to survive. But simply curbing translation and transcription is not enough; nucleotides are still being generated and will build up if their synthesis is not put on pause. Cells can build nucleotides in one of two ways: either by salvaging existing materials or starting completely from scratch. PurF kicks off the first step in the latter process leading to the A and G nucleotides. However, when ppGpp binds to PurF, it causes the enzyme to change its shape, which prevents it from doing its job, thus reducing nucleotide production in the cell.

“This is the first time that an enzyme involved in that specific pathway or function has been identified as a ppGpp target,” Wang says. “If you limit the consumption of nucleotides but not their production, the nucleotide pool is going to explode, which isn’t good for the cell. So we’ve shown that ppGpp actually addresses this problem as well.”

In addition to PurF and other enzymes required for nucleotide production, the researchers noticed that ppGpp also binds to many GTPase enzymes involved in translation. This could indicate a failsafe mechanism slowing down translation by striking multiple, similar enzymes in an almost redundant manner in the face of starvation.

As Wang continues to refine his method, he aims to increase its specificity and ensure his capture compounds bind to the exact same proteins they would inside a live cell. He also hopes to screen for ppGpp binding proteins in other bacteria, including pathogens that rely on ppGpp to survive within their hosts and propagate conditions like tuberculosis.

“This is an exciting chemical approach to better understand the function of a long-studied conserved signaling molecule in bacteria,” says Jue Wang, professor of bacteriology at the University of Wisconsin at Madison, who was not involved with the study. “Their findings and techniques are highly relevant to many other bacteria, and will greatly improve knowledge of how bacteria use this critical signaling molecule to mediate everything from surviving in the human gut to causing disease.”

Adds Laub: “We are still discovering new nucleotide-based signaling molecules in bacteria even today, and every single one of them could eventually be derivatized in a similar way to identify their binding partners.”

This research was supported by a fellowship from the Jane Coffin Childs Memorial Fund for Medical Research and a grant from the National Institutes of Health.

How returning to a prior context briefly heightens memory recall
Picower Institute
December 11, 2018

Whether it’s the pleasant experience of returning to one’s childhood home over the holidays or the unease of revisiting a site that proved unpleasant, we often find that when we return to a context where an episode first happened, specific and vivid memories can come flooding back. In a new study in Neuron, scientists in MIT’s Picower Institute for Learning and Memory report the discovery of a mechanism the brain may be employing to make that phenomenon occur.

“Suppose you are driving home in the evening and encounter a beautiful orange twilight in the sky, which reminds you of the great vacation you had a few summers ago at a Caribbean island,” said study senior author Susumu Tonegawa, Picower Professor of Neuroscience at MIT. “This initial recall could be a general recall of the vacation. But moments later, you may get reminded of details of some specific events or situations that took place during the vacation which you had not been thinking about.”

At the heart of that second stage of recall, where specific details are suddenly vividly available, is a change in the electrical excitability of “engram cells,” or the ensemble of neurons that together encode a memory through the specific pattern of their connection. In the new study Tonegawa’s lab, led by postdoc Michele Pignatelli and former member Tomas Ryan, now at Trinity College Dublin, showed that after mice formed a memory in a context, the engram cells encoding that memory in a brain region called the hippocampus would temporarily become much more electrically excitable if the mice were placed back in the same context again. So for instance, if they were given a little shock in a specific context one day, then the engram cells would be much more excitable for about an hour after they were put back in that same context the next day.

The specific change in the engram cells’ electrical properties has some direct implications for learning and behavior that hadn’t been appreciated before. Importantly, during that hour after returning to the initial context, because of the engrams’ elevated excitability, mice proved better able to learn from a shock in that context and better able to distinguish between that and distinct contexts even if they shared some similar cues. The increase in excitability therefore allowed them both to learn to avoid places where danger happened very recently and to continue to function normally in places that happen to have some irrelevant resemblance. And because the effect was short-lived, it didn’t oblige them to remain overly attuned for very long.

“The short-term reactivation increases the future recognition capability of specific cues,” Pignatelli and Tonegawa’s team wrote. “Engram cell excitability may be crucial for survival by facilitating rapid adaptive behavior without permanently altering the fundamental nature of the long-term engram.”

Tonegawa added that “while the survival interpretation may be an evolutionary origin of this multi-step episodic memory recall” it likely also applies to positive episodic memories, like the vacation sunset experience, just as much.