Author: Raleigh McElvery

How some tissues can “breathe” without oxygen
Eva Frederick | Whitehead Institute
December 2, 2021

Humans need oxygen molecules for a process called cellular respiration, which takes place in our cells’ mitochondria. Through a series of reactions called the electron transport chain, electrons are passed along in a sort of cellular relay race, allowing the cell to create ATP, the molecule that gives our cells energy to complete their vital functions.

At the end of this chain, two electrons remain, which are typically passed off to oxygen, the “terminal electron acceptor.” This completes the reaction and allows the process to continue with more electrons entering the electron transport chain.

In the past, however, scientists have noticed that cells are able to maintain some functions of the electron transport chain, even in the absence of oxygen. “This indicated that mitochondria could actually have partial function, even when oxygen is not the electron acceptor,” said Whitehead Institute postdoctoral researcher Jessica Spinelli. “We wanted to understand, how does this work? How are mitochondria capable of maintaining these electron inputs when oxygen is not the terminal electron acceptor?”

In a paper published December 2 in the journal Science, Whitehead Institute scientists and collaborators led by Spinelli have found the answer to these questions. Their research shows that when cells are deprived of oxygen, another molecule called fumarate can step in and serve as a terminal electron acceptor to enable mitochondrial function in this environment. The research, which was completed in the laboratory of former Whitehead Member David Sabatini, answers a long-standing mystery in the field of cellular metabolism, and could potentially inform research into diseases that cause low oxygen levels in tissues, including ischemia, diabetes and cancer.

A new runner in the cellular relay

The researchers began their investigation into how cells can maintain mitochondrial function without oxygen by using mass spectrometry to measure the quantities of molecules called metabolites that are produced through cellular respiration in both normal and low-oxygen conditions. When cells were deprived of oxygen, researchers noticed a high level of a molecule called succinate.

When you add electrons to oxygen at the end of the electron transport chain, it picks up two protons and becomes water. When you add electrons to fumarate, it becomes succinate. “This led us to think that maybe this accumulation of succinate that’s occurring could actually be caused by fumarate being used as an electron acceptor, and that this reaction could explain the maintenance of mitochondrial functions in hypoxia,” Spinelli said.

Usually, the fumarate-succinate reaction runs the other direction in cells — a protein complex called the SDH complex takes away electrons from succinate, leaving fumarate. For the opposite to happen, the SDH complex would need to be running in reverse. “Although the SDH complex is known to catalyze some fumarate reduction, it was thought that it was thermodynamically impossible for this SDH complex to undergo a net reversal,” Spinelli said. “Fumarate is used as an electron acceptor in lower eukaryotes, but they use a totally different enzyme and electron carrier, and mammals are not known to possess either of these.”

Through a series of assays, however, the researchers were able to ascertain that this complex was indeed running in reverse in cultured cells, largely due to accumulation of a molecule called ubiquinol, which the researchers observed to build up under low-oxygen conditions.

With their hypothesis confirmed, “We wanted to get back to our initial question and ask, does that net reversal of the SDH complex maintain mitochondrial functions which are happening when cells are exposed to hypoxia?” said Spinelli. “So, we knocked out SDH complex and then we demonstrated through a number of means that loss of both oxygen and fumarate as an electron acceptors was sufficient to [bring the electron transport chain to a halt].”

All this work was done in cultured cells, so the next step for Spinelli and collaborators was to study whether fumarate could serve as a terminal electron acceptor in mouse models.

When they tried this, the team uncovered something interesting: some, but not all, of the mice’s tissues were able to successfully reverse the activity of the SDH complex and perform mitochondrial functions using fumarate as a terminal electron acceptor.

“What was really cool to see is that there were three tissues  — the kidney, the liver, and the brain — which on a bulk tissue scale, are operating the SDH complex in a backwards direction, even at physiological oxygen levels,” said Spinelli. In other words, even in normal conditions, these tissues were reducing both fumarate and oxygen to maintain their functions, and when deprived of oxygen, fumarate could take over as a terminal electron acceptor.

In contrast, tissues such as the heart and the skeletal muscle are able to perform minimal fumarate reduction without reversing the SDH complex, but not to the extent that they could effectively retain mitochondrial function when deprived of oxygen.

“We think there’s a lot of exciting work downstream of this to figure out how exactly this process is regulated differently in different tissues — and understanding in disease settings whether the SDH complex is operating in the net reverse direction,” Spinelli said.

In particular, Spinelli is interested in studying the behavior of the SDH complex in cancer cells.
“Certain regions of solid tumors have very low levels of oxygen, and certain regions have high levels of oxygen,” Spinelli said. “It’s interesting to think about how those cells are surviving in that microenvironment — are they using fumarate as an electron acceptor to enable cell survival?”

Study reveals a protein’s key contribution to heterogeneity of neurons

Tomosyn’s tight regulation of neurotransmitter release distinguishes functions of two neuron classes at the fly neuromuscular junction

Picower Institute
November 29, 2021

The versatility of the nervous system comes from not only the diversity of ways in which neurons communicate in circuits, but also their “plasticity,” or ability to change those connections when new information has to be remembered, when their circuit partners change, or other conditions emerge. A new study by neuroscientists at The Picower Institute for Learning and Memory of MIT shows how just one protein situated on the front lines of neural connections, or synapses, can profoundly change how some neurons communicate and implement plasticity.

The team found that expression of the tomosyn protein is a major determining factor in whether the “presynaptic” neurons that sending signals to control muscle contraction will be “phasic,” meaning they quickly release a lot of the neurotransmitter glutamate across synapses to drive communication, or will be “tonic,” meaning they will apportion glutamate in measured doses, keeping some in reserve. Because tonic neurons have those reserves, the study shows, they can step up glutamate release when receptors across the synapse begin to falter, a plasticity known as presynaptic homeostatic potentiation (PHP). Phasic neurons, with little or no tomosyn-mediated reserve, cannot respond similarly.

“If you break the synapse on the postsynaptic side, the presynaptic neuron will recognize that and generate more output to keep the overall synaptic response the same. This critical type of adaptive plasticity requires tomosyn,” said Troy Littleton, senior author of the new study in eLife and Menicon Professor of Neuroscience in The Picower Institute and MIT’s Departments of Biology and Brain and Cognitive Sciences. “Diversity in the ability of different neurons to express this form of plasticity depends on whether they normally express the protein or not.”

Understanding Tomosyn’s role in neurons is important not only for defining the fundamental workings of synapses and plasticity mechanisms, a long-term goal of Littleton’s lab, but also because like flies, humans make tomosyn proteins and have tonic and phasic classes of neurons.

A decoy diversion

Before the study, tomosyn was known to become involved in the “SNARE” molecular machinery of presynaptic neurons. SNARE proteins dock packets, or vesicles, of neurotransmitters such as glutamate on the membrane of neurons so they can be released across the synapse. Tomosyn was also suspected to be a target of an enzyme considered important for learning and memory and plasticity, Littleton said.

Picower Fellow and former graduate student Chad Sauvola led the new study in Littleton’s lab to determine exactly what tomosyn does. He picked up on work started by co-author Nicole Aponte-Santiago, a fellow former graduate student, who had made (but not yet tested) mutations of the tomosyn gene in her research on tonic and phasic neuron plasticity.

When Sauvola started recording synaptic transmission from neurons with the tomosyn mutations, which were designed to disable the protein, he saw that the synapses engaged in much more glutamate transmission, with the muscles having much larger responses than normal. The loss of normal tomosyn apparently took the brakes off of glutamate release. Notably, he could repair the effects of the mutation by swapping in the human tomosyn protein, suggesting conservation of the protein’s property across species.

To learn how tomosyn works, Sauvola studied its structure and found the protein prevented synaptic vesicles from docking to the membrane by acting as a decoy to sequester SNARE proteins on the plasma membrane. He confirmed this in electron microscopy of neurons, with synapses lacking tomosyn showing 50 percent more vesicles at the membrane than those with tomosyn present. He also purposely stimulated synapses to encourage glutamate release and found that while normal tomosyn normally kept a lid on activity in wildtype animals, the mutants could not properly brake the amount of synaptic transmission.

Side by side panels show lots of gray circles above a dark gray line. The left "control" panel. shows 4 circles on the line while the right tomosyn mutant panel shows six circles on the line
Electron microscope images show a normal tonic synapse on the left with four vesicles docked to the cell membrane (see arrows), and a tomosyn mutant tonic synapse on the right with six vesicles on the membrane.

A stark difference

Given the difference in glutamate release behavior between tonic and phasic neurons, Sauvola decided to examine tomosyn levels in those cell types. The weaker tonic neurons turned out to have more than twice as much tomosyn as the stronger phasic neurons, suggesting that tomosyn levels could account for the difference in glutamate release style.

To determine if tomosyn had such a pivotal role, Sauvola did more stimulation experiments in the two neuronal types. After stimulation in normal animals, phasic neurons emitted much more glutamate than tonic neurons, as expected. However,  in the tomosyn mutants, the two neuronal classes behaved similarly, with tonic neurons releasing more similarly to their phasic neuronal counterparts.

Enabling plasticity

If tomosyn was holding back vesicle release of glutamate specifically in tonic neurons, then that might account for why only tonic neurons are able to exhibit PHP plasticity. Sure enough, when Sauvola disrupted glutamate receptors in muscle cells to induce the PHP response, he found that tonic neurons lacking tomosyn, just like control phasic neurons, could not trigger this form of plasticity. But when he looked at the response in normal tonic neurons, he found that synapse by synapse there were major increases in glutamate release – even synapses that showed very little propensity beforehand seemed to gain substantial capability to release synaptic signals.

“That’s really an amazing discovery that I hadn’t anticipated,” Littleton said. “It’s very surprising to see that these weak synapses could act much more mature on a very rapid timescale.”

On a dark gray background are two wavy strips of multi colored dots. The top strip is much bluer while the bottom strip has dots of warmer colors
These maps of synapse active zone release probability in a tonic neuron show low probabilities (cooler colors) under normal circumstances and much higher release probabilities (warmer colors) amid presynaptic homeostatic potentiation.

One of the next steps for the lab will be to figure out what molecular interaction causes tomosyn to ease off the brakes when PHP is needed, Littleton said. Another future direction will be to look at other neuron types, especially in the brain, to see how tomosyn levels vary and how that affects their synaptic output.

But the new results definitively show that tomosyn’s ability to prevent SNARE binding of vesicles and resulting glutamate release makes a dramatic difference in neural communication style between tonic and phasic neurons.

In addition to Sauvola, Littleton and Aponte-Santiago, the paper’s other authors are Yulia Akbergenova and Karen Cunningham.

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

Stem Cell Research Zeroes in on Cancer

Collaborators investigate colon health with novel tools

Deborah Halber | Spectrum
November 9, 2021

In a building at the edge of the Massachusetts General Hospital (MGH) complex, Ömer Yilmaz, MD, and a group of pathology residents gather around a microscope. A resident reads from a chart: a growth was found in the intestine of a patient who had complained of abdominal pain.

Yilmaz, an MIT cancer researcher and a gastrointestinal pathologist, hoped a closer look at the tumor would reveal a noncancerous collection of fat cells or lymphoid cells.

It had taken a couple of days to prepare the biopsy. Somewhere in the hospital, the patient and her family were anxiously awaiting a diagnosis. Yilmaz leaned forward and adjusted the focus on the microscope.

On the tracks of cancer

If the long, twisting tube of the human digestive tract were stretched out straight, it would extend 30 feet, and its absorptive surface area is roughly comparable to the size of a tennis court. A significant chunk of that tube is the large intestine, an intricate place rife with microscopic structures called niches and crypts, evoking an underground cavern or the ocean floor. Besides the skin, the intestines are the body’s primary barrier against external invaders.

Yilmaz, an associate professor of biology at the Koch Institute for Integrative Cancer Research, believes certain cancers and diseases such as inflammatory bowel disease originate with a breakdown of the intestine’s protective barrier. Diet appears to affect intestinal stem cells; these cells can morph into a variety of cell types, and changes in stem cells can lead to cancer, but no one understands exactly how this occurs.

That’s where Yilmaz’s partnership with MIT biomedical engineer and chemist Alex Shalek comes in. Yilmaz and Shalek are both members of the MIT Stem Cell Initiative, which focuses on fundamental biological questions about benign and cancerous adult stem cells.

Shalek, a core member of the Institute for Medical Engineering and Science (IMES), a member of the Koch Institute, and an associate professor of chemistry, develops experimental and computational tools that provide researchers with detailed snapshots of what’s going on inside living cells at a moment in time. Some of these tools, Yilmaz hoped, would enable him to see how intestinal cells react when they encounter an influx of fat or are deprived of food for hours or days.

“In the past, people would have taken a piece of gut that had many different cell types and said, ‘What changes, on average, under different dietary conditions?’” Shalek says. His tools give him and Yilmaz more precise information, providing a window into the discrete molecular responses of individual cells within the colon.

The role of stem cells

Growing up in Battle Creek, Michigan, Yilmaz spent all his free time trailing after his father, a physician who had immigrated from Turkey. He’d make hospital rounds with his dad, visiting the pathology and radiology labs. As Yilmaz grew older, the two would talk about the mechanisms underlying disease.

After completing his MD/PhD at the University of Michigan, Yilmaz did his residency in pathology, the study of disease, at MGH. He began working at the Whitehead Institute with MIT biology professor David M. Sabatini, a pioneer in elucidating the mechanisms under-lying the regulation of growth and metabolism in mammals. Yilmaz had long been fascinated with stem cells’ seemingly miraculous ability to become any kind of cell the body needed. In adults, stem cells are relatively rare, best studied in bone marrow.

When scientists first found stem cells in the intestine in 2007, Yilmaz shifted his research focus. “As soon as intestinal stem cells were identified, I became interested in understanding how they are regulated by diet and aging,” he says.

“We know obesity elevates cancer risk in a wide range of tissues, including the colon, but we don’t know exactly how. And fasting regimens have been known to improve organ and tissue health, but this, too, is not well understood.”

To better study the transition from healthy to diseased cells in the colon, Yilmaz’s team generated colon tumors in mice that closely resemble human tumors. These colon tumors from mice or humans can be grown in culture, creating miniature three-dimensional tumors called organoids.

Subjecting the organoids to different conditions, Yilmaz and Sabatini found that in mice, age-related loss of stem cell function can be reversed by a 24-hour fast. Other studies looked at the type of high-fat diet leading to obesity. Yilmaz determined that a high-fat diet boosted the population of intestinal stem cells and generated even more cells that behaved like stem cells. These stem cells and stem-like cells are more likely to give rise to intestinal tumors.

What’s happening inside

In the microenvironment of the digestive system, the single layer of epithelial cells that line the colon die after only a few days of ferrying nutrients into the bloodstream and lymphatic system.

Stem cells sheltered in protected spaces with fanciful names like the crypts of Lieberkühn generate a hundred grams of new intestinal tissue every day. The source of all the epithelial cells as well as the cells of the villi, a velvety layer of fingerlike projections that line the intestine, stem cells repair and replace tissue continually assaulted by stomach acid, pancreatic enzymes, bile, fats, and bacteria.

Nearby cells guard the stem cells by secreting agents that fight off harmful bacteria, fungi, and viruses and help regulate the composition of the microbiome.

Most of the body’s stem cells, like those deep within bone marrow, are not nearly as prolific as intestinal stem cells, likely because there’s a risk associated with the stem cells’ ability to rapidly replace themselves: mutations.

At the heart of a cell’s behavior is its messenger RNA, or mRNA, the technology used in the Moderna and Pfizer Covid-19 vaccines. These mRNA vaccines teach cells how to make a protein that triggers an immune response to the virus. Each mRNA transcript, a single strand of RNA carrying a specific genetic instruction from the DNA in the nucleus to the cell’s protein-making machinery, determines which protein gets made to help support the cell’s activity.

“From a snapshot of all of the cell’s mRNA, its transcriptome, we can see how it is trying to respond to change,” Shalek says.

Shalek’s tools help him and Yilmaz measure the properties of multiple types of intestinal cells—immune cells, stem cells, and epithelial cells, to name a few—at once to see precisely how these otherwise invisible, minute features collectively orchestrate tissue-wide responses to external signals.

Sequencing a cell’s mRNA makeup requires smashing the cell open and collecting all of its transcripts. Shalek jokingly likened the process to an alien invader beaming human specimens up to a spaceship and investigating what’s happening inside them.

One of the methods Shalek helped develop tags each mRNA within a cell so that it can be traced back to its cell of origin even after it’s been ripped apart. The inexpensive, portable system, called Seq-Well, looks like an ice cube tray. Around the size of a stick of gum, it contains roughly a hundred thousand miniature wells, each approximately 50-by-50-by-50 microns.

Each cell is deposited into its own well, which contains a bead coated with uniquely barcoded DNA molecules; those DNA molecules are designed to latch onto mRNA and ignore the rest of the cell’s components. The wells are sealed and the cells broken apart. The beads are then extracted, processed, and analyzed, providing a record of each cell’s intentions in its last living moments.

The fact that the system can look simultaneously at thousands of individual cells of any type allows Shalek and Yilmaz to check the effect of nutrients on epithelial cells, immune cells, and stem cells all at once.

The Shalek lab is also developing screening tools that are particularly useful for exposing the Yilmaz lab’s organoids to hundreds of nutrients or drugs at one time, potentially reducing the effort needed to identify substances that boost or hinder stem cell function.

Already, Yilmaz and Shalek have used Seq-Well to identify an enzyme that could be a potential future target for a drug that would counter the negative effects of a high-fat diet on intestinal stem cells. More broadly, Yilmaz says, their collaboration is helping develop a very nuanced understanding of a very complex organ.

“Understanding that complexity is what has really driven our collaboration,” Yilmaz says. “Alex has developed the tools that enable us to dissect out individual cell populations and start to understand how environmental factors impact gene expression.”

“Scientists have spent the past 40 years delineating the genetic drivers of colon cancer, and we still have more to learn. But we’ve now entered the era in which we want to understand the impact of environmental and host factors,” Yilmaz says.

Yilmaz hopes to identify nutrients and metabolites that can enhance stem cell function to repair damage after injury, or to identify mechanisms that dampen tumor formation. In addition, biomarkers such as levels of certain substances in the blood could be a key to early intervention, he says.

“Can we identify which obese patients are more prone to developing colon cancer? If so, can we identify therapies that go after weaknesses in their tumors?”

Battling colon cancer

During the time Yilmaz spends at MGH, he looks at slide after slide of biopsied cells. Normal epithelial cells line up in a single, orderly row. After 15 years in medicine, the twisted appearance of diseased cells still shocks him. “You know, in most cases, the number one predictor of how bad a tumor is going to behave isn’t its genetic signature,” he says. “It’s how deep they invade into their organ of origin, whether they have spread to distant organs, and how bad they look under the microscope.” The cells of this patient’s tumor are misshapen, haphazardly stacked on top of each other.

The patient is in her forties. Yilmaz recalled that when he was a resident, colon cancer in a 40-year-old or 30-year-old was a rarity. He now sees such cases almost weekly. Colorectal cancer is among the top three leading causes of cancer-related deaths in the United States, according to the American Cancer Society. It’s expected to cause around 53,000 deaths during 2021. Yilmaz writes up his diagnosis: invasive cancer of the sigmoid colon. The patient’s oncologist will consult with Yilmaz, radiologists, and the surgical team to come up with a treatment plan.

Ultimately, Yilmaz wants to develop strategies to prevent and reduce the growth of tumors in the intestinal tract. The fact that increasingly younger patients are being diagnosed highlights, for him, the importance of diet. “It’s very worrisome,” he says. “We’re at the beginning of a trend where we’re going to see more and more young people afflicted with what can be a fatal disease if not caught early.” Diet could be an important place to start.

He says, “If you can prevent cancer, that’s the best treatment.”

How sea stars get their symmetry
Greta Friar | Whitehead Institute
November 4, 2021

In a paper published Nov. 4 in the journal Current Biology, Zak Swartz, a postdoctoral researcher at Whitehead Institute, along with researchers in the lab of Whitehead Institute Member Iain Cheeseman and collaborators at the Massachusetts Institute of Technology (MIT), the University of Miami, and the Marine Biological Laboratory Embryology Course delve into the origins of the initial polarity in an animal’s first cell, which establishes an axis of symmetry for the developing organism and underlies the first steps of development. Their research reveals how a specific protein, called Dishevelled, localizes in a cell to help create this polarity.

All multicellular organisms begin as a single cell — the oocyte, precursor cell to the egg — which carries within it a “plan” for the fully developed, complex creature it will become. “How that multifunctional body plan is created is one of the deepest questions in developmental biology,” said Swartz.

“Sea stars, and a huge diversity of other animals, have an incredibly complex body plan, none of which is possible without the polarity of the initial cell,” said Cheeseman. “This work shows how the polarity originates as early as the meiotic divisions in the developing oocyte through an unexpected strategy to break its symmetry and achieve the asymmetric distribution of developmental factors.”

To study the intricate process of body patterning, Cheeseman Lab researchers used a type of sea star called the bat star, or Patiria miniata. These colorful animals are radially symmetric as adults — they usually have five arms, sometimes more — but as larvae they are bilaterally symmetric like humans.

The sea star larvae’s mirror-image symmetry is established when they are egg cells, called oocytes. A key step in the development of this organization involves a protein called Dishevelled, which localizes to the vegetal, or “bottom” end of the oocyte (which will define the posterior end of the embryo) as the cell gets ready to divide into two daughter cells.

Dishevelled — so named because a mutation in the homologous protein in fruit flies lends their tiny hairs a messy, tousled look  — is a component of a common signaling pathway called the Wnt pathway, which is found in many creatures throughout the animal kingdom. The pathway serves various purposes in the cells, from body patterning to cell proliferation. “The Wnt pathway is evolutionarily ancient,” Swartz said. “Jellyfish use it, sea stars use it, people use it, and I think that’s really quite profound.”

In the sea stars, the pathway provides a link between the initial asymmetry of the oocyte and the polarity of the resulting embryo. Dishevelled serves as a messenger on the inside of the sea star’s cells, relaying external signals that are then transmitted through a molecular pathway to the cells’ nuclei.

The researchers used time-lapse imaging to visualize how Dishevelled moved around the oocyte as the cell went through different phases of its development. When the sea star oocyte was in a non-dividing phase, Dishevelled could be found distributed uniformly in small aggregations throughout the cytoplasm.

As the oocyte got ready to divide, however, Dishevelled aggregations dissolved and then reformed at the bottom of the cell at the furthest point from the nucleus.  This provided a clear difference between the two ends of the oocyte.

Swartz was curious about how exactly the protein was localizing to the bottom of the oocyte. There were a number of protein transport options to investigate, so he began systematically ruling them out; the protein was not transported by the cell’s cytoskeleton (“You can think of these like little railroad tracks,” Swartz said), nor was it buoyed along on cytoplasmic currents, nor repelled by some factor at the “top” of the oocyte.

At this interval, Swartz reached out to two collaborators in MIT’s physics department, who helped design experiments to further probe the behavior of Dishevelled in the oocytes. “That’s when we started to consider the idea of dissolution and reassembly, which is kind of the punchline of the paper,” Swartz said. “You can think of it like salt crystals dissolving in water — rather than taking a pre-assembled thing and physically transporting it down [to the bottom of the oocyte], the idea is that these Dishevelled assemblies start out everywhere, get dissolved into their individual components, and then selectively reform in the vegetal region.”

The exact mechanism of this dissolution and reformation is not yet clear. Swartz was able to show that the reformation could not take place in the absence of another Wnt pathway protein called Frizzled, but because Frizzled is not exclusive to the bottom of the oocyte, it is not the only thing driving the reassembly.

In the future, Swartz plans to investigate whether the Dishevelled aggregates are formed in precise structures, or whether they group together as phase-separated droplets such as the RNA molecules studied in Whitehead Institute Member Ankur Jain’s lab or the protein molecules involved in transcription from Whitehead Institute Member Richard Young’s lab. “I’m interested in the broader composition of these structures,” he added. “Do they only contain Dishevelled, or are there other ingredients?”

Regardless of how the assemblies form, the new information on how Dishevelled localizes shines a light on a previously mysterious step in how the Wnt pathway plays a role in early body patterning in sea stars.

“It’s quite striking that Dishevelled localization seems to be an important feature in the Wnt pathway in sea stars, but also in distantly related vertebrates,” Swartz said. “My feeling is that the ability to activate this pathway in selective parts of the early embryo by interpreting polarity built into the oocyte may be a really critical feature of the evolution of the animal body plan.”