Research Area: Cell Biology
The clinically-trained cell biologist exploits the liver’s unique capacities in search of new medical applications.
Grace van Deelen | Department of Biology
December 15, 2021
Why is the liver the only human organ that can regenerate? How does it know when it’s been injured? What can our understanding of the liver contribute to regenerative medicine? These are just some of the questions that new assistant professor of biology Kristin Knouse and her lab members are asking in their research at the Koch Institute for Integrative Cancer Research. Knouse sat down to discuss why the liver is so unique, what lessons we might learn from the organ, and what its regeneration might teach us about cancer.
Q: Your lab is interested in questions about how body tissues sense and respond to damage. What is it about the liver that makes it a good tool to model those questions?
A: I’ve always felt that we, as scientists, have so much to gain from treasuring nature’s exceptions, because those exceptions can shine light onto a completely unknown area of biology and provide building blocks to confer such novelty to other systems. When it comes to organ regeneration in mammals, the liver is that exception. It is the only solid organ that can completely regenerate itself. You can damage or remove over 75 percent of the liver and the organ will completely regenerate in a matter of weeks. The liver therefore contains the instructions for how to regenerate a solid organ; however, we have yet to access and interpret those instructions. If we could fully understand how the liver is able to regenerate itself, perhaps one day we could coax other solid organs to do the same.
There are some things we already know about liver regeneration, such as when it begins, what genes are expressed, and how long it takes. However, we still don’t understand why the liver can regenerate but other organs cannot. Why is it that these fully differentiated liver cells — cells that have already assumed specialized roles in the liver — can re-enter the cell cycle and regenerate the organ? We don’t have a molecular explanation for this. Our lab is working to answer this fundamental question of cell and organ biology and apply our discoveries to unlock new approaches for regenerative medicine. In this regard, I don’t necessarily consider myself exclusively a liver biologist, but rather someone who is leveraging the liver to address this much broader biological problem.
Q: As an MD/PhD student, you conducted your graduate research in the lab of the late Professor Angelika Amon here at MIT. How did your work in her lab lead to an interest in studying the liver’s regenerative capacities?
A: What was incredible about being in Angelika’s lab was that she had an interest in almost everything and gave me tremendous independence in what I pursued. I began my graduate research in her lab with an interest in cell division, and I was doing experiments to observe how cells from different mammalian tissues divide. I was isolating cells from different mouse tissues and then studying them in culture. In doing that, I found that when the cells were isolated and grown in a dish they could not segregate their chromosomes properly, suggesting that the tissue environment was essential for accurate cell division. In order to further study and compare these two different contexts — cells in a tissue versus cells in culture — I was keen to study a tissue in which I could observe a lot of cells undergoing cell division at the same time.
So I thought back to my time in medical school, and I remembered that the liver has the ability to completely regenerate itself. With a single surgery to remove part of the liver, I could stimulate millions of cells to divide. I therefore began exploiting liver regeneration as a means of studying chromosome segregation in tissue. But as I continued to perform surgeries on mice and watch the liver rapidly regenerate itself, I couldn’t help but become absolutely fascinated by this exceptional biological process. It was that fascination with this incredibly unique but poorly understood phenomenon — alongside the realization that there was a huge, unmet medical need in the area of regeneration — that convinced me to dedicate my career to studying this.
Q: What kinds of clinical applications might a better understanding of organ regeneration lead to, and what role do you see your lab playing in that research?
A: The most proximal medical application for our work is to confer regenerative capacity to organs that are currently non-regenerative. As we begin to achieve a molecular understanding of how and why the liver can regenerate, we put ourselves in a powerful position to identify and surmount the barriers to regeneration in non-regenerative tissues, such as the heart and nervous system. By answering these complementary questions, we bring ourselves closer to the possibility that, one day, if someone has a heart attack or a spinal cord injury, we could deliver a therapy that stimulates the tissue to regenerate itself. I realize that may sound like a moonshot now, but I don’t think any problem is insurmountable so long as it can be broken down into a series of tractable questions.
Beyond regenerative medicine, I believe our work studying liver regeneration also has implications for cancer. At first glance this may seem counterintuitive, as rapid regrowth is the exact opposite of what we want cancer cells to do. However, the reality is that the majority of cancer-related deaths are attributable not to the rapidly proliferating cells that constitute primary tumors, but rather to the cells that disperse from the primary tumor and lie dormant for years before manifesting as metastatic disease and creating another tumor. These dormant cells evade most of the cancer therapies designed to target rapidly proliferating cells. If you think about it, these dormant cells are not unlike the liver: they are quiet for months, maybe years, and then suddenly awaken. I hope that as we start to understand more about the liver, we might learn how to target these dormant cancer cells, prevent metastatic disease, and thereby offer lasting cancer cures.
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?”
December 2, 2021
November 18, 2021
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November 3, 2021
Researchers glean a more complete picture of a complex structure called the nuclear pore complex by studying it directly inside cells.
Raleigh McElvery | Department of Biology
October 13, 2021
Context matters. It’s true for many facets of life, including the tiny molecular machines that perform vital functions inside our cells.
Scientists often purify cellular components, such as proteins or organelles, in order to examine them individually. However, a new study published today in the journal Nature suggests that this practice can drastically alter the components in question.
The researchers devised a method to study a large, donut-shaped structure called the nuclear pore complex (NPC) directly inside cells. Their results revealed that the pore had larger dimensions than previously thought, emphasizing the importance of analyzing complex molecules in their native environments.
“We’ve shown that the cellular environment has a significant impact on large structures like the NPC, which was something we weren’t expecting when we started,” says Thomas Schwartz, the Boris Magasanik Professor of Biology at MIT and the study’s co-senior author. “Scientists have generally thought that large molecules are stable enough to maintain their fundamental properties both inside and outside a cell, but our findings turn that assumption on its head.”
In eukaryotes like humans and animals, most of a cell’s DNA is stored in a rounded structure called the nucleus. This organelle is shielded by the nuclear envelope, a protective barrier that separates the genetic material in the nucleus from the thick fluid filling the rest of the cell. But molecules still need a way to come in and out of the nucleus in order to facilitate important processes, including gene expression. That’s where the NPC comes in. Hundreds — sometimes thousands — of these pores are embedded in the nuclear envelope, creating gateways that allow certain molecules to pass.
The study’s first author, former MIT postdoc Anthony Schuller, compares NPCs to gates at a sports stadium. “If you want to access the game inside, you have to show your ticket and go through one of these gates,” he explains.
The NPC may be tiny by human standards, but it’s one of the largest structures in the cell. It’s comprised of roughly 500 proteins, which has made its structure challenging to parse. Traditionally, scientists have broken it up into individual components to study it piecemeal using a method called X-ray crystallography. According to Schwartz, the technology required to analyze the NPC in a more natural environment has only recently become available.
Together with researchers from the University of Zurich, Schuller and Schwartz employed two cutting-edge approaches to solve the pore’s structure: cryo-focused ion beam (cryo-FIB) milling and cryo-electron tomography (cryo-ET).
An entire cell is too thick to look at under an electron microscope. But the researchers sliced frozen colon cells into thin layers using the cryo-FIB equipment housed at MIT.nano’s Center for Automated Cryogenic Electron Microscopy and the Koch Institute for Integrative Cancer Research’s Peterson (1957) Nanotechnology Materials Core Facility. In doing so, the team captured cross-sections of the cells that included NPCs, rather than simply looking at the NPCs in isolation.
“The amazing thing about this approach is that we’ve barely manipulated the cell at all,” Schwartz says. “We haven’t perturbed the cell’s internal structure. That’s the revolution.”
What the researchers saw when they looked at their microscopy images was quite different from existing descriptions of the NPC. They were surprised to find that the innermost ring structure, which forms the pore’s central channel, is much wider than previously thought. When it’s left in its natural environment, the pore opens up to 57 nanometers — resulting in a 75 percent increase in volume compared to previous estimates. The team was also able to take a closer look at how the NPC’s various components work together to define the pore’s dimensions and overall architecture.
“We’ve shown that the cellular environment impacts NPC structure, but now we have to figure out how and why,” Schuller says. Not all proteins can be purified, he adds, so the combination of cryo-ET and cryo-FIB will also be useful for examining a variety of other cellular components. “This dual approach unlocks everything.”
“The paper nicely illustrates how technical advances, in this case cryo-electron tomography on cryo-focused ion beam milled human cells, provide a fresh picture of cellular structures,” says Wolfram Antonin, a professor of biochemistry at RWTH Aachen University in Germany who was not involved in the study. The fact that the diameter of the NPC’s central transport channel is larger than previously thought hints that the pore could have impressive structural flexibility. “That may be important for the cell to adapt to increased transport demands,” Antonin explains.
Next, Schuller and Schwartz hope to understand how the size of the pore affects which molecules can pass through. For instance, scientists only recently determined that the pore was big enough to allow intact viruses like HIV into the nucleus. The same principle applies to medical treatments: only appropriately-sized drugs with specific properties will be able to access the cell’s DNA.
Schwartz is especially curious to know whether all NPCs are created equal, or if their structure differs between species or cell types.
“We’ve always manipulated cells and taken the individual components out of their native context,” he says. “Now we know this method may have much bigger consequences than we thought.”
Education
- PhD, 2016, Stanford University School of Medicine
- BA, 2008, Molecular Biology, Princeton University
Research Summary
Our bodies are tuned to detect and respond to cues from the outside world and from within through exquisite collaborations between cells. For example, the cells lining our airways communicate with sensory neurons in response to chemical and mechanical signals, and evoke key reflexes such as coughing. This cellular collaboration protects our airways from damage and stabilizes breathing, but can become dysregulated in disease. Despite their vital importance to human health, fundamental questions about how sensory transduction is accomplished at these sites remain unsolved. We use the mammalian airways as a model system to investigate how physiological insults are detected, encoded, and addressed at essential barrier tissues — with the ultimate goal of providing new ways to treat autonomic dysfunction.
Awards
- Warren Alpert Distinguished Scholars Award, 2021
- Life Sciences Research Foundation Fellowship, 2018
Whitehead Institute
July 30, 2021
To create eggs and sperm, cells must rewire the process of cell division. Mitosis, the common type of cell division that our bodies use to grow everything from organs to fingernails and to replace aging cells, produces two daughter cells with the same number of chromosomes and approximately the same DNA sequence as the original cell. Meiosis, the specialized cell division that makes egg and sperm in two rounds of cell division, creates four granddaughter cells with new variations in their DNA sequence and half as many chromosomes in each cell. Meiosis uses most of the same cellular machinery as mitosis to achieve this very different outcome; only a few key molecular players prompt the rewiring from one type of division to another. One such key player is the protein Meikin, which is found exclusively in cells undergoing meiosis.
New research from Whitehead Institute Member Iain Cheeseman, graduate student Nolan Maier and collaborators Professor Michael Lampson and senior research scientist Jun Ma at the University of Pennsylvania demonstrates how Meikin is elegantly controlled, and sheds light on how the protein acts to serve multiple roles over different stages of meiosis. The findings, which appear in Developmental Cell on July 30, reveal that Meikin is precisely cut in half midway through meiosis. Instead of this destroying the protein, one half of the molecule, known as C-Meikin, goes on to play a critical role as a previously hidden protein actor in meiosis.
“Cells have this fundamental process, mitosis, during which they have to divide chromosomes evenly or it will cause serious problems like cancer, so the system has to be very robust,” Maier says. “What’s incredible is that you can add one or two unique meiotic proteins like Meikin and dramatically change the whole system very quickly.”
Helping chromosomes stick together
During both mitosis and meiosis, sister chromatids — copies of the same chromosome — pair up to form the familiar “X” shape that we recognize as a chromosome. In mitosis, each chromatid—each half of the X — is connected to a sort of cellular fishing line and these lines reel the chromatids to opposite ends of the cell, where the two new cells are formed around them. However, in the first round of division in meiosis, the sister chromatids stick together, and one whole “X” is reeled into each new cell. Meikin helps to achieve this different outcome by ensuring that, while the chromosomes are being unstuck from each other in preparation for being pulled apart, each pair of sister chromatids stays glued together in the right place. Meikin also helps ensure that certain cellular machinery on the sister chromatids is fused so that they will connect to the same line and be reeled together to the same side of the cell.
More specifically, when chromosomes are first paired up, they are glued together by adhesive molecules in three regions: the centromere, or center of the X, where Meikin localizes; the region around the center; and the arms of the X. In the first round of meiosis, Meikin helps to keep the glue in the region around the center intact, so the sister chromatids will stick together. Simultaneously, Meikin helps to prime the center region to be unglued, while a separate process unglues the arms. This ungluing allows the chromosomes to separate and be prepared for later stages of meiosis.
Cheeseman and Maier initially predicted that Meikin’s role ended after meiosis I, the first round of meiotic cell division. In meiosis II, the second round of cell division, the cells being created should end up with only one sister chromatid each, and so the chromatids must not be kept glued together. Maier found that near the end of meiosis I, Meikin is cleaved in two by an enzyme called Separase, the same molecule that cleaves the adhesive molecules gluing together the chromosomes. At first, this cleavage seemed like the end of Meikin and the end of this story.
A hidden role for a hidden proteinHowever, unexpectedly, the researchers found that cells lacking Meikin during the second half of meiosis do not divide properly, prompting them to take another look at what happens to Meikin after it gets cleaved. They found that Separase cleaves Meikin at a specific point — carving it with the precision of a surgeon’s scalpel — to create C-Meikin, a previously unknown protein that turns out to be necessary for meiosis II. C-Meikin has many of the same properties as the intact Meikin molecule, but it is just different enough to take on a different role: helping to make sure that the chromosomes align properly before their final division.
“There’s a lot of protein diversity in cells that you would never see if you don’t go looking for it, if you only look at the DNA or RNA. In this case, Separase is creating a completely different protein variant of Meikin than can function differently in meiosis II,” says Cheeseman, who is also a professor of biology at Massachusetts Institute of Technology. “I’m very excited to see what we might discover about other hidden protein forms in cell division.”
Recombining ideas
Answering the question of Meikin’s role and regulation throughout meiosis required a close collaboration and partnership between Maier and Lampson lab researcher Ma – the Lampson lab being experts on studying meiosis using mouse models. Working with mouse oocytes (immature egg cells), Ma was able to reveal the behaviors and critical contributions of Meikin cleavage in meiotic cells in mice. Both labs credit the close exchange with helping them to get a deeper understanding of how cells rewire for meiosis.
“It was a pleasure working together to understand how some of the specialized meiotic functions that are necessary for making healthy eggs and sperm are controlled,” Lampson says.
Finally, once cells have completed these specialized meiotic divisions, the researchers found that it was critical for oocytes to fully eliminate Meikin. The researchers determined that, after meiosis two, C-Meikin is degraded by another molecule (the anaphase-promoting complex or APC/C)—this time for good. With Meikin gone and the rewiring of cell division reversed, eggs and sperm are ready for mitosis; should they fuse and form an embryo, that is the next cell division they will undergo. The researchers note that the way Meikin is regulated by being broken down — first into C-Meikin and then completely — may help cells to organize their timing during meiosis. Breaking apart a protein is an irreversible step that creates a clear demarcation between before and after in a multi-step process.The researchers hope that by uncovering the intricacies of meiosis, they may shed light on what happens when the creation of eggs and sperm goes wrong, and so perhaps contribute to our understanding of infertility. Cheeseman also hopes that by studying how mitotic processes are rewired for meiosis, his lab can gain new insights into the original wiring of mitosis.