Research Area: Cell Biology

A Summer of Protein Degradation and the Beauty of Basic Science

MSRP-Bio student Elizabeth Bond worked in the Baker Lab, investigating the macromolecular machines that roam the cell and gobble up unneeded proteins.

Raleigh McElvery
September 25, 2018

Elizabeth Bond’s greatest summer accomplishment is proudly displayed as the background image on her phone. To the casual observer, it looks like columns of black blobs, but to Bond this stained protein gel signifies that, after two long weeks, she successfully isolated her protein of interest. The snapshot also underscores that she’s found her “people” — the kind who, as she describes, “will freak out with you over a great looking gel.”

A rising senior at UMass Amherst, Bond joined 18 fellow MIT Summer Research Program in Biology (MSRP-Bio) students — collaborating in labs across the biology department and various MIT-affiliated institutes for 10 weeks. Together, they attended seminars, lectures, Q&A sessions, meals, and field trips while living in dorms and bonding over science and life in general.

“You’re with a group of other college students looking towards the future, and you’re all stressing out about what comes next,” she says. “That’s amazing because you’re able to talk about your different insecurities and anxieties. You have a built-in support system that you might not get by staying at your home institution over the summer.”

Bond grew up not too far from MIT in the quiet town of Boxford, Massachusetts. Before setting foot on campus, she expected MIT to be cutthroat and competitive. Instead, she found “a bunch of nerds who are willing to help other nerds learn, make mistakes, and be human beings.” The researchers she met were supportive and eager to share their insights, scientific or otherwise. “In addition to being really interesting, these conversations helped me feel that I fit in with a group of very intelligent scientists,” she says.

As a biochemistry major, Bond appreciates basic science because it allows her to probe biological phenomena with no immediate goal other than to understand the underlying mechanisms. “Maybe 10 years down the line my research will help someone’s translational work, but right now I can pursue knowledge for its own sake,” she says. “The beauty of basic science is that you’re able to study things because they’re cool, while also contributing to the body of work your lab family began before you.”

At UMass Amherst, she serves as an undergraduate research assistant, investigating AAA+ proteases — the same protein degradation machines she studied all summer in Tania Baker’s lab, mentored by graduate student Kristin Zuromski. Back home, Bond examines these proteases in bacteria, using a combination of microbiology and computational biology. As a member of the Baker lab, she studied these macromolecular machines leveraging biochemical approaches.

She likens AAA+ proteases to Pac-Men from the classic arcade game, roaming the cell and gobbling up misfolded, excess, or unneeded proteins. One of the AAA+ proteases studied in the Baker lab is ClpAP, which is comprised of the AAA+ unfoldase ClpA and its partner peptidase ClpP. Bond’s protein of interest this summer was ClpA, a hexameric protein depicted as a cylinder with a central channel. ClpA unfolds and threads protein substrates through its channel, which contains pore loop structures that protrude from the chamber and play important roles in the function of ClpA. From there, the proteins enter ClpP, where they are degraded into small peptide fragments.

There are two types of pore loops in ClpA, D1 and D2, but their respective roles in the recognition, unfolding, and movement of proteins for degradation are not fully characterized. Bond hoped to discern their roles relative to one another.

She introduced a mutation into the gene that encodes ClpA, switching one amino acid for another in the D2 pore loop, in a region thought to be critical for recognizing proteins targeted for degradation. This mutation would, in theory, lead to a variant of ClpA where the D1 pore loop retained normal activity, but the D2 pore loop was unable to function. She used chemical crosslinking to generate a ClpA dimer variant that was half wildtype and half mutant in the D2 pore loops, and monitored the ability of the assembled hexameric AAA+ protease to function.

By observing the degradation activity of this crosslinked ClpA variant containing three active and three inactive D2 pore loops in an alternating order, she hoped to get a better sense of the role the D2 pore loops play in ClpAP protease function.

Although there is still more to be done to answer this particular question, reflecting on the summer Bond feels her project went “surprisingly well,” despite being more challenging than she initially anticipated — primarily due to multi-week protein purification experiments and performing many procedures simultaneously. She arrived with the sole intention of bolstering her biochemistry knowledge, and left with a greater appreciation for the breadth of scientific fields she could pursue.

“MSRP-Bio gave me the chance to talk with students and faculty members working in multiple branches of science,” she says. “I study bacteria, but I can learn a lot from someone researching roundworms or cancer cells, or using computational approaches to biology. Those conversations prompted me to think more critically about my own research.”

Besides feeling integrated into the tightly-knit Baker lab, her favorite aspect of the summer was the bond she formed within her MSRP-Bio cohort. In addition to freaking out over protein gels, they started their own journal club, and they discussed personal struggles, family, where they came from, and where they want to go.

“A lot of students of color who come from underrepresented groups in science, like I do, have this anxiety about not being smart enough or not fitting in,” she says. “The program allows you to bond over these shared feelings and that is part of what makes it really amazing for students who are trying to do great things, but do not often feel fully represented.”

At the beginning of the summer, Bond hadn’t fully admitted to herself that she wanted to apply to grad school. “It was easier for me to be ambiguous about what I wanted to do, because it was scary to admit that grad school was something I might actually want,” she recalls. After a summer at MIT, she’s gained the confidence to apply and state her ambitions out loud.

“My project’s been amazing and great, but now I want to have my own body of work,” she says. “It’s something I have this great urge to do — and, because of MSRP-Bio, I’m ready for it.”

Photo credit: Raleigh McElvery
Parasite’s riff on essential enzyme highlights unique biology
Nicole Giese Rura | Whitehead Institute
September 18, 2018

Cambridge, Mass. — The primary currency of energy in cells—adenosine triphosphate (ATP)—is essential for their survival and without it, cellular processes would seize. In the apicomplexan Toxoplasma gondii (T. gondii), a parasite that Whitehead Member Sebastian Lourido studies, key components of the ATP synthase—the enzyme responsible for ATP production—have remained elusive. While investigating indispensable proteins with unknown functions, Lourido and Diego Huet, a postdoctoral researcher in Lourido’s lab, identified a critical component of the enzyme. While highly conserved from yeast to humans, it proved to be considerably different in T. gondii. The findings, published online September 11 in the journal eLife, underscore the unique biology of these parasites and highlight differences between them and their human hosts.

More closely related to plants than to animals, the single-celled apicomplexans are among the most common and deadly human pathogens. According to the World Health Organization, every year these diseases sicken hundreds of millions, kill hundreds of thousands—primarily children—and cost billions of dollars to treat. Species of apicomplexans cause malaria (Plasmodium spp.), cryptosporidiosis (Cryptosporidium spp.), and toxoplasmosis (T. gondii).

Using a CRISPR-based genetic screen that they had adapted to T. gondii, Lourido and Huet had previously identified about 200 genes in T. gondii that are fitness-conferring and specific to apicomplexans. Of that cadre, a few were localized to the mitochondria, where cells manufacture ATP, the cellular currency of energy. Because those genes have not been annotated previously, and the proteins encoded by them have no known function, Huet ran their protein sequences through a database that compared them to protein sequences with known structures.

One of the proteins came back with an interesting hit: it shares structural similarity, but not sequence similarity, with an integral part of the ATP synthase. Most of the protein subunits that compose the apicomplexan ATP synthase have been identified, but key components of the stator—a portion of the enzyme essential for its function—was not yet known.

When Huet experimentally removed the function of the stator subunit in T. gondii, the parasites’ growth stalled, their mitochondria were misshapen and shrunken, and energy production halted—all traits typical of interrupted ATP synthase function.

Because the apicomplexan ATP synthase varies so much from its hosts’ version, those differences, like the unusual stator, could serve as future drug targets. But for Lourido, who is also an assistant professor of biology at Massachusetts Institute of Technology (MIT), the unique stator protein emphasizes how unique and extraordinary apicomplexan organisms are compared to us and their other hosts.

This work was supported by the National Institutes of Health (NIH grants 1DP5OD017892, R21AI123746, and K99AI137218).

* * *

Sebastian Lourido’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.

* * *

Full Citation:

“Identification of cryptic subunits from an apicomplexan ATP synthase”

eLife, online September 11, 2018.  DOI: 10.7554/eLife.38097

Diego Huet (1) , Esther Rajendran (2) , Giel G van Dooren (2) , Sebastian Lourido (1,3*).

1. Whitehead Institute for Biomedical Research, Cambridge, United States

2. Research School of Biology, Australian National University, Canberra, Australia

3. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States

Detangling DNA replication

Researchers identify an essential protein that helps enzymes relax overtwisted DNA so each strand can be copied during cell division.

Raleigh McElvery | Department of Biology
September 18, 2018

DNA is a lengthy molecule — approximately 1,000-fold longer than the cell in which it resides — so it can’t be jammed in haphazardly. Rather, it must be neatly organized so proteins involved in critical processes can access the information contained in its nucleotide bases. Think of the double helix like a pair of shoe laces twisted together, coiled upon themselves again and again to make the molecule even more compact.

However, when it comes time for cell division, this supercoiled nature makes it difficult for proteins involved in DNA replication to access the strands, separate them, and copy them so one DNA molecule can become two.

Replication begins at specific regions of the chromosome where specialized proteins separate the two strands, pulling apart the double helix as you would the two shoe laces. However, this local separation actually tangles the rest of the molecule further, and without intervention creates a buildup of tension, stalling replication. Enter the enzymes known as topoisomerases, which travel ahead of the strands as they are being peeled apart, snipping them, untwisting them, and then rejoining them to relieve the tension that arises from supercoiling.

These topoisomerases are generally thought to be sufficient to allow replication to proceed. However, a team of researchers from MIT and the Duke University School of Medicine suggests the enzymes may require guidance from additional proteins, which recognize the shape characteristic of overtwisted DNA.

“We’ve known for a long time that topoisomerases are necessary for replication, but it’s never been clear if they were sufficient on their own,” says Michael Laub, an MIT professor of biology, Howard Hughes Medical Institute Investigator, and senior author of the study. “This is the first paper to identify a protein in bacteria, or eukaryotes, that is required to localize topoisomerases ahead of replication forks and to help them do what they need to do there.”

Postdoc Monica Guo ’07 and former graduate student Diane Haakonsen PhD ’16 are co-first authors of the study, which appeared online in the journal Cell on Sept. 13.

Necessary but not sufficient

Although it’s well established that topoisomerases are crucial to DNA replication, it has now becoming clear that we know relatively little about the mechanisms regulating their activity, including where and when they act to relieve supercoiling.

These enzymes fall into two groups, type I and type II, depending on how many strands of DNA they cut. The researchers focused on type II topoisomerases found in a common species of freshwater bacteria, Caulobacter crescentus. Type II topoisomerases in bacteria are of particular interest because a number of antibiotics target them in order to prevent DNA replication, treating a wide variety of microbial infections, including tuberculosis. Without topoisomerases, the bacteria can’t grow. Since these bacterial enzymes are unique, poisons directed at them won’t harm human topoisomerases.

For a long time, type II topoisomerases were generally assumed adequate on their own to manage the overtwisted supercoils that arise during replication. Although researchers working in E. coli and other, higher organisms have pinpointed additional proteins that can activate or repress these enzymes, none of these proteins were required for replication.

Such findings hinted that there might be similar interactions taking place in other organisms. In order to understand the protein factors involved in compacting Caulobacter DNA — regulating topoisomerase activity specifically — the researchers screened their bacteria for proteins that bound tightly to supercoiled DNA. From there, they honed in on one protein, GapR, which they observed was essential for DNA replication. In bacteria missing GapR, the DNA became overtwisted, replication slowed, and the bacteria eventually died.

Surprisingly, the researchers found that GapR recognized the structure of overtwisted DNA rather than specific nucleotide sequences.

“The vast majority of DNA-binding proteins localize to specific locations of the genome by recognizing a specific set of bases,” Laub says. “But GapR basically pays no attention to the actual underlying sequence — just the shape of overtwisted DNA, which uniquely arises in front of replication forks and transcription machinery.”

The crystal structure of the protein bound to DNA, solved by Duke’s Maria Schumacher, revealed that GapR recognizes the backbone of DNA (rather than the bases), forming a snug clamp that encircles the overtwisted DNA. However, when the DNA is relaxed in its standard form, it no longer fits inside the clamp. This might signify that GapR sits on DNA only at positions where topoisomerase is needed.

An exciting milestone

Although GapR appears to be required for DNA replication, it’s still not clear precisely how this protein promotes topoisomerase function to relieve supercoiling.

“In the absence of any other proteins, GapR is able to help type II topoisomerases remove positive supercoils faster, but we still don’t quite know how,” Guo says. “One idea is that GapR interacts with topoisomerases, recognizing the overtwisted DNA and recruiting the topoisomerases. Another possibility is that GapR is essentially grabbing onto the DNA and limiting the movement of the positive supercoils, so topoisomerases can target and eliminate them more quickly.”

Anthony Maxwell, a professor of biological chemistry at the John Innes Centre who was not involved with the study, says the buildup of DNA supercoils is a key problem in both bacterial replication and transcription.

“Identifying GapR and its potential role in controlling supercoiling in vivo is an exciting milestone in understanding the control of DNA topology in bacteria,” he says. “Further work will be required to show how exactly these proteins cooperate to maintain bacterial genomic integrity.”

According to Guo, the study provides insight into a fundamental process — DNA replication — and the ways topoisomerases are regulated, which could extend to eukaryotes.

“This was the first demonstration that a topoisomerase activator is required for DNA replication,” she says. “Although there’s no GapR homolog in higher organisms, there could be similar proteins that recognize the shape of the DNA and aid or position topoisomerases.”

This could open up a new field of drug research, she says, targeting activators like GapR to increase the efficacy of existing topoisomerase poisons to treat conditions like respiratory and urinary tract infections. After all, many topoisomerase inhibitors have become less effective due to antibiotic resistance. But only time will tell; there is still much to learn in order to untangle the complex process of DNA replication, along with its many twists and turns.

The research was funded by NIH grants, the HHMI International Predoctoral Fellowship, and the Jane Coffin Childs Memorial Fellowship.

A Summer of Science

Victor Rivera-Santana, a chemistry major at the University of Puerto Rico at Mayagüez, visited MIT Biology for 10 weeks to investigate protein form and function.

August 30, 2018

Victor Rivera-Santana grew up on the western edge of Puerto Rico, in what he refers to as an “atmosphere of science.” His mother is a professor of animal science at the University of Puerto Rico at Mayagüez, and in elementary school he would attend her lectures about the effects of environment and hormones on animal behavior. Three years ago, Rivera-Santana enrolled there as an undergraduate, and has been studying chemistry ever since — with the exception of this past summer, when he became a full-fledged member of the MIT Biology Department for 10 weeks during MIT’s Summer Research Program in Biology (MSRP­-Bio).

Rivera-Santana remembers being drawn to basic research because of its simple, pure, and noble nature, stemming from the creativity of the researcher. “Science almost always has an application, so the fact that researchers in basic science are not looking for an application per se doesn’t mean their work won’t have one in the future,” he says. “The researcher fulfills his or her own curiosity, and afterwards someone else can find a way to put that into practice in society.”

A rising senior, Rivera-Santana chose chemistry because he enjoyed analyzing the minute building blocks of life, but wasn’t sure which field he would ultimately pursue. With chemistry, he could engineer the major to encompass biology and physics as well, which would give him “a taste of everything.”

At first he didn’t know what post-graduation life might hold. However, two weeks into the MSRP-Bio program he’d made up his mind: a PhD. “I like the people, I like the passion, and most importantly I like the research — everything is so interesting it’s hard to pick,” he says.

Rivera-Santana applied to MSRP-Bio early last January because he had it on good authority from three independent sources that this was the program for him. First a good friend and former MSRP-Bio student suggested it, then his professor, and finally his father.

He had two main expectations coming in. First, that everyone would be intimidating and aloof. “Boy was I wrong,” he says. “The MIT faculty are really accessible and engage you as a potential researcher. You can stop them as they’re walking down the hall, or ask them questions during the scheduled Q&A sessions.”

Second, he expected everyone would be hardworking, irrespective of their area of focus. “I was very pleased to find that’s the case,” he says. “I have not met one person at MIT who would not go the extra mile to do their job correctly.”

Rivera-Santana worked in Thomas Schwartz’s lab, investigating an aggregate of proteins known as the nuclear pore complex (NPC), which is embedded in the nuclear envelope and controls the passage of proteins, RNAs, and even ribosomal subunits between the cytoplasm and the nucleus. Although the NPC is vital to cellular survival, its structure is not yet fully understood.

The Schwartz lab goes bit by bit, studying each of its components and their interactions with one another. Rivera-Santana concentrated on one NPC protein in particular, Nup93, parsing its role and design. He hopes this work will eventually help scientists understand the complex as a whole, “because as the name says, it’s complex.”

Rivera-Santana studied four different variants of Nup93, working to express each variant by itself in bacteria. Most of his days in lab went by “either very slowly or very quickly.” He would spend the beginning of the week growing the bacteria to express his proteins — a relatively low-key process since the bacteria “essentially take care of themselves.” The latter half of the week, though, when he began the purification process, was more fast-paced. It involved extracting and purifying the proteins from the bacterial cytosol, while at the same time taking steps to prevent the proteins from becoming damaged, such as keeping them at low temperatures by performing the purification steps in the cold room.

“Purifying is the really challenging part, but it’s also the most fun,” Rivera-Santana says. “I had to work at four degrees Celsius, and I’m from Puerto Rico so you can just imagine how I bear the temperature,” he adds.

Looking back, his most exciting summer experience was purifying his first protein. “I just felt this bundle of joy well up inside me,” he says. “When I ran it through the gel to check its identity and I saw that beautiful blob of ‘ink’ that told me I had my protein, I just felt so happy.”

While Rivera-Santana thoroughly enjoyed his experiences in lab, he was also thrilled to meet other budding researchers and explore Boston’s museums and brick buildings. His proudest moment was cooking his first meal for his new MSRP-Bio friends (a classic Puerto Rican dish: rice, beans, chicken, and plantains). He enjoys putting smiles on people’s faces, “especially when those grins are full of food.” After all, MSRP-Bio isn’t just about being at MIT; it’s also about meeting people and being part of the community.

He also learned he could live by himself, thousands of miles from his family. And he’s prepared to do it again next summer, perhaps in the same lab. “I’m definitely considering doing MSRP-Bio again,” he says. “And I’m certainly also thinking about MIT for graduate school.”

Until then, the three things he’ll miss the most — in no particularly order — are his MSRP-Bio cohort, his lab mentor, and the tasty East Coast cherries.

Photo credit: Raleigh McElvery
Exploring cancer metabolism

Matthew Vander Heiden seeks new cancer treatments that exploit tumor cells’ abnormal metabolism.

Anne Trafton | MIT News Office
August 28, 2018

Nearly 100 years ago, the German chemist Otto Warburg discovered that cancer cells metabolize nutrients differently than most normal cells. His discovery launched the field of cancer metabolism research, but interest in this area waned; by the 1970s most cancer scientists had shifted their focus to the genetic mutations that drive cancer development.

In the past decade or so, interest in cancer metabolism has resurged, and the first drugs that target cancer cells’ abnormal metabolism were approved to treat leukemia in 2017.

“Cancer metabolism is a very sophisticated field at this point,” says Matthew Vander Heiden, an associate professor of biology at MIT. “We have a lot better understanding of what nutrients cancer cells use and what determines how those nutrients are used. This has led to different ways to think about drugs.”

Vander Heiden, who is also a member of MIT’s Koch Institute for Integrative Cancer Research, is one of the people responsible for the recent surge in cancer metabolism research. As a graduate student and postdoc, he published some of the first studies of how cancer cells alter their metabolism, and now his lab at MIT is devoted to the topic.

“All of the time that I was in grad school and working as a postdoc, I was never working in a lab that was dedicated to studying metabolism. So my vision, if someone gave me a job, was to set up a lab that could really be built in a way that would allow us to ask questions about metabolism,” he says.

Metabolism and cancer

Vander Heiden grew up in a small town in Wisconsin, and unlike most of his high school classmates, he headed out of state for college, to the University of Chicago. He was interested in science, so decided on a pre-med track. A work-study job in a plant biology lab led him to discover that he also enjoyed doing research.

“At that point I already had this idea I was going to go to medical school, but then the idea of MD/PhD came up, and I ended up going down that path,” Vander Heiden says.

While in the MD/PhD program at the University of Chicago Medical School, he worked in the lab of Craig Thompson, now president of Memorial Sloan Kettering Cancer Center. At that time, Thompson was studying the biochemical regulation of apoptosis, the programmed cell death pathway. For his PhD thesis, Vander Heiden investigated the function of a protein called Bcl-x, which is a regulator of apoptosis found in the membranes of mitochondria — cell organelles responsible for generating energy.

“That project really got me thinking about how the mitochondria work and how metabolism works,” Vander Heiden recalls. “At the time, I came to the realization that we don’t understand cell metabolism anywhere near as well as we thought we did, and someone should really study this.”

After finishing his degrees, he spent five years doing clinical training, then decided to pursue research in cancer metabolism.

“Altered metabolism has been known about in cancer for 100 years, but few people were studying it,” Vander Heiden says. “The challenge was finding a lab that would allow me to study metabolism and cancer, which in 2004-2005 was not such an obvious thing to do.”

He ended up going to Harvard Medical School to work with Lewis Cantley, who studies signaling pathways in cells and was receptive to the idea of exploring cancer metabolism. There, Vander Heiden began studying an enzyme called pyruvate kinase M2 (PKM2), which is involved in regulation of glycolysis, a biochemical process that cells use to break down sugar for energy.

In 2008, Vander Heiden, Cantley, and others at Harvard Medical School reported that when cells shift between normal and Warburg (cancer-associated) metabolism, they start using PKM2 instead of PKM1, the enzyme that adult cells normally use for glycolysis. Cantley and Craig Thompson have since founded a company, Agios Pharmaceuticals, that is developing potential drugs that target PKM2, as well as other molecules involved in cancer metabolism.

While at Harvard, Vander Heiden also worked on a paper that contributed to the eventual development of drugs that target cancer cells with a mutation in the IDH gene. These drugs, the first modern FDA-approved cancer drugs that target metabolism, shut off an alternative pathway used by cancer cells with the IDH mutation.

New drug targets

In 2010, Vander Heiden became one of the first new faculty members hired after the creation of MIT’s Koch Institute, where he set up a lab focused on metabolism, particularly cancer metabolism.

His research has yielded many insights into the abnormal metabolism of cancer cells. In one study, together with other MIT researchers, he found that tumor cells turn on an alternative pathway that allows them to build lipids from the amino acid glutamine instead of the glucose that healthy cells normally use. He also found that altering the behavior of PKM2 to make it act more like PKM1 could stop tumor cell growth.

Studies such as these can offer insights that may help researchers to develop drugs that starve tumor cells of the nutrients they need, offering a new way to fight cancer, Vander Heiden says.

“If one wants to develop drugs that target metabolism, one really needs to focus on the context in which it’s happening, which is the environment of the cell plus the genetics of the cell,” he says. “That is what defines the sensitivity to drugs.”

Tissue architecture affects chromosome segregation

Biologists discover that the environment surrounding a cell plays an integral role in its ability to accurately segregate its chromosomes.

Ashley Junger | Koch Institute
August 24, 2018

All growth and reproduction relies on a cell’s ability to replicate its chromosomes and produce accurate copies of itself. Every step of this process takes place within that cell.

Based on this observation, scientists have studied the replication and segregation of chromosomes as a phenomenon exclusively internal to the cell. They traditionally rely on warm nutritional cultures that promote growth but bear little resemblance to the cell’s external surroundings while in its natural environment.

New research by a group of MIT biologists reveals that this long-held assumption is incorrect. In a paper published this week, they describe how some types of cells rely on signals from surrounding tissue in order to maintain chromosome stability and segregate accurately.

Kristin Knouse, a fellow at the Whitehead Institute, is the lead author of the paper, which was published online in the journal Cell on Aug. 23. Angelika Amon, the Kathleen and Curtis Marble Professor in Cancer Research in the Department of Biology and a member of the Koch Institute for Integrative Cancer Research, is the senior author.

“The main takeaway from this paper is that we must study cells in their native tissues to really understand their biology,” Amon says. “Results obtained from cell lines that have evolved to divide on plastic dishes do not paint the whole picture.”

When cells replicate, the newly duplicated chromosomes line up within the cell and cellular structures pull one copy to each side. The cell then divides down the middle, separating one copy of each chromosome into each new daughter cell.

At least, that’s how it’s supposed to work. In reality, there are sometimes errors in the process of separating chromosomes into daughter cells, known as chromosome mis-segregation. Some errors simply result in damage to the DNA. Other errors can result in the chromosomes being unevenly divided between daughter cells, a condition called aneuploidy.

These errors are almost always harmful to cell development and can be fatal. In developing embryos, aneuploidy can cause miscarriages or developmental disorders such as Down syndrome. In adults, chromosome instability is seen in a large number of cancers.

To study these errors, scientists have historically removed cells from their surrounding tissue and placed them into easily controlled plastic cultures.

“Chromosome segregation has been studied in a dish for decades,” Knouse says. “I think the assumption was … a cell would segregate chromosomes the same way in a dish as it would in a tissue because everything was happening inside the cell.”

However, in previous work, Knouse had found that reported rates for aneuploidy in cells grown in cultures was much higher than the rates she found in cells that had grown within their native tissue. This prompted her and her colleagues to investigate whether the surroundings of a cell influence the accuracy with which that cell divided.

To answer this question, they compared mis-segregation rates between five different cell types in native and non-native environments.

But not all cells’ native environments are the same. Some cells, like those that form skin, grow in a very structured context, where they always have neighbors and defined directions for growth. Other cells, however, like cells in the blood, have greater independence, with little interaction with the surrounding tissue.

In the new study, the researchers observed that cells that grew in structured environments in their native tissues divided accurately within those tissues. But once they were placed into a dish, the frequency of chromosome mis-segregation drastically increased. The cells that were less tied to structures in their tissue were not affected by the lack of architecture in culture dishes.

The researchers found that maintaining the architectural conditions of the cell’s native environment is essential for chromosome stability. Cells removed from the context of their tissue don’t always faithfully represent natural processes.

The researchers determined that architecture didn’t have an obvious effect on the expression of known genes involved in segregation. The disruption in tissue architecture likely causes mechanical changes that disrupt segregation, in a manner that is independent of mutations or gene expression changes.

“It was surprising to us that for something so intrinsic to the cell — something that’s happening entirely within the cell and so fundamental to the cell’s existence — where that cell is sitting actually matters quite a bit,” Knouse says.

Through the Cancer Genome Project, scientists learned that despite high rates of chromosome mis-segregation, many cancers lack any mutations to the cellular machinery that controls chromosome partitioning. This left scientists searching for the cause of the increase of these division errors. This study suggests that tissue architecture could be the culprit.

Cancer development often involves disruption of tissue architecture, whether during tumor growth or metastasis. This disruption of the extracellular environment could trigger chromosome segregation errors in the cells within the tumor.

“I think [this paper] really could be the explanation for why certain kinds of cancers become chromosomally unstable,” says Iain Cheeseman, a professor of biology at MIT and a member of the Whitehead Institute, who was not involved in the study.

The results point not only to a new understanding of the cellular mechanical triggers and effects of cancers, but also to a new understanding of how cell biology must be studied.

“Clearly a two-dimensional culture system does not faithfully recapitulate even the most fundamental processes, like chromosome segregation,” Knouse says. “As cell biologists we really must start recognizing that context matters.”

This work was supported by the National Institutes of Health, the Kathy and Curt Marble Cancer Research Fund, and the Koch Institute Support (core) Grant from the National Cancer Institute.

Ankur Jain

Education

  • PhD, 2013, University of Illinois, Urbana-Champaign
  • BTech, 2007,  Biotechnology and Biochemical Engineering, Indian Institute of Technology Kharagpur

Research Summary

We study how biomolecules in a cell self-organize. In particular, we are interested in understanding how membrane-free cellular compartments such as RNA granules form and function. Our lab develops new biochemical and biophysical techniques to investigate these compartments and to understand their dysfunction in human disease.

Awards

  • Young Alumni Achiever’s Award, Indian Institute of Technology Kharagpur, 2019
  • NIH K99/R00 Pathway to Independence Award, 2017
  • Pew Scholar in the Biomedical Sciences, 2022
The Y chromosome: Holding steadfast in a sea of change
Nicole Davis
August 2, 2018

The human Y chromosome is, in many ways, a study in contrasts. For decades, scientists have struggled to dissect its evolution in part because it does not have a genetic partner (or homolog), as all of the other human chromosomes do. That solitary existence means the Y chromosome is subject to some unusual evolutionary pressures. For example, it does not swap genetic material with a homologous chromosome — a practice known as recombination that other chromosomes follow — along the lion’s share of its length. However, its lack of recombination presents a unique opportunity: Because so much of its own genetic material stays put, scientists can trace the history of individual human Y chromosomes much further back in time than other chromosomes — in fact, they can go as far back as the data will allow.

That is precisely the approach taken by a team of Whitehead Institute researchers, led by Whitehead Institute Director David Page, who is also a professor of biology at the Massachusetts Institute of Technology and a Howard Hughes Medical Institute investigator. Their work is published in the August 2nd online issue of the American Journal of Human Genetics. Page and his colleagues, including graduate student and first author Levi Teitz, set out to examine a series of regions on the Y chromosome called amplicons — vast stretches of DNA, from tens of thousands to millions of nucleotides in length, which are present in two or more copies per chromosome. While the DNA contained in amplicons is often highly repetitive, it also houses biologically important genes. Although the precise functions of many of these genes remains to be determined, some have been found to play important roles in the development of sperm cells and testicular cancer. However, the amplicons vary drastically among species, so scientists cannot look to other organisms such as mice or chimpanzees to help reconstruct their past.

Page’s team zeroed in on these amplicons. Specifically, they looked at how the number of amplicon copies varies from one person’s Y chromosome to another. The researchers developed sophisticated computational tools to analyze DNA sequencing data collected from more than 1,200 males as part of the 1000 Genomes Project. What they discovered was quite surprising. Although the amplicons are quite variable, they found that overall, the configuration of amplicon copies on the Y chromosome has been painstakingly maintained over the last 300,000 years of human evolution. That means that despite the high level of mutation the chromosome experiences, evolutionary forces work to counteract this change and preserve its ancestral structure.

More work is needed to determine which aspects of the amplicons’ structure are important for chromosome biology, and in turn proper male development and fertility. However, the efforts of Teitz, Page, and their colleagues shed new light on the unusual tricks the solo chromosome uses to maintain its genomic integrity.

This research is supported by the National Institutes of Health and the Howard Hughes Medical Institute.

 

Written by Nicole Davis

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David Page’s primary affiliation is with Whitehead Institute for Biomedical Research, where his laboratory is located and all his research is conducted. He is also a Howard Hughes Medical Institute Investigator and a Professor of Biology at the Massachusetts Institute of Technology.

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Full citation:

“Selection Has Countered High Mutability to Preserve the Ancestral Copy Number of Y Chromosome Amplicons in Diverse Human Lineages”

American Journal of Human Genetics, online August 2, 2018.

Levi S. Teitz (1,2), Tatyana Pyntikova (1), Helen Skaletsky (1,3), and David C. Page (1,2,3).

1. Whitehead Institute, Cambridge, MA 02142, USA

2. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA

3. Howard Hughes Medical Institute, Whitehead Institute, Cambridge, MA 02142, USA

A tale of two projects

Graduate student Julie Monda has spent five years investigating two divergent aspects of cell division, revealing some unexpected results and new research questions.

Raleigh McElvery
July 23, 2018

To sixth year graduate student Julie Monda, dividing cells are among the most beautiful things she’s ever seen. Watching the tiny, delicate spheres split into identical versions of themselves also provides her with a visual readout for her experiments — will the process continue if she removes a certain piece of a certain protein? Will the genetic material still distribute equally between the two cells? Which molecules are crucial for cell division, and how are they regulated?

Our cells are constantly dividing in order to grow and repair themselves, although some (like skin cells) do so more often than others, say, in the brain. This process, known as mitosis, is the primary focus of Iain Cheeseman’s lab, situated in the Whitehead Institute for Biomedical Research. Most of the research in the Cheeseman lab involves the kinetochore, a group of proteins located on the chromosome where the arms join. During mitosis, long, fibrous structures, known as microtubules, attach to the kinetochore to pull apart the duplicated chromosomes as the parent cell splits in half, ensuring each daughter cell receives an exact copy of the parent’s genetic blueprint.

Before she arrived at MIT Biology in the fall of 2012, Monda worked as a research technician at St. Jude Children’s Research Hospital in Memphis, Tennessee in the lab of Brenda Schulman PhD ’96 . As she recalls, she always “preferred performing hands-on research techniques at the lab bench over being in a classroom.” So she surprised even herself when she chose MIT’s graduate program in biology precisely because it requires all first-year students to take a full course load their fall semester before beginning lab rotations.

“That structure seemed useful given that I studied biochemistry as an undergraduate at the University of Tulsa, and the degree requirements were weighted more towards chemistry than biology,” she says. “Plus, when you’re only taking classes, you spend more time interacting with your classmates. It creates a close-knit community that extends throughout your entire graduate career and beyond.”

Monda ultimately selected the Cheeseman lab because it married her interests in biochemistry and cell biology.

“The research in this lab focuses on various elements of kinetochore function and cell division, but everyone is generally working on their own distinct questions,” she explains. “I knew I would have an area that was mine to explore. It’s both exciting and challenging because no one else is thinking about your projects to the extent that you are.”

Monda’s story is a tale of two projects: one focused on the interface between the kinetochore and the array of microtubules known as the mitotic “spindle,” and another project that ended up taking both her and the lab in a slightly new direction.

The first, concerning kinetochore-microtubule interactions, represented a collaboration with former lab technician Ian Whitney. For this endeavor, Monda investigated a protein complex called Ska1, found at the outer kinetochore.

The Ska1 complex is located where the kinetochore and microtubule meet. Ska1’s role, Monda explains, is to allow the kinetochores to remain attached to the spindle during chromosome segregation, even as the microtubules that compose the spindle begin to disassemble (as they must do).

“We wanted to know how the kinetochore hangs onto this polymer that is essentially falling apart,” Monda explains. “Long story short, we ended up defining specific surfaces within the Ska1 complex that are important for holding on to the microtubule as it shrinks, and — as we were surprised to note — also as it grows”

Although Ska1 only requires a single point of contact to bind a microtubule, Monda and Whitney pinpointed multiple surfaces on Ska1 that are required to allow it to remain associated with the microtubules as they disassemble and reassemble themselves.

While her Ska1 project was very much in line with the types of questions that the Cheeseman lab traditionally pursues, Monda also worked on another endeavor that “began as a side project and slowly evolved into a more full-time effort.” This project involves a motor protein called dynein, which helps to align the chromosomes and position the spindle during mitosis.

Dynein piqued Monda’s interest because of its role in mitosis, as well as its importance throughout the entire cell cycle. Motor proteins are molecules powered by the release of chemical energy that move along surfaces, sometimes transporting cargo, sometimes performing other essential tasks. Dynein is a motor protein that walks in one direction along microtubules, even when the microtubules latch onto the kinetochore to yank apart the chromosomes during mitosis.

But dynein doesn’t act alone. There are a number of additional proteins that also play a key role in coordinating its activity and localization. Monda is studying two of these accessory regulatory proteins, Nde1 and NdeL1, which bind to dynein and help promote some of its functions. She wanted to understand how Nde1 and NdeL1 interact with dynein to activate it. Although Nde1 and NdeL1 are nearly identical in function, Monda discovered that Nde1 (but not NdeL1) binds to another complex: the 26S proteasome.

The proteasome degrades proteins within the cell, influencing virtually all aspects of cellular function, including DNA synthesis and repair, transcription, translation, and cell signaling. Given its ubiquity, it has remained a point of interest among the scientific community for years. And yet, before Monda’s research, the interaction between Nde1 and the proteasome had apparently gone unnoticed. Researchers have long studied Nde1 in relation to dynein, but it’s possible that the interaction between Nde1 and the proteasome represents a new function for Nde1 unrelated to dynein regulation. In fact, Monda’s finding may have implications for understanding the development of the human brain.

“It’s clear that patients with mutations in Nde1 have much more severe neurodevelopmental defects than scientists would have predicted,” Monda says, “so it’s possible that this new interaction between Nde1 and the proteasome could help to explain why Nde1 is so important in the brain.”

Her most recent results have been published in Molecular Biology of the Cell.

“I’ve found some exciting results over the past few years,” Monda says, “and even though a lot of my research has gone in a direction that’s not strictly mitosis-related, Iain has been great about allowing me to follow the science wherever it leads. We want to know what these proteins are actually doing, both in terms of this new interaction and also more broadly within the cell.”

Monda intends to submit and defend her thesis this summer, and assume a postdoctoral position at the University California, San Diego in the fall. Although she’s been watching cells divide for years now, the process still retains its grandeur.

“Often times biologists investigate questions at scales where we can’t really see what we’re studying as we study it,” she says. “But having this visual readout makes it more tangible; I feel like I can better appreciate what exactly it is that I’m trying to understand, as well as the beauty and complexity of the processes that sustain life.”

Sharpening the edges of cancer chemotherapy
Nicole Davis | Whitehead Institute
July 11, 2018

Cambridge, MA — Tackling unsolved problems is a cornerstone of scientific research, propelled by the power and promise of new technologies. Indeed, one of the shiniest tools in the biomedical toolkit these days is the genome editing system known as CRISPR/Cas9. Whitehead Institute Member David Sabatini and his colleagues pioneered the use of this tool as a foundation for large-scale genetic screens in human cells, turning up a treasure trove of new insights into cellular metabolism, in both normal cells and cancer cells.

When Naama Kanarek, a postdoc in Sabatini’s laboratory, pondered how to apply these state-of-the-art CRISPR/Cas9 screens to her own research, her thoughts turned to a classic cancer chemotherapy drug, methotrexate, which has been in clinical use for nearly seven decades. Often used to treat a form of pediatric leukemia, known as acute lymphoblastic leukemia (ALL), the drug, when deployed as part of a multifaceted treatment plan, can be highly effective. But its power comes at a cost. Because methotrexate can damage not only cancer cells but also healthy tissues, it must be administered with great care. For children who receive high doses of the drug, a mainstay of ALL treatment, that can mean several days spent in the hospital with rigorous clinical monitoring.

In other forms of cancer, methotrexate’s efficacy is more uncertain. For example, in pediatric osteosarcoma, only 65 percent of patients respond. Unfortunately, there is currently no way for doctors to pinpoint who will and who will not.

“From a scientific standpoint, methotrexate is quite special because it was the first metabolic drug to be developed, but much of its biology remains to be discovered — particularly what drives these different responses in patients,” Kanarek says. “So, this is really one of these old, classic questions that has been lingering in the field for some time. We thought we could learn something new.”

And they did. In the July 11 online issue of the journal Nature, Kanarek, Sabatini, and their colleagues report the findings of a CRISPR/Cas9 screen for factors involved in methotrexate sensitivity. The team’s work yielded a surprising set of discoveries that point to the breakdown of histidine — one of several amino acids used by the body to construct proteins — as a critical gatekeeper of cancer cells’ vulnerability to methotrexate. The researchers’ findings not only help illuminate the biology of a well-known cancer chemotherapy, but also suggest a simple dietary supplement that could help broaden its therapeutic window and reduce its toxicity.

 “This study is an example of the power of modern genomic tools to shine a bright light on longstanding questions in human biology,” says senior author David Sabatini, a Member of Whitehead Institute, a professor of biology at Massachusetts Institute of Technology and investigator with the Howard Hughes Medical Institute (HHMI). “While cancer chemotherapies can be quite effective, their biological effects are often poorly understood. By laying bare their biology, we may be able to devise ways to utilize them more wisely.”

ATTACK THE CANCER, NOT THE PATIENT

The history of methotrexate stretches back to the 1940s, a time when strikingly little was known about the origins of cancer much less how best to treat it. The birth of methotrexate as a chemotherapeutic agent was sparked by the astute observations of Sidney Farber, a pediatric pathologist at Boston Children’s Hospital who cared for children with a variety of maladies, including ALL. In the course of caring for patients with ALL, Farber recognized that cancer cells depended on the nutrient folic acid for their own proliferation. That gave him the idea of using folate antagonists to treat ALL. Methotrexate was developed in 1949 precisely for this purpose and was subsequently shown to induce remission in children with ALL. Fast forward to today, and the drug has evolved into a significant tool in oncologists’ toolkit.

“Methotrexate is a major part of the backbone of chemotherapy treatment across many human cancers,” says Loren Walensky, a pediatric hematologist/oncologist at the Dana-Farber Cancer Institute who is not a study co-author but served as an early adviser on the project and will also play a deeper role in planning future follow-up studies. “It is also used outside of the cancer field for the treatment of several autoimmune diseases.”

He added, “But as with all chemotherapy, the critical issue is how to best use it to inflict maximal damage on the cancer without irreparably harming the patient.”

Kanarek explains how new genetic tools are allowing insights into the sensitivity of cancer cells to methotrexate.

The basic mechanics of methotrexate are fairly well known. The drug inhibits dihydrofolate reductase (DHFR), an enzyme that generates the functional form of folate, known as tetrahydrofolate (THF). THF is essential for preparing the raw materials needed to make nucleic acids, such as DNA, which carries cells’ genetic information, and RNA, a close chemical relative involved in making proteins. “Proliferating cells must duplicate their DNA, so they need a lot of THF,” Kanarek explains. “But even cells that are not dividing need to make RNA, and that requires THF, too.”

The results of Kanarek’s CRISPR/Cas9 screen now bring greater clarity to this molecular picture. She and her colleagues uncovered another enzyme, called FTCD, which is involved in the breakdown of histidine. Interestingly, FTCD also requires THF for its function — though not nearly as much as the main target of methotrexate, DHFR. Despite the differential demands of the two enzymes, they both draw from the same, shared pool of THF.

“Under normal conditions, this pool is sufficiently full, so there is no competition for resources, even in rapidly dividing cells,” Kanarek says.

But when the amount of THF becomes limiting — as it does in cells that are treated with methotrexate — the story is quite different, the Whitehead Institute team discovered. In that case, the activity of FTCD poses serious problems, because there isn’t enough THF in the pool to support both cell proliferation and histidine breakdown. When that happens, the cells die.

That got Kanarek thinking more about histidine: Could the nutrient provide a way to tinker with FTCD activity and, by virtue of the cancer cells’ own metabolism, make them more vulnerable to methotrexate?

To explore this question, the researchers used mouse models of leukemia, engineered by transplanting human leukemia cells under the skin of immunocompromised mice. A subset of the mice received injections of methotrexate together with histidine. This one-two punch, Kanarek hypothesized, should ramp up the function of FTCD and more rapidly drain the THF pool, thereby making the cells more sensitive to the cancer-killing effects of methotrexate.

That is precisely what the team observed. Notably, these experiments involved lower than normal doses of methotrexate, suggesting the cells had indeed been made more sensitive to the cancer drug. Moreover, the studies included a human leukemia cell line, called SEM, which harbors a specific genetic mutation that is associated with a particularly poor prognosis in patients — further underscoring the power of the histidine degradation pathway to weaken cells’ defenses.

Now, Kanarek and her colleagues are working to extend these initial findings with additional preclinical studies and, together with Walensky, determine how to best evaluate the potential benefits of histidine supplementation in cancer patients. Their ultimate goal: to pursue clinical trials that will assess histidine’s ability to improve the effectiveness of methotrexate in humans.

In addition to making cancer cells more vulnerable to methotrexate, the Whitehead Institute team’s research also holds promise for another therapeutic challenge: identifying which patients will or will not respond to the drug.

Two other enzymes cooperate with FTCD in breaking down histidine. The levels of one of the enzymes, known as HAL, appears to correlate with cells’ sensitivity to methotrexate: That is, cancer cells with high levels of HAL tend to be more sensitive to the drug. More work is needed to determine whether this correlation extends to a broader swath of patient samples and if it has predictive value in the clinic. Nevertheless, Kanarek and her colleagues are already beginning work on this front. Together with Abner Louissaint, Jr., a hematopathologist at Massachusetts General Hospital who also served as an early adviser on the Nature study, the Whitehead Institute team will launch a second clinical study to examine whether HAL levels can predict methotrexate response in patients with lymphoma.

“Being able to understand who is going to respond to methotrexate and who is not, and how to achieve a therapeutic benefit while mitigating the drug’s potential side effects, could have a profound impact on patient care,” Walensky says. “The insights from this study bring an entirely new dimension to our understanding of a decades-old and critically important cancer medicine. And as a physician and a scientist, that’s truly exciting.”

Written by Nicole Davis

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David Sabatini’s primary affiliation is with Whitehead Institute for Biomedical Research, where his laboratory is located and all his research is conducted. He is also a Howard Hughes Medical Institute investigator and a professor of biology at Massachusetts Institute of Technology.

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Full citation:

“Histidine catabolism is a major determinant of methotrexate sensitivity”

Nature, online on July 11, 2018.

Naama Kanarek (1,2,3,4), Heather R. Keys (1), Jason R. Cantor (1,2,3,4), Caroline A. Lewis (1), Sze Ham Chan (1), Tenzin Kunchok (1), Monther Abu-Remaileh (1,2,3,4), Elizaveta Freinkman (1), Lawrence D. Schweitzer (4), and David M. Sabatini (1,2,3,4).

  1. Whitehead Institute for Biomedical Research and Massachusetts Institute of Technology, Department of Biology, 455 main Street, Cambridge, Massachusetts 02142, USA
  2. Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
  3. Koch Institute for Integrative Cancer Research and Massachusetts Institute of Technology, Department of Biology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA
  4. Broad Institute of Harvard and Massachusetts Institute of Technology, 415 main Street, Cambridge, Massachusetts 02142, USA