Research Area: Cell Biology

Germ cells move like tiny bulldozers
Eva Frederick | Whitehead Institute
September 15, 2022

During fruit fly embryo formation, primordial germ cells — the stem cells that will later form eggs and sperm — must travel from the far end of the embryo to their final location in the gonads. Part of the primordial germ cell migration is passive; the cells are simply pushed into place by the movements of other cells. But at a certain point in development, the primordial germ cells must move on their own.

“A lot of the background in this field has been established by studying  how cells move in culture, and there’s this model that they move by using their cytoskeleton to push out their membranes to crawl,” said Benjamin Lin, a postdoctoral researcher in the lab of Whitehead Institute Director Ruth Lehmann. “We weren’t so sure they were actually moving that way in vivo.”

Now, in a new paper published September 14 in Science Advances, Lehmann who is also a professor of biology at the Massachusetts Institute of Technology, and researchers at Whitehead Institute and the Skirball Institute at New York University School of Medicine show that germ cells in growing fly embryos are in fact using a different method of movement which depends on a process called cortical flow, similar to the way bulldozers move on rotating treads. The research also reveals a new player in the pathway that governs this germ cell movement. “This work brings us one step closer to understanding the regulatory network that guides the germ cells on their long and complex journey across an ever-changing cellular landscape,” Lehmann said.

The research could also provide researchers with a new model for studying this type of cell movement in other situations — for example, cancer cells have been shown to move via cortical flow under certain conditions. ”We think there are more general implications for this mode of migratory behavior that go beyond primordial germ cells and apply to other migratory cells as well,” said Lin.

Balloon-shaped cells 

The first clue that Lehmann and Lin found that germ cells might not move the way scientists thought came from a simple observation. “When we began to study how these primordial germ cells move in the embryo, we saw that the cells actually remain shaped like a balloon while they’re moving and they don’t actually change their shape at all,” said Lin. “It’s really different from the crawling model.”

But if the cells weren’t moving by crawling, how were they moving through the embryo? To find out more, the researchers developed new techniques to image the germ cells in live fly embryos, and were able to watch clusters of a protein called actin moving backwards in each cell, as the cell itself was moving forward.

“There’s this thin layer of actin cytoskeleton just under the membrane of cells called the cortex, and they actually moved by making that cortex ‘flow,’” said Lin. “It’s like if you think of the tread of a bulldozer that’s moving backwards as the bulldozer is moving forward. The cells move that cortex backwards to generate friction to move the cell forward.”

Lin hypothesizes that this method of movement is especially well-suited to germ cells moving through a crowded embryo with many different cell types because instead of depending on recognizing specific proteins to “grab” in order to pull themselves through the embryo, it allows the germ cells to move independently. “Everything is pretty individualistic for primordial germ cells,” he said. “They don’t actually signal to each other at all, all the signaling is within each cell… And germ cells have to move through so many different tissues that they need a universal method of movement.”

A new role for a known protein

The researchers also found new information about how the cells control this form of motility. “We found that a protein called AMPK can control this pathway, which was really unexpected,” Lin said. “ Most people know it as a protein that senses energy. We found that this protein was important for helping these cells navigate. It’s one of these upstream players that can control how fast the cell goes, and in which direction.”

In the future, the researchers hope to map the entire pathway that allows germ cells to get to the right place at the right time in development. They also hope to learn more about the mechanisms behind cortical flow. “We want to figure out what is important for establishing these flows,” Lin said. “Our findings here could have implications not just for germ cells, but for other migrating cells as well.”

Notes

Benjamin Lin, Jonathan Luo, Ruth Lehmann. “An AMPK phosphoregulated RhoGEF feedback loop tunes cortical flow–driven amoeboid migration in vivo.” Science Advances, September 14, 2022. DOI: 10.1126/sciadv.abo0323

Hot off the press: parasite researchers melt down proteins to understand their roles in infection
Eva Frederick | Whitehead Institute
August 31, 2022

Much like humans, plants, and bacteria, the single-celled parasite Toxoplasma gondii (T. gondii) uses calcium as a messenger to coordinate important cellular processes. But while the messenger is the same, the communication pathways that form around calcium differ significantly between organisms.

“Since Toxoplasma parasites are so divergent from us, they have evolved their own sets of proteins that are involved in calcium signaling pathways,”  said Alice Herneisen, a graduate student in the lab of Whitehead Institute Member Sebastian Lourido.

Lourido and his lab study the molecular mechanisms that allow the single-celled parasite T. gondii and related pathogens to be so widespread and potentially deadly — and calcium signaling is an important part of the parasite’s process of invading its hosts. “Calcium governs this very important transition from the parasites replicating inside of host cells to parasites leaving those cells and seeking out new ones to infect,” said Lourido. “We’ve been really interested in how calcium plays into the regulation of proteins inside the parasite.”

A paper published August 17 in eLife provides some insight. In the paper, Herneisen, Lourido and collaborators used an approach called thermal profiling to broadly survey which parasite proteins are involved in calcium signaling in T. gondii. The new work reveals that an unexpected protein plays a role in parasite calcium pathways, and provides new targets that scientists could potentially use to stop the spread of the parasite. The data will also serve as a resource that other Toxoplasma researchers can use to find out whether their own proteins of interest interact with calcium pathways in parasite cells.

The heat is on

When studying calcium pathways in humans, researchers can often draw parallels from work in mice. “But parasites are very different from us,” said Lourido. “All of the principles that we’ve learned about calcium signaling in humans or mice can’t be readily translated over to parasites.”

So to study these mechanisms in Toxoplasma, the researchers had to start from scratch to determine which proteins were involved. That’s where the thermal profiling method came in. The method is based on the observation that proteins are designed to work well at specific temperatures, and when it becomes too hot for them, they melt. Consider eggs: when the proteins in egg whites and egg yolks are heated in a frying pan, the proteins begin to melt and congeal. “When we think about a protein melting, what we mean is the proteins unraveling,” said Lourido. “When proteins unravel, they expose side chains that bind to each other. They stop being individual proteins that are well-folded, and become a mesh.”

Small changes to the chemical structure of a protein — such as the changes resulting from binding a small molecule such as calcium — can alter the melting point of a protein. Researchers can then trace these alterations using proteomic methods. “Proteins that are binding calcium are changing in response to calcium, and are ultimately changing their thermal stability,” Herneisen said. “That’s kind of the language of proteins, alterations in their thermal stability.”

The thermal profiling method works by applying heat to parasite cells and graphing how each of the parasite’s proteins responds to changes in temperature under different conditions (for example, the presence or absence of calcium). In a 2020 paper, the researchers used the thermal profiling method to investigate the role of a protein called ENH1 in calcium signaling.

In their new paper, Lourido and Herneisen investigated the effect of calcium on all proteins in the parasite using two approaches. The researchers combined parasites with specific amounts of calcium, applied heat, and then performed proteomics techniques to track how the calcium affected the melting behavior of each protein. If a protein’s melting point was higher or lower than usual, the researchers could deduce that that protein was changed either by calcium itself or by another player in a calcium signaling pathway.

They then treated the parasites with a chemical that caused them to release stored calcium in a controlled manner and measured how a protein modification called phosphorylation changed over time. Together, these methods allowed them to infer how proteins might sense and respond to calcium within the signaling network.

Their approach provided data on nearly every expressed protein in the parasite cells, but the researchers zeroed in on one particular protein called Protein Phosphatase 1 (or PP1). The protein is ubiquitous across many species, but has never previously been implicated in calcium signaling pathways. They found that the protein was concentrated at the front end of the parasite. This region of the parasite cell is involved in motility and host invasion.

The protein’s role in the parasites — and in the other organisms in which it appears — is to remove the small molecules called phosphates from phosphorylated proteins. “This is a modification that can often change the activity of individual proteins, because it’s this big charge that’s been covalently stuck onto the surface of the protein,” Lourido said. “This ends up being a principle through which many, many different biological processes are regulated.”

How exactly PP1 interacts with calcium remains to be seen. When the researchers depleted PP1 in parasite cells, they found that the protein is somehow involved in helping the parasite take in calcium necessary for movement. It’s unclear whether or not it actually binds calcium or is involved in the pathway through another mechanism.

Because parasites use calcium signaling to coordinate life cycle changes such as entering or leaving  host cells, insights into the key players in calcium pathways could be a boon to public health. “These are kind of the pressure points or the hubs that would be ideal to target in order to prevent the spread and pathogenesis of these parasites,” Herneisen said.

Herneisen and collaborators focused primarily on PP1, but there are many other proteins to investigate using the data from this project. “I think part of the reason why I wanted to release this paper is so that the field could take the next steps,” she said. “I’m just one person — it would be great if 20 other people find that the protein that they were studying is calcium responsive, and they can chase down the exact reason for that or how it is involved in this greater calcium signaling network. This was exciting for us with regards to PP1, and I’m sure other researchers will make their own connections.”

Notes

Alice L. Herneisen,  Zhu-Hong Li, Alex W. Chan, Silvia NJ Moreno, and Sebastian Lourido. “Temporal and thermal profiling of the Toxoplasma proteome implicates parasite Protein Phosphatase 1 in the regulation of Ca2+-responsive pathways”. eLife, August 17, 2022. DOI: https://doi.org/10.7554/eLife.80336

A key process in asymmetric cell division preserves the immortality of the germline
Eva Frederick | Whitehead Institute
July 27, 2022

During cell division, chromosomes are replicated into two copies — one for each daughter cell. These copies, called sister chromatids, are usually considered identical. In fact, it’s the two pairs of sister chromatids that make up the symmetrical X shape usually shown when visualizing chromosomes.

A 2013 paper from the lab of Whitehead Institute Member Yukiko Yamashita showed that in the case of asymmetric cell division — such as when a stem cell is dividing into two different kinds of daughter cells (i.e. a stem cell and a differentiating daughter)  — sister chromatids of sex chromosomes actually may carry distinct information, and the dividing cell “chooses” which of the daughters receive a specific copy.

What that “choice” means, and how it’s executed, has been a mystery — until now. A new paper from Yamashita, who is also a professor of biology at the Massachusetts Institute of Technology and an investigator with the Howard Hughes Medical Institute, published in Science Advances on July 27, illuminates the mechanisms that underlie nonrandom sister chromatid segregation, and suggests that the whole process may serve as a way to maintain the amount of ribosomal DNA (or rDNA) that is passed on to subsequent generations. “Tying together these two processes — rDNA copy number maintenance and nonrandom chromatid segregation — is an unexpected and exciting advance in our understanding of how germ cells are able to maintain their immortality,” said Yamashita.

George Watase, a postdoctoral scholar in the Yamashita Lab, led the study. Watase began his research intent on discovering the genetic underpinnings of nonrandom segregation of X and Y chromosomes in the fruit fly Drosophila melanogaster. As he surveyed the genome for genes that were essential to nonrandom segregation, it became apparent that ribosomal DNA was key for the process.

When rDNA was left intact, the sister chromatid  with more rDNA was preferentially chosen by the daughter stem cell instead of the differentiating daughter cell. When rDNA was removed from X and Y chromosomes, however, Watase found that the sister chromatids segregated randomly to the daughter cells.

Ribosomal DNA, or rDNA, is composed of a long stretch of repeats of certain base pairs. The rDNA provides the instructions and material to make ribosomes, which are essential for cells to create proteins. “Most genes exist only as a single copy, but in the case of rDNA we have hundreds of copies in our genome,” Watase said. “The reason for this is that we need a massive amount of ribosomes to synthesize proteins to maintain our cells’ viability.”

As organisms age, most of their cells naturally lose some of those rDNA repeats, including germline stem cells.  However, germline cells are sometimes called “immortal” — while all other cells in the body are made anew with each generation and die when an organism dies, germline cells such as sperm and eggs must carry DNA between generations. THerefore, the stem cells that produce sperm and eggs thus cannot keep losing rDNA repeats, and must bypass the mortality of other cells, by maintaining  a high number of rDNA repeats over time.

By isolating proteins that bind to rDNA, Watase discovered one specific gene, the protein product of which bound to rDNA and somehow assigned the sister chromatid with more rDNA repeats to the daughter cell that was destined to remain a germline stem cell.

This particular gene had not been described before, and Watase and Yamashita were now tasked with naming it. Fruit fly genes are named after what happens to the animal when the gene is removed. When this new gene was knocked down, the germ cells of subsequent generations gradually lost the immortality that separates germline stem cells from their differentiated counterparts.

Watase wasn’t sure how to convey the intricacies of this outcome. In the end, it was Watase’s wife who came up with the perfect name: Indra. In Hindu scriptures, Indra, the lord of all deities, was given a garland of fragrant flowers by a sage called Durvasa. Indra placed the garland on the trunk of his elephant, but the animal was irritated by the smell of the flowers and threw the garland down, trampling it underfoot. When Durvasa saw this, he became enraged and cursed Indra, taking away his immortality.

The name also opened up a world of possibilities for naming future genes that are important in nonrandom sister chromatid segregation. “People sometimes pull from Roman or Greek myths when naming genes, but not as many people use names from Hindu myths,” he said. “And since this is new biology, if we identify additional related genes in the future, we can use names from Hindu myths again.”

Watase and Yamashita’s study opens new avenues for future research. For example, the paper focused primarily on male fruit flies and the production of sperm via asymmetric division. Indra is expressed in the female germline as well, and when the gene is knocked down in females, the resulting phenotype is much more severe. “There must be some mechanism in female germ cells to avoid rDNA copy number reduction,” said Watase. “We just don’t know what that mechanism is.”

In the future, Watase and Yamashita also hope to elucidate how exactly Indra is interacting with cell division machinery to influence which chromatid ends up in the stem cell and which in the differentiating cell, and beyond this mechanism, how the stem cell “selects” the longer chromatid.

“Many biologists study germ cells, but few specifically study how they maintain their immortality,” said Yamashita. “This study is a step towards understanding this fascinating property of germ cells. It’s a really fascinating area and we really have to keep digging deeper into this phenomenon.”

Investigating how cell orientation drives tissue growth during development

In a new study published July 7, 2022 in eLife, Adam Martin’s lab at the MIT Department of Biology identified the mechanical forces and molecular cues that help the spindles in cells located in the portion of the embryo destined to become the fly’s head to assume the same orientation.

Raleigh McElvery
July 7, 2022

Raleigh McElvery

During development, virtually all multicellular creatures must build themselves up from a ball of cells to a multilayered, fully-functioning organism. In the case of a fruit fly embryo, in order go from blob to organism, the cells must coordinate their divisions along the same axis to drive tissue growth in a specific direction — eventually going on to form the three germ layers known as the endoderm, ectoderm, and mesoderm.

Scientists have long studied how cells orient themselves to divide in a specific direction. It’s clear that this orientation is determined by a bundle of tiny fibers inside the cell called the spindle, which helps segregate the chromosomes so they can be distributed between the two daughter cells as the parent cell splits. When the spindles in neighboring cells are parallel with one another, then the cells will divide along the same axis.

In a new study published in eLife on July 7, 2022, Adam Martin’s lab at the MIT Department of Biology identified the mechanical forces and molecular cues that help the spindles in cells located in the portion of the embryo destined to become the fly’s head to assume the same orientation.

According to Martin, an associate professor of biology and the study’s senior author, his lab was among a handful of labs to take interest in this coordinated spindle orientation in the early fruit fly embryo — and identified the molecular, mechanical, and molecular cues that orient the spindle in a living organism.

“Cell divisions in the fly embryo was thought to be regulated independently of cell shape changes and morphogenetic movements,” Martin says. “However, we found that forces associated with cell invagination actually oriented cell divisions through a novel mechanism that we describe.” First author Jaclyn Camuglia, he explains, spearheaded the project from start to finish.

Prior to Camuglia’s work, researchers knew that spindle orientation was controlled by a complex of multiple proteins, including one protein in fruit flies called Pins (known as LGN in vertebrates). The entire protein complex, including Pins, is coupled to motor proteins that help to rotate the spindle. However, it was still unclear what mechanical forces were orienting Pins to dictate spindle rotation.

“It’s a very striking phenomenon when you look into the microscope at a fly embryo and see all the spindles orienting together,” Camuglia says. “We wanted to know: How are these spindles oriented and why is that directionality important?”

The fly is an ideal organism for probing cell division, because it has a very consistent and predictable cell cycle. During development, cells divide a set number of times, pause briefly, and then start up again in very discrete pockets across the embryo. The researchers wanted to know how the cells in a subset of those pockets in the fly’s head were coordinating their divisions.

Camuglia found that, in healthy embryos, Pins was always recruited to the end of the cell along the anterior-posterior axis (from the fly’s head to rear). By cutting the embryo with a laser, treating it with chemical inhibitors, disrupting the connections between cells, and depleting certain transcription factor proteins, she was able to characterize the mechanical forces that directed Pins to the anterior-posterior axis and thus rotated the spindle in the proper direction. As it turns out, these forces stem from other large-scale tissue movements that occur at the same time to force the embryo to fold in on itself and eventually form the fly’s muscles.

The researchers don’t yet know the role that these coordinated divisions along the anterior-posterior axis play in the developing fruit fly head — or what might happen during development if the divisions were to go awry. But Martin and Camuglia suspect this process facilitates tissue elongation, and helps compensate for any cells that are lost from the tissue during the division process as the embryo folds in on itself.

“This study is unique because it connects mechanical forces to the specific molecular cues like Pins that coordinate cell division,” Camuglia explains. “The factors that shape a developing embryo are critical to understand, because all organisms — from fruit flies to humans — experience these driving forces.” This intersection between physics and biology, she says, is what makes the study so exciting.”

Video: Groups of cells divide in a coordinated and oriented manner in the fruit fly embryo.
Top Image: Enrichment of cortical cues at one end of the cell coordinates the orientation of the mitotic spindle. Credit: Jaclyn Camuglia

Citation:
“Morphogenetic forces planar polarize LGN/Pins in the embryonic head during Drosophila gastrulation”
eLife, online 07/07/2022, DOI: https://doi.org/10.7554/eLife.78779
Jaclyn Camuglia, Soline Chanet, and Adam C Martin

Lab-grown fat cells help scientists understand type 2 diabetes
Eva Frederick | Whitehead Institute
June 16, 2022

In research published June 17 in the journal Science Advances, researchers in the lab of Whitehead Institute Founding Member Rudolf Jaenisch present a way to create fat cells that can be modified to display different levels of insulin sensitivity.

The cells accurately model healthy insulin metabolism, as well as insulin resistance, one of the key hallmarks of type 2 diabetes. “This system, I think, will be really useful for studying the mechanisms of this disease,” said Jaenisch, who is also a professor of biology at the Massachusetts Institute of Technology (MIT).

“It’s really exciting,” said Max Friesen, a postdoctoral researcher in Jaenisch’s lab and a first author of the study. “This is the first time that you can actually use a human stem cell-derived [fat cell] to show a real insulin response.”

Body fat — also known as adipose tissue — is essential for regulating your body’s metabolism and plays an important role in the storage and release of energy. When fat cells called adipocytes encounter the hormone insulin, they suck up sugar from the blood and store it for future use.

But over many years, factors such as genetics, stress, certain diets, or polluted air or water can cause this process to go awry, leading to type 2 diabetes. In this disease, adipocytes, as well as cells in the muscles and liver, become resistant to insulin and therefore unable to regulate the levels of sugar in the blood.

Tools to model diabetes in the lab generally rely on mice or on cells in a petri dish or test tube. Both these systems have their own problems. Mice, although they are comparable with humans in some respects, have a completely different metabolism and do not experience human diabetes comorbidities like heart attacks. And cell culture has, in the past, failed to replicate key markers of diabetes in a way that is comparable to human tissues.

That’s why Friesen and Andrew Khalil, another postdoc in Jaenisch’s lab, set out to create a new model. The researchers started with human pluripotent stem cells. These cells are the shapeshifters of the body — given the right conditions, they can assume the specific characteristics of almost any human cell type. The Jaenisch Lab has used them in the past to replicate liver cells, brain cells, and even cancerous tumors.

They decided to try to optimize an existing method for differentiating pluripotent cells into fat cells. The protocol created cells that looked like adipocytes, but these cells did not recreate the conditions of healthy insulin signaling or insulin resistance seen in the human body in type 2 diabetes. When healthy adipocytes encounter insulin in the human body, they respond by taking up glucose out of the bloodstream. These lab-made fat cells weren’t doing that, unless the researchers cranked up insulin levels to a thousand times higher than levels ever seen in humans. “Taking up glucose [in response to normal levels on insulin] is really the main function of an adipocyte, so if the model fails to do that, anything downstream in terms of disease research is not going to work either,” Friesen said.

Friesen and Khalil wondered if the lab-grown adipocytes’ low sensitivity to insulin could be a product of the conditions in which they grew. “We thought that maybe this happens because we’re feeding them an artificial culture medium, with all kinds of extra supplements that might be inhibiting their metabolic response,” Friesen said.

Friesen and Khalil decided to use a method called the Design of Experiments approach, which allows researchers to tease out the contributions of different factors to a specific outcome. Informed by this approach, they created nearly 30 different media compositions, each with slightly different levels of key ingredients such as glucose, insulin, the growth factor IGF-1, and albumin, a protein found in blood serum.

The medium that worked best had concentrations of insulin and glucose that were similar to the levels in the human body. When grown in this new medium, the cells responded to much lower concentrations of insulin, just like cells in the body. “So this is our healthy adipocyte,” Friesen said. “Next we wanted to see if we could make a disease model out of this — to make it an insulin-resistant adipocyte like you would see in the progression to type 2 diabetes.”

To desensitize the cells, they flooded the media with insulin for a short period of time. This caused the cells to become less sensitive to the hormone, and respond similarly to diabetic or pre-diabetic fat cells in a living person.

The researchers could then study how the cells responded to the change — such as what genes the insulin resistant cells expressed that healthy cells did not — in order to tease out the underlying genetics of insulin resistance. “We saw small changes in a lot of genes that are metabolism regulated, so that seems to be pointing to a deficiency of the metabolism or mitochondria of the insulin-resistant cells,” Friesen said. “That’s one thing we want to pursue in the future — figure out what is wrong with their metabolism, and then hopefully how to fix it.”

Now that they have created this new model for studying insulin resistance in fat cells, the researchers hope to develop similar procedures for other cells affected in diabetes.  “It seems that with some modifications, we can apply this method to other tissues as well,” Friesen said. “In the future, this will hopefully lead to a unified system for all stem cell-derived tissues, including liver, skeletal muscle, and other cell types, to get a really robust insulin response.”

Ankur Jain Named as Pew Scholar in Biomedical Sciences
Merrill Meadow | Whitehead Institute
June 13, 2022

The Pew Charitable Trusts has selected Whitehead Institute Member Ankur Jain to be a 2022 Pew Scholar in the Biomedical Sciences. The Pew program provides funding to young investigators of outstanding promise who work in areas of science relevant to the advancement of human health.

Jain, who joined the Whitehead Institute faculty in 2019, is one of 22 scientists selected to receive this year’s honor, chosen from among 197 nominations submitted by leading U.S. academic and research institutions. “I am grateful to the Pew Trusts for funding our work, and thrilled to be a part of the Pew community,” says Jain, who is also an assistant professor of biology and the Thomas D. and Virginia W. Cabot Career Development Professor at Massachusetts Institute of Technology.

The Pew award will provide research support for the next four years, enabling him to study the role of evolutionarily ancient metabolites called polyamines, which are essential for cell growth and survival.

“Polyamine concentrations within cells are carefully regulated, and disruptions in polyamine production are known to be associated with conditions ranging from cancer and aging to neurological disorders such as Parkinson’s disease” Jain explains. “But, despite being studied for more than a century, the specific role polyamines play in both healthy and diseased cells remains obscure. This is due, in part, to a lack of technologies effective in probing polyamines.”

Jain’s lab will harness the cell’s own polyamine detection machinery to build new tools to inspect polyamines. Those tools will allow his team to measure and track polyamines in individual cells, study how cells maintain their polyamine content, and explore how changing polyamine levels affect cellular functions. “Ultimately, this work could provide the basis for novel strategies for treating cancer or promoting healthy aging,” Jain observes.

Previously, Jain received a 2017 NIH Pathway to Independence Award and was named a 2019 Packard Fellow for Science and Engineering. He is the third current Whitehead Member to be named a Pew Scholar, following in the steps of Mary Gehring (2010) and Jing-Ke Weng (2014). Former Whitehead Fellow Fernando Camargo, now professor of stem cell and regenerative biology at Harvard University, also became a Pew Scholar in 2010.

Launched in 1985, the Pew Scholars in the Biomedical Sciences program supports top U.S. scientists at the assistant professor level and has, since inception, provided nearly 1000 young investigators with  funding for research projects that, though seemingly risky, have the potential to benefit human health. Pew Scholars are selected by a national advisory committee of eminent scientists, who evaluate candidates on the basis of proven creativity.

More information about Jain’s selection, the 2022 class of Pew Scholars, and the Pew Scholars program is available here.

New CRISPR-based map ties every human gene to its function

Jonathan Weissman and collaborators used their single-cell sequencing tool Perturb-seq on every expressed gene in the human genome, linking each to its job in the cell.

Eva Frederick | Whitehead Institute
June 9, 2022

The Human Genome Project was an ambitious initiative to sequence every piece of human DNA. The project drew together collaborators from research institutions around the world, including MIT’s Whitehead Institute for Biomedical Research, and was finally completed in 2003. Now, over two decades later, MIT Professor Jonathan Weissman and colleagues have gone beyond the sequence to present the first comprehensive functional map of genes that are expressed in human cells. The data from this project, published online June 9 in Cell, ties each gene to its job in the cell, and is the culmination of years of collaboration on the single-cell sequencing method Perturb-seq.

The data are available for other scientists to use. “It’s a big resource in the way the human genome is a big resource, in that you can go in and do discovery-based research,” says Weissman, who is also a member of the Whitehead Institute and an investigator with the Howard Hughes Medical Institute. “Rather than defining ahead of time what biology you’re going to be looking at, you have this map of the genotype-phenotype relationships and you can go in and screen the database without having to do any experiments.”

The screen allowed the researchers to delve into diverse biological questions. They used it to explore the cellular effects of genes with unknown functions, to investigate the response of mitochondria to stress, and to screen for genes that cause chromosomes to be lost or gained, a phenotype that has proved difficult to study in the past. “I think this dataset is going to enable all sorts of analyses that we haven’t even thought up yet by people who come from other parts of biology, and suddenly they just have this available to draw on,” says former Weissman Lab postdoc Tom Norman, a co-senior author of the paper.

Pioneering Perturb-seq

The project takes advantage of the Perturb-seq approach that makes it possible to follow the impact of turning on or off genes with unprecedented depth. This method was first published in 2016 by a group of researchers including Weissman and fellow MIT professor Aviv Regev, but could only be used on small sets of genes and at great expense.

The massive Perturb-seq map was made possible by foundational work from Joseph Replogle, an MD-PhD student in Weissman’s lab and co-first author of the present paper. Replogle, in collaboration with Norman, who now leads a lab at Memorial Sloan Kettering Cancer Center; Britt Adamson, an assistant professor in the Department of Molecular Biology at Princeton University; and a group at 10x Genomics, set out to create a new version of Perturb-seq that could be scaled up. The researchers published a proof-of-concept paper in Nature Biotechnology in 2020.

The Perturb-seq method uses CRISPR-Cas9 genome editing to introduce genetic changes into cells, and then uses single-cell RNA sequencing to capture information about the RNAs that are expressed resulting from a given genetic change. Because RNAs control all aspects of how cells behave, this method can help decode the many cellular effects of genetic changes.

Since their initial proof-of-concept paper, Weissman, Regev, and others have used this sequencing method on smaller scales. For example, the researchers used Perturb-seq in 2021 to explore how human and viral genes interact over the course of an infection with HCMV, a common herpesvirus.

In the new study, Replogle and collaborators including Reuben Saunders, a graduate student in Weissman’s lab and co-first author of the paper, scaled up the method to the entire genome. Using human blood cancer cell lines as well noncancerous cells derived from the retina, he performed Perturb-seq across more than 2.5 million cells, and used the data to build a comprehensive map tying genotypes to phenotypes.

Delving into the data

Upon completing the screen, the researchers decided to put their new dataset to use and examine a few biological questions. “The advantage of Perturb-seq is it lets you get a big dataset in an unbiased way,” says Tom Norman. “No one knows entirely what the limits are of what you can get out of that kind of dataset. Now, the question is, what do you actually do with it?”

The first, most obvious application was to look into genes with unknown functions. Because the screen also read out phenotypes of many known genes, the researchers could use the data to compare unknown genes to known ones and look for similar transcriptional outcomes, which could suggest the gene products worked together as part of a larger complex.

The mutation of one gene called C7orf26 in particular stood out. Researchers noticed that genes whose removal led to a similar phenotype were part of a protein complex called Integrator that played a role in creating small nuclear RNAs. The Integrator complex is made up of many smaller subunits — previous studies had suggested 14 individual proteins — and the researchers were able to confirm that C7orf26 made up a 15th component of the complex.

They also discovered that the 15 subunits worked together in smaller modules to perform specific functions within the Integrator complex. “Absent this thousand-foot-high view of the situation, it was not so clear that these different modules were so functionally distinct,” says Saunders.

Another perk of Perturb-seq is that because the assay focuses on single cells, the researchers could use the data to look at more complex phenotypes that become muddied when they are studied together with data from other cells. “We often take all the cells where ‘gene X’ is knocked down and average them together to look at how they changed,” Weissman says. “But sometimes when you knock down a gene, different cells that are losing that same gene behave differently, and that behavior may be missed by the average.”

The researchers found that a subset of genes whose removal led to different outcomes from cell to cell were responsible for chromosome segregation. Their removal was causing cells to lose a chromosome or pick up an extra one, a condition known as aneuploidy. “You couldn’t predict what the transcriptional response to losing this gene was because it depended on the secondary effect of what chromosome you gained or lost,” Weissman says. “We realized we could then turn this around and create this composite phenotype looking for signatures of chromosomes being gained and lost. In this way, we’ve done the first genome-wide screen for factors that are required for the correct segregation of DNA.”

“I think the aneuploidy study is the most interesting application of this data so far,” Norman says. “It captures a phenotype that you can only get using a single-cell readout. You can’t go after it any other way.”

The researchers also used their dataset to study how mitochondria responded to stress. Mitochondria, which evolved from free-living bacteria, carry 13 genes in their genomes. Within the nuclear DNA, around 1,000 genes are somehow related to mitochondrial function. “People have been interested for a long time in how nuclear and mitochondrial DNA are coordinated and regulated in different cellular conditions, especially when a cell is stressed,” Replogle says.

The researchers found that when they perturbed different mitochondria-related genes, the nuclear genome responded similarly to many different genetic changes. However, the mitochondrial genome responses were much more variable.

“There’s still an open question of why mitochondria still have their own DNA,” said Replogle. “A big-picture takeaway from our work is that one benefit of having a separate mitochondrial genome might be having localized or very specific genetic regulation in response to different stressors.”

“If you have one mitochondria that’s broken, and another one that is broken in a different way, those mitochondria could be responding differentially,” Weissman says.

In the future, the researchers hope to use Perturb-seq on different types of cells besides the cancer cell line they started in. They also hope to continue to explore their map of gene functions, and hope others will do the same. “This really is the culmination of many years of work by the authors and other collaborators, and I’m really pleased to see it continue to succeed and expand,” says Norman.

Tracing a cancer’s family tree to its roots reveals how tumors grow

Family trees of lung cancer cells reveal how cancer evolves from its earliest stages to an aggressive form capable of spreading throughout the body.

Greta Friar | Whitehead Institute
May 5, 2022

Over time, cancer cells can evolve to become resistant to treatment, more aggressive, and metastatic — capable of spreading to additional sites in the body and forming new tumors. The more of these traits that a cancer evolves, the more deadly it becomes. Researchers want to understand how cancers evolve these traits in order to prevent and treat deadly cancers, but by the time cancer is discovered in a patient, it has typically existed for years or even decades. The key evolutionary moments have come and gone unobserved.

MIT Professor Jonathan Weissman and collaborators have developed an approach to track cancer cells through the generations, allowing researchers to follow their evolutionary history. This lineage-tracing approach uses CRISPR technology to embed each cell with an inheritable and evolvable DNA barcode. Each time a cell divides, its barcode gets slightly modified. When the researchers eventually harvest the descendants of the original cells, they can compare the cells’ barcodes to reconstruct a family tree of every individual cell, just like an evolutionary tree of related species. Then researchers can use the cells’ relationships to reconstruct how and when the cells evolved important traits. Researchers have used similar approaches to follow the evolution of the virus that causes Covid-19, in order to track the origins of variants of concern.

Weissman and collaborators have used their lineage-tracing approach before to study how metastatic cancer spreads throughout the body. In their latest work, Weissman; Tyler Jacks, the Daniel K. Ludwig Scholar and David H. Koch Professor of Biology at MIT; and computer scientist Nir Yosef, associate professor at the University of California at Berkeley and the Weizmann Institute of Science, record their most comprehensive cancer cell history to date. The research, published today in Cell, tracks lung cancer cells from the very first activation of cancer-causing mutations. This detailed tumor history reveals new insights into how lung cancer progresses and metastasizes, demonstrating the wealth of understanding that lineage tracing can provide.

“This is a new way of looking at cancer evolution with much higher resolution,” says Weissman, who is a professor of biology at MIT, a member of the Whitehead Institute for Biomedical Research, and an investigator with Howard Hughes Medical Institute. “Previously, the critical events that cause a tumor to become life-threatening have been opaque because they are lost in a tumor’s distant past, but this gives us a window into that history.”

In order to track cancer from its very beginning, the researchers developed an approach to simultaneously trigger cancer-causing mutations in cells and start recording the cells’ history. They engineered mice such that when their lung cells were exposed to a tailor-made virus, that exposure activated a cancer-causing mutation in the Kras gene and deactivated tumor suppressing gene Trp53 in the cells, as well as activating the lineage tracing technology. The mouse model, developed in Jacks’ lab, was also engineered so that lung cancer would develop in it very similarly to how it would in humans.

“In this model, cancer cells develop from normal cells and tumor progression occurs over an extended time in its native environment. This closely replicates what occurs in patients,” Jacks says. Indeed, the researchers’ findings closely align with data about disease progression in lung cancer patients.

The researchers let the cancer cells evolve for several months before harvesting them. They then used a computational approach developed in their previous work to reconstruct the cells’ family trees from their modified DNA barcodes. They also measured gene expression in the cells using RNA sequencing to characterize each individual cell’s state. With this information, they began to piece together how this type of lung cancer becomes aggressive and metastatic.

“Revealing the relationships between cells in a tumor is key to making sense of their gene expression profiles and gaining insight into the emergence of aggressive states,” says Yosef, who is a co-corresponding author on both the current work and the previous lineage tracing paper.

The results showed significant diversity between subpopulations of cells within the same tumor. In this model, cancer cells evolved primarily through inheritable changes to their gene expression, rather than through genetic mutations. Certain subpopulations had evolved to become more fit — better at growth and survival — and more aggressive, and over time they dominated the tumor. Genes that the researchers identified as commonly expressed in the fittest cells could be good candidates for possible therapeutic targets in future research. The researchers also discovered that metastases originated only from these groups of dominant cells, and only late in their evolution. This is different from what has been proposed for some other cancers, in which cells may gain the ability to metastasize early in their evolution. This insight could be important for cancer treatment; metastasis is often when cancers become deadly, and if researchers know which types of cancer develop the ability to metastasize in this stepwise manner, they can design interventions to stop the progression.

“In order to develop better therapies, it’s important to understand the fundamental principles that tumors adopt to develop,” says co-first author Dian Yang, a Damon Runyon Postdoctoral Fellow in Weissman’s lab. “In the future, we want to be able to look at the state of the cancer cells when a patient comes in, and be able to predict how that cancer’s going to evolve, what the risks are, and what is the best treatment to stop that evolution.”

The researchers also figured out important details of the evolutionary paths that cancer subpopulations take to become fit and aggressive. Cells evolve through different states, defined by key characteristics that the cell has at that point in time. In this cancer model the researchers found that early on, cells in a tumor quickly diversified, switching between many different states. However, once a subpopulation landed in a particularly fit and aggressive state, it stayed there, dominating the tumor from that stable state. Furthermore, the ultimately dominant cells seemed to follow one of two distinct paths through different cell states. Either of those paths could then lead to further progression that enabled cancers to enter aggressive “mesenchymal” cell states, which are linked to metastasis.

After the researchers thoroughly mapped the cancer cells’ evolutionary paths, they wondered how those paths would be affected if the cells experienced additional cancer-linked mutations, so they deactivated one of two additional tumor suppressors. One of these affected which state cells stabilized in, while the other led cells to follow a completely new evolutionary pathway to fitness.

The researchers hope that others will use their approach to study all kinds of questions about cancer evolution, and they already have a number of questions in mind for themselves. One goal is to study the evolution of therapeutic resistance, by seeing how cancers evolve in response to different treatments. Another is to study how cancer cells’ local environments shape their evolution.

“The strength of this approach is that it lets us study the evolution of cancers with fine-grained detail,” says co-first author Matthew Jones, a graduate student in the Weissman and Yosef labs. “Every time there is a shift from bulk to single-cell analysis in a technology or approach, it dramatically widens the scope of the biological insights we can attain, and I think we are seeing something like that here.”

Helping drugs play nice in the human body
Whitehead Institute
March 25, 2022

For the hundreds of thousands of people diagnosed with breast cancer each year, surgery to remove the cancerous tissue is often the best option — but this relatively simple procedure comes with some drawbacks. In more than a few cases, the surgical removal of a tumor can lead to an increased risk of the cancer reemerging in other locations in the body.

In a 2018 study, a postdoc in the lab of Whitehead Institute Member Bob Weinberg discovered that, at least in mice, this phenomenon was due to a bodily butterfly effect: the creation of a wound site in one place in the body, which necessitated subsequent wound healing, caused immune system changes affecting distant parts of the body.

These changes occurred as bone marrow cells responded to the wounding with a flood of inflammatory cells that entered into the wound site and, at the same time,  scattered throughout the body. These dispersed inflammatory cells weakened the ability of the immune system to control the outgrowth of a distantly located metastatic tumor.  Without this immune control, which otherwise could keep the metastasis at a very small size,  the metastasis would grow out aggressively.

Hence, wounding in one part of the body provoked metastasis outgrowth at a distant site. This suggested, among other things, that the outgrowth of metastatic tumors, which is often seen in women who have recently undergone a mastectomy,  might be actively provoked by the post-surgical wound-healing process.

Weinberg’s work also presented a way to potentially avoid this effect, using a preventative measure that’s probably sitting in your bathroom cabinet right now: the cheap and common class of drugs known as NSAIDS, which includes ibuprofen and aspirin. When mice were given NSAIDS before and after tumor removal surgeries, they experienced a fivefold lower rate of cancer recurrence at the site of metastasis than a control group given opioids. These NSAIDs could therefore be used in place of the opioids, which are often used to treat post-surgical pain.

The human body is full of undiscovered connections like this one and adding in foreign substances further complicates matters. While a treatment might work well in a Petri dish, researchers describe whole -body metabolism as “a whole different kettle of fish.”

The way drugs move through the body and interact with internal systems is called pharmacokinetics. When a person is given a medicine — either orally, through a chemotherapy method, or via injection — that drug must be able to find its way to its target in a high enough concentration to have an effect, and then when its purpose is served, it must be able to leave the body safely and not build up to a harmful amount.

Much like Weinberg’s work on NSAIDS in breast cancer, Whitehead Institute’s basic research has led to other surprising discoveries about drug activities in the human body. Read on to learn about research that is changing the way new drugs are designed, making existing treatments less toxic, and more.

Concentration is key

When it comes to the action of drugs in the human body, concentration is key. Just ask Rick Young, a Whitehead Institute Member and professor of biology at MIT. In 2018, Young’s lab, which had previously studied the regulatory circuitry involved in transcription (the copying of DNA into RNA), shifted its focus after discovering tiny droplets within cells that concentrate the molecular materials needed to transcribe the DNA.

The droplets, called transcriptional condensates, were the newest in a slew of recent discoveries of other such groupings of cellular components. Some of these aggregations facilitate RNA splicing while others help to form ribosomes.

For Young, the discovery of transcription-related condensates sparked an interest in how these droplets were affecting the action of drugs. Previous theories held that transcription was able to take place in cells because there was a sufficient concentration of necessary proteins, such as RNA polymerase and other accessory proteins. As the Young lab showed,  these collaborating cellular players were actually being concentrated in the condensates,

In 2020, Young and Ann Boija and Isaac Klein, two postdocs in his lab, took their investigation a step further, analyzing the mechanism by which several cancer drugs are concentrated in cellular condensates, and how that concentration could affect their action in individual cells and thus in the body. They found that cancer drugs sort themselves into specific types of condensates, independently of their targets, which can allow them to build up into high concentrations in these localized areas within cells.

“This could have enormous implications for the way we discover and develop drugs,” said Rick Young.  “If drugs had properties that had them partitioning into a condensate where their target lives, then they would enjoy two properties of condensates: they would be compartmentalized, and they would be at much higher concentrations than if they diffuse through the cell.”

Young’s work on condensates led him to co-create a pharmaceutical company called Dewpoint Therapeutics, with the goal of reformulating treatments for cancer or neurological conditions such as amyotrophic lateral sclerosis by targeting biomolecular condensates. Whitehead Institute Founding Member Rudolf Jaenisch serves as a scientific advisor.

Trouble in parasites

While researchers in Young’s lab investigate how drugs could be more efficiently targeted, Sebastian Lourido’s lab is taking a different tack — why do some drugs stop working as time progresses?

The malaria drug artemisinin was developed in China in the 1970s, and completely changed the way the world treated malaria. In the following decades, however, the parasites that cause malaria, several species within the genus Plasmodium, have slowly grown less susceptible to the drug.

In a paper published in September of 2020, Whitehead Institute Member Lourido and collaborators identified two parasite genes that were negatively impacting the actions of the drug in the parasite’s cells.

Researchers liken artemisinin to a “ticking time bomb,” which needs another molecule, called heme, to light its fuse. Heme, a small molecule that is one component of hemoglobin, helps transport electrons and deliver oxygen to tissues. When heme encounters artemisinin, it activates the drug, allowing the creation of small, toxic chemical radicals. These proceed to react with the parasites proteins, fats, and metabolites, eventually leading to its death.

In order to understand how some parasites were becoming less vulnerable to the drug, Lourido, along with researchers Clare Harding, Boryana Petrova and Saima Sidik, ran a genetic screen on a related parasite, Toxoplasma gondii. The screen allowed them to assess which mutations in the parasites’ genomes were beneficial for their survival and which ones were harmful.

The screen revealed two genes that affected how susceptible the parasites were to treatment with artemisinin. One, called Tmem14c, seemed to be protecting the parasites. The gene is analogous to a gene that transports heme out of mitochondria where it is generated. Lourido hypothesized that when the  Tmem14c protein is working properly, it helps the cells shuttle heme and its building blocks and get them where they need to go in the cell. When this gene is knocked out or mutated, heme can build up in the parasite cells, making them more likely to activate the artemisinin “bomb.”

Another gene, when mutated, made the parasites less sensitive to artemisinin. The gene, called DegP2, encodes a protein that plays a role in heme metabolism, so when it was mutated, less heme was available in the cells to activate the drug.

This knowledge provides useful insights for treatment methods, said Lourido. For example, healthcare providers should take into consideration the fact that heme is key in artemisinin’s action, and avoid combining the drug with other treatments that might lower the amount of heme in parasite cells. “Understanding how different pathways within the cell participate to render parasites susceptible to these antiparasitic drugs helps us better pair them with other compounds that are going to be synergistic and not work against our own goal of defeating parasites,” Lourido said.

Taking the edge off toxic treatments 

Another application of fundamental pharmacokinetics research involves mitigating the harmful effects of drugs. Consider the chemotherapy drug methotrexate. Methotrexate was the very first targeted drug ever made. Developed more than 60 years ago by Dr. Sidney Farber, the drug acts by inhibiting a key molecule in the metabolic process that builds DNA and RNA, thereby interfering with basic functions of the cell and with DNA synthesis, repair and replication, helping to destroy cancerous cells in the body.

Methotrexate is still a widely used component of chemotherapy cocktails, especially for pediatric leukemia. In the human body, though, methotrexate is like a bull in a china shop. It is very effective at knocking back cancer, but the drug’s life-threatening side effects, including gut, liver, kidney and brain damage, often lead doctors to terminate their patients’ treatment early, or seriously compromise the survivors’ quality of life.

The drug was much-studied in the 70s, but research trailed off in the subsequent decades due to limits on the existing technologies. Nearly fifty years later, Naama Kanarek, then a postdoctoral researcher in the lab of former Whitehead Institute Member David Sabatini, decided to take  a fundamental research approach to studying the effects of methotrexate, in hopes that she might discover some way to make the drug less toxic.

“We now have access to genetic tools that allow us to address long standing questions in a way that was not possible before,” said Kanarek, who now runs her own lab at Boston Children’s Hospital. “We can use a CRISPR screen, and instead of focusing on what is known, we can ask what is unknown about the drug. We can find new genes that are involved in the response of cells to the drug that were not found before simply because the tools were not there.”

The screen revealed one gene in particular that seemed to be playing a role in how sensitive cancer cells were to methotrexate, the researchers reported in Nature in July of 2018. The cells’ sensitivity is important, Kanarek said, because if the cells can be made more vulnerable to methotrexate, the duration of treatment or required dose could be reduced. “If we can reduce dose because we can improve efficacy, then we can reduce toxicity without compromising on the cure rates and that is good news to the patients,” Kanarek said.

The gene in question, called FTCD, encodes an enzyme involved in the breakdown of the amino acid histidine. When the gene was knocked out, cancer cells were less sensitive to methotrexate. When the pathway was boosted with the addition of extra histidine, cells became more sensitive.

Former Whitehead Institute Director Susan Lindquist, who passed away 2016, performed similar work on the natural product amphotericin B, a drug which is used to treat some fungal infections. The drug is especially useful because fungi have not yet developed a resistance to it, as they have with so many other treatments. But amphotericin B also has some serious drawbacks; it can cause kidney damage, heart failure, and other serious and even fatal side effects.

These side effects mostly happened because amphotericin B works by binding to a chemical group called a sterol. In fungi, it binds to molecules called ergosterols in the cell wall, destabilizing the cells. Unfortunately humans also have a prevalent sterol: cholesterol. When amphotericin B binds to cholesterol in human cell membranes, it can damage human cells.

Using chemical synthesis methods, Lindquist and colleagues at Whitehead Institute and elsewhere were able to tweak the structure of the drug to bind only to ergosterol molecules and not cholesterol, bypassing most of the harmful side effects.

Why fundamental research 

Drug development is often an extremely targeted pursuit, but for Whitehead Institute scientists, their advances have mostly come from a simple curiosity about the cellular mechanisms. For example, Rick Young didn’t set out to study condensates, but an inquiry into the fundamentals of transcription led him in this entirely new direction.

Such fundamental research has the potential to branch in any number of different ways. “Fundamental science can lead in directions that you would not foresee,” said the Institute’s Associate Director of Intellectual Property Shoji Takahashi. Basic research into drug behavior is essential and can contribute to life-changing therapies down the line.