AudioHelicase Special: The Conductors of the Cell’s Symphony

In this special episode of AudioHelicase podcast, we’re taking a look at how our researchers are identifying the key players that help conduct the cell’s symphony — the proteins and molecules that direct many other parts of the cell on when and how to do their jobs. The episode features highlights from interviews with Whitehead Institute Members David Sabatini, Mary Gehring, and Iain Cheeseman.

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EDITED TRANSCRIPT

Conor Gearin, host: Welcome to AudioHelicase, the podcast from Whitehead Institute where we unwind the science and the people behind some of the Institute’s most exciting discoveries.

Our bodies are made up of trillions of cells, but each of those cells is made up of millions of proteins and other biological molecules that carry out specific functions to keep the cell alive. And when those molecules malfunction, it can lead to disease. If we imagine the cell as an orchestra, with many musicians each with a small job to do, then what conducts all of these musicians and keeps them organized and harmonized as they play a symphony? 

I’m Conor Gearin, digital media specialist at Whitehead Institute. And in this special episode, we’re taking a look at how our researchers are identifying the key players that help conduct the cell’s symphony — the proteins and molecules that direct many other parts of the cell on when and how to do their jobs. 

We’ll hear highlights from interviews with Whitehead Institute Members David Sabatini, Mary Gehring, and Iain Cheeseman. Sabatini studies mTOR, the keystone factor in a cellular pathway that helps a cell collect information about its environment and then change its growth and metabolism to adapt. Gehring uses plants to uncover how chemical modifications to the genome without changing the DNA sequence itself can act as a master regulator of gene expression. And Cheeseman focuses on the kinetochore, a protein complex at the center of the complicated dance of cell division. 

Whitehead Institute researchers are showing that like a conductor, these molecules tune the cell’s behavior to the needs of the occasion. And the knowledge of how cells give order to complicated processes could ultimately prove crucial to learning how to restore healthy function in diseased cells.  

In order to survive, cells need to adjust their metabolism based on the amount of nutrients available around them. But how do they connect information about their environment to their growth and metabolism? Communications Director Lisa Girard spoke with Whitehead Institute Member David Sabatini about a protein called mTOR, which stands for the mechanistic target of rapamycin. The central importance of mTOR to regulating cellular growth means that it’s a key player in cancer, diabetes, and a number of other diseases.

[Listen to the full episode with Sabatini here.]

David Sabatini: Pretty much all the work in the lab falls under the broad umbrella of the study of growth and metabolism. In particular how metabolites and nutrients impact different physiological processes. And to a large extent, that stems from our early work on the mTOR pathway, which is a protein that I discovered when I was a student and then kept working on when I came to the Whitehead. And our realization that this is a major integrator of nutrition to metabolism and eventually leading us not only to studying that pathway, but to study metabolism per se, and in particular how metabolic processes impact diseases like cancer, or diabetes. How does, for example, fasting and feeding affect stem cells in the gut. Or how does it affect the aging process.

Gearin: Sabatini’s work on mTOR goes back to his graduate student days when he was studying a molecule called rapamycin—a drug that was isolated from a bacterium on Rapa Nui, also known as Easter Island. People began taking rapamycin clinically after preliminary studies showed immunosuppressive and anticancer effects, but how the drug worked and what pathways it affected in the cell remained unknown.

Sabatini: So when I was a graduate student at Hopkins with Sol Snyder, I became interested in this molecule rapamycin, which at the time was actually not that well studied. But there was a significant mostly clinical and preclinical body of work—largely abstracts—showing that it did interesting things, like immunosuppression, anticancer effects, antifungal effects, and yet we didn’t know how it worked. And so I set about trying to figure that out, and that eventually led to the discovery of the protein mTOR.

What we started to figure out, and by we, I mean the larger community, is that this protein is really at the center of many, many, key processes. And in fact, you could almost argue that it is one of the most tied-in signaling networks there is. And the reason likely for that is that this pathway, that we now call the mTOR pathway, is a pathway that is really involved in sensing the environment, largely the nutrient state of the environment and then tying it to physiology. But, clearly in our evolutionary history, most animals, even now some fraction of the human population, knowing whether you’re fed or fasted, and in particular, what nutrients you have, which ones you don’t, it’s not hard to imagine how this would impact all of your physiology. In fact, you can connect any physiological system to whether the animal is in a fed or fasted state. So if you’re one of the pathways that makes that connection, then almost by definition, you’re going to be connected to most physiological processes.

Girard: From mTOR’s position in this sensing hub, it is emerging to play a role in a number of diseases.

Sabatini: So we now know that mTOR is implicated in a large number of diseases. The best connection, probably, is still cancer. And there is really two sides to this. All cancer cells have to grow. And so this is the pathway that drives that growth response. And so by definition, a cancer cell almost has to activate this. And so, there are estimates in the literature that 60-80% of cancer-causing mutations will lead to the activation of this system. And so this has led to quite a bit of excitement of using rapamycin as an anticancer agent, where rapamycin and other molecules that inhibit mTOR are starting to have a significant impact are in cancers where it’s really the pathway itself that’s hyperactivated, like either through mTOR or direct regulators, rather than more upstream components that lead to turning on potentially of any downstream processes. So there I think there’s clear evidence and that’s having an impact already.

Gearin: While rapamycin shows promise, prolonged treatment with rapamycin can have unwanted side effects. To address this problem, Sabatini’s lab seeking to develop a new generation of drugs that would affect mTOR and fight against cancer-causing processes while avoiding the limitations of rapamycin. 

In order to find a new way of drugging a specific protein, it helps to know what that protein actually looks like. A study led by postdoc Kacper Rogala described a new structure for mTOR complex 1, a regulatory protein complex which includes the mTOR protein. The study reveals how mTOR complex 1 docks with a second complex called Rag-Ragulator, which then anchors to the lysosome, an organelle that breaks down and recycles materials in the cell. Once on the lysosome, the mTOR complex 1 can become activated. The structure is both more detailed than any previous work, and it reveals a new mechanism for how the complex docks with the lysosome.

With an experimentally determined three-dimensional structure of mTOR doing its job on the lysosome, Sabatini’s lab can start engineering drugs that act on it with high specificity without affecting other important cellular pathways. mTOR’s central role in the cell makes it an attractive target for new drugs, and Sabatini’s lab is giving us a clearer picture of what that target looks like and how it works. 

Gearin: There’s more than one way to regulate a gene. While some features of our cells are hard-coded into the DNA sequence, altering the chemical structure around DNA can change how the cell reads a gene and affect whether or not it gets activated. In general, these modifications of the genome are called epigenetics, and dysregulated epigenetics has been linked to many disorders, including Fragile X syndrome and Rett syndrome. Remarkably, some of these epigenetic alternations can be inherited. Whitehead Institute Member Mary Gehring uses plants as a model system to investigate how epigenetics are passed from one generation to the next. And she has found a particular gene, called ROS1, that plays a special role in ensuring that the genome has the right amount of epigenetic changes. Gehring spoke with Lisa Girard.

[Listen to the full episode with Gehring here.]

Lisa Girard: Which aspects of epigenetics in plants is your lab currently working on?

Gehring: We’re broadly interested in understanding epigenetics dynamics during plant growth and development. A couple of key areas that we’re interested in is how is epigenetic information inherited from one generation to the next. We know in plants that a lot of epigenetic information appears to be inherited between generations, and so what factors influence that? Whether that’s specific features of the genome or the epigenome or specific genes and proteins that are important. That’s one broad set of questions.  

Another broad area is really understanding what’s happening to the epigenome during reproductive development in plants, because we know that it’s really at the reproductive stage of the life cycle that we see remodeling of the epigenome in this extra-embryonic lineage called the endosperm—this tissue that’s also formed by fertilization, like the embryo, that’s in seeds, and it supports the embryo’s development during seed development. Endosperm is not only a good model, but really the only tissue where imprinting takes place in plants, so that’s why we’re looking at it.

Girard:  So you mentioned imprinting. Can you tell a little bit more about what imprinting is?  

Gehring: Imprinting is when alleles or copies of genes are expressed differently depending on whether they are inherited through the male parent, through the dad, or through the female parent, the mom. There is a set of genes in plants that are imprinted. There’s somewhere around 100 or 200 genes that are imprinted. And about 50 of those are primarily expressed from the copy that’s inherited from the father and the rest are primarily expressed from the copy that’s inherited from the mother.  We’re really interested in imprinting, because it is an example of epigenetic regulation. For an imprinted gene, the two copies may have very similar sequence or even identical sequence for some of the things we look at, yet, they’re expressed differently. Even though they’re in the same cells, they’re in the same nucleus, the copy on the maternally inherited chromosome is not expressed in the same way as the copy on the paternally inherited chromosome.

Girard: What role does methylation—or the addition of methyl groups to certain parts of the genome—play in imprinting?

Gehring: Well, we’ve worked a lot on DNA methylation, in the context of imprinting and understanding whether or not differential DNA methylation between the maternal and paternal alleles is important for imprint expression. And it clearly is. So we know that a signature of imprinted genes, particularly the paternally expressed imprinted genes, is that the maternally inherited copy is less methylated than the paternally inherited copy. So we have a difference in gene expression and a difference in methylation. And that difference in methylation often is not happening right in the coding sequence of the gene, it’s happening upstream or downstream in 5’ or 3’ regulatory regions.

Girard: Can you tell us about an interesting example of an imprinted gene you’ve come across so far?

Gehring: So, in terms of thinking about how methylation patterns are inherited between generations, one thing we’ve found recently is that regulation of this DNA demethylase gene, ROS1, is crucial for maintaining methylation patterns across generations—so for maintaining fidelity and epigenetic inheritance. So we showed a few years ago that ROS1, which is an enzyme that removes methylcytosine from the genome, that it is itself regulated it’s expression is regulated by DNA methylation, so it has a methylated region 5’ of the gene, and that methylation actually promotes expression of ROS1, and demethylation reduces its expression. That’s the opposite of what you typically associate with methylation, although actually that’s also what we find for paternally expressed imprinted genes in the endosperm. 

It does look like you really need ROS1 to be under tight control that’s tied to the methylation status of the genome in order to maintain the proper balance of methylation and demethylation activities. And if ROS1, its demethylase is just expressed, regardless of what’s going on with methylation at its 5’ region, and thus elsewhere in the genome, what you find is this epigenome that gets progressively worse across generations, at least in more gene-rich regions of the genome. We actually found that the genome is actually really plastic, and we found in heterochromatic regions, that whereas if you initially disrupt this ROS1 regulatory mechanism, you lose methylation at those regions, they can actually bounce back in later generations. There’s something else when you disrupt this mechanism at ROS1 over generational time, there’s something else that kicks in to try and restore the proper methylation patterning.

Gearin: The gene ROS1 gives order and balance to the complex landscape of methylation across the genome. And in a recent study, Gehring and postdoc Satyaki Rajavasireddy found a methylation pathway regulated by a small RNA molecule that determines whether Arabidopsis seeds are viable. Importantly, methylation activity in the paternal parent is sufficient to determine whether or not the seeds are viable or not, regardless of the input from the maternal parent. With discoveries like this, Gehring’s lab is helping identify the key players that give direction to all the possible ways to methylate DNA and affect the activation of genes. The findings give us a clearer grasp of how cells use epigenetics to tune the expression of their genes to just the right amounts.

Gearin: There are few processes more delicate or more essential to our bodies than cell division, known as mitosis in normal cell division and meiosis in sex cell division. For bodies to grow, cells need to divide through mitosis to build tissues. And one cell dividing into two daughter cells has to split its genome of 46 chromosomes evenly between the two new nuclei. In this complicated dance routine, what makes sure those 46 chromosomes perform the right steps and end up where they’re needed? Enter the kinetochore, the dance master of cell division. This huge protein complex is made up of over 100 individual proteins and attaches itself to each chromosome, precisely directing them to the right destination. Iain Cheeseman talked to Lisa Girard about his work understanding on cell division and the kinetochore.  

[Listen to the full episode with Cheeseman here.]

Girard: Iain, in terms of the big picture, why is cell division so important?

Iain Cheeseman: All of us started as one cell. The number of cells in the human body is about 30 trillion. If you think about the number of divisions that you had to go through all along the way: one cell becomes two, two cells become four, four cells become eight. It’s astronomical the number of times they have to do this. Even in an adult human, there’s about 50 billion cells dividing every day just to replenish the tissues we have.

Every single one of those, you have to take all 46 different chromosomes—23 from your mom, 23 from your dad—and make sure that every new cell that is made gets the entire set. So actually even small deviations from that normal number of 46 can be a huge challenge. One chromosome too many or one chromosome too few can kill that cell. It can make it work not as well.

Girard: What actually needs to happen during mitosis?

Cheeseman: As mitosis begins you take these long linear stretches of DNA, and you compact it up into pieces that are actually something you can move and distribute.

And then you have to have a physical apparatus, which is able to capture them and move them around, and that is a structure called the mitotic spindle. That’s made of microtubule polymers that are long rods capable of growing and shrinking. Essentially like you’re casting a fishing line out, and it’s this process of essentially having a long rod that can extend and shrink back again, until it finds the chromosomes, and then once it’s captured them, that growth and shrinkage can help facilitate its ability to move within a cell.

The goal of our lab is to take all of this beautiful imaging and microscopy that has been done using light microscopy from the earliest days through to electron microscopy that really probed more deeply into the cell and say, ok, how do you assemble a protein machinery that’s capable of doing these things? That division after division is capable of physically capturing the DNA, distributing it, and then doing it with such a low rate of error as to not be a problem and allow our bodies to develop and continue to function.

Girard: So how did you become interested in the kinetochore?

Cheeseman: I really like protein machineries. Before I even realized that the kinetochore existed, what I first fell in love with in science was the physical players that have to do stuff. The things in cells where you have protein assemblies that build up to actually achieve something, move something, do something, or accomplish something for the cell. I really like protein machineries, and I really like how they’re precisely controlled. The kinetochore is I think by far the best example.

One of my favorite features of the kinetochore is just how dynamic it is. The kinetochore is a machine that has those 110-120 proteins but exists in that form for only really a narrow window. In a human cell growing in culture, it takes about 24 hours for that cell to divide. To go from one cell to two takes about 24 hours. Most of that time is about it growing, about it replicating its DNA—DNA replication might take about 8 hours for example—you’ve got to copy that genetic material first, which can take a while. The actual process of mitosis, where you’re dividing and segregating your chromosomes, takes less than an hour. So it’s a pretty small portion of the whole cell cycle from one cell to two cells.

Girard: So it seems like a lot really depends on the kinetochore properly functioning or things can really go terribly wrong.

Cheeseman:  I think the nice thing about the machines that exist is that they have been well optimized to work well. You have the physical things in place to segregate the chromosomes and you have a lot of players that come in add robustness, add security to that. And you have the regulatory components so that if there is a problem, you can recognize that and correct it. I think it’s still fascinating that we exist. You think about all of the things that could’ve gone wrong to go from the one cell that we started with to who we are today. The more you learn about how intricate and beautiful the biology is the more you realize how often it could’ve gone wrong.

Gearin: Cheeseman’s research has implications for understanding cancer, and it’s also shedding light on long-standing mysteries of how our bodies work. Scientists have wanted to know how egg cell precursors called oocytes retain their ability to divide more many years before they develop into viable eggs. In a recent study led by postdoc Zak Swartz, Cheeseman took a look at another component of the cell division apparatus called the centromere, which is the small DNA area that anchors the rope-like fibers that separate chromosomes during cell division. A number of proteins form that anchor, and the most important of them is called CENP-A. If that protein is lost, the cell loses its ability to divide. It was thought that the protein remains static in cells that go dormant but later divide. Using sea star oocytes as a model because of their similarities to human oocytes, Swartz and Cheeseman showed that CENP-A is instead continuously replaced in oocytes. This slow but steady replenishment of the centromere’s key component lets a cell remain dormant for many years until it needs to divide.

The cell can look like a crowded and confusing place through a microscope, with many organelles and millions of proteins, all with different roles to play. But Whitehead Institute scientists are showing that the cell’s many parts aren’t just a crowd going every which way. There are crucial organizers in the cell that help conduct the noise and turn it into a melody. Without those conductors, the cell can turn down a path towards disease. Restoring proper functions to the cell in cancer and other deadly diseases could depend on working with the conductors of the cell’s orchestra. 

Learn more about the latest research from the Institute at wi.mit.edu. Find past episodes of AudioHelicase and stay tuned for new ones by subscribing on iTunes and SoundCloud. Thanks for listening.

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Music: “Versailles” by Pierce Murphy (CC-BY 4.0), “The Little Robot” by Forget the Whale (CC-BY-NC 4.0)