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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.

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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

Biologists glean insight into repetitive protein sequences

A computational analysis reveals that many repetitive sequences are shared across proteins and are similar in species from bacteria to humans.

Anne Trafton | MIT News Office
September 13, 2022

About 70 percent of all human proteins include at least one sequence consisting of a single amino acid repeated many times, with a few other amino acids sprinkled in. These “low-complexity regions” are also found in most other organisms.

The proteins that contain these sequences have many different functions, but MIT biologists have now come up with a way to identify and study them as a unified group. Their technique allows them to analyze similarities and differences between LCRs from different species, and helps them to determine the functions of these sequences and the proteins in which they are found.

Using their technique, the researchers have analyzed all of the proteins found in eight different species, from bacteria to humans. They found that while LCRs can vary between proteins and species, they often share a similar role — helping the protein in which they’re found to join a larger-scale assembly such as the nucleolus, an organelle found in nearly all human cells.

“Instead of looking at specific LCRs and their functions, which might seem separate because they’re involved in different processes, our broader approach allows us to see similarities between their properties, suggesting that maybe the functions of LCRs aren’t so disparate after all,” says Byron Lee, an MIT graduate student.

The researchers also found some differences between LCRs of different species and showed that these species-specific LCR sequences correspond to species-specific functions, such as forming plant cell walls.

Lee and graduate student Nima Jaberi-Lashkari are the lead authors of the study, which appears today in eLife. Eliezer Calo, an assistant professor of biology at MIT, is the senior author of the paper.

Large-scale study

Previous research has revealed that LCRs are involved in a variety of cellular processes, including cell adhesion and DNA binding. These LCRs are often rich in a single amino acid such as alanine, lysine, or glutamic acid.

Finding these sequences and then studying their functions individually is a time-consuming process, so the MIT team decided to use bioinformatics netbet sports betting app— an approach that uses computational methods to analyze large sets of biological data — to evaluate them as a larger group.

“What we wanted to do is take a step back and instead of looking at individual LCRs, to try to take a look at all of them and to see if we could observe some patterns on a larger scale that might help us figure out what the ones that have assigned functions are doing, and also help us learn a bit about what the ones that don’t have assigned functions are doing,” Jaberi-Lashkari says.

To do that, the researchers used a technique called dotplot matrix, which is a way to visually represent amino acid sequences, to generate images of each protein under study. They then used computational image processing methods to compare thousands of these matrices at the same time.

Using this technique, the researchers were able to categorize LCRs based on which amino acids were most frequently repeated in the LCR. They also grouped LCR-containing proteins by the number of copies of each LCR type found in the protein. Analyzing these traits helped the researchers to learn more about the functions of these LCRs.

As one demonstration, the researchers picked out a human protein, known as RPA43, that has three lysine-rich LCRs. This protein is one of many subunits that make up an enzyme called RNA polymerase 1, which synthesizes ribosomal RNA. The researchers found that the copy number of lysine-rich LCRs is important for helping the protein integrate into the nucleolus, the organelle responsible for synthesizing ribosomes.

Biological assemblies

In a comparison of the proteins found in eight different species, the researchers found that some LCR types are highly conserved between species, meaning that the sequences have changed very little over evolutionary timescales. These sequences tend to be found in proteins and cell structures that are also highly conserved, such as the nucleolus.

“These sequences seem to be important for the assembly of certain parts of the nucleolus,” Lee says. “Some of the principles that are known to be important for higher order assembly seem to be at play because the copy number, which might control how many interactions a protein can make, is important for the protein to integrate into that compartment.”

The researchers also found differences between LCRs seen in two different types of proteins that are involved in nucleolus assembly. They discovered that a nucleolar protein known as TCOF contains many glutamine-rich LCRs that can help scaffold the formation of assemblies, while nucleolar proteins with only a few of these glutamic acid-rich LCRs could be recruited as clients (proteins that interact with the scaffold).

Another structure that appears to have many conserved LCRs is the nuclear speckle, which is found inside the cell nucleus. The researchers also found many similarities between LCRs that are involved in forming larger-scale assemblies such as the extracellular matrix, a network of molecules that provides structural support to cells in plants and animals.

The research team also found examples of structures with LCRs that seem to have diverged between species. For example, plants have distinctive LCR sequences in the proteins that they use to scaffold their cell walls, and these LCRs are not seen in other types of organisms.

The researchers now plan to expand their LCR analysis to additional species.

“There’s so much to explore, because we can expand this map to essentially any species,” Lee says. “That gives us the opportunity and the framework to identify new biological assemblies.”

The research was funded by the National Institute of General Medical Sciences, National Cancer Institute, the Ludwig Center at MIT, a National Institutes of Health Pre-Doctoral Training Grant, and the Pew Charitable Trusts.

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 NetBet sporta 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

Scientists identify a plant molecule that sops up iron-rich heme

The peptide is used by legumes to control nitrogen-fixing bacteria; it may also offer leads for treating patients with too much heme in their blood.

Anne Trafton | MIT News Office
August 11, 2022

Symbiotic relationships between legumes and the bacteria that grow in their roots are critical for plant survival. Without those bacteria, the plants would have no source of nitrogen, an element that is essential for building proteins and other biomolecules, and they would be dependent on nitrogen fertilizer in the soil.

To establish that symbiosis, some legume plants produce hundreds of peptides that help bacteria live within structures known as nodules within their roots. A new study from MIT reveals that one of these peptides has an unexpected function: It sops up all available heme, an iron-containing molecule. This sends the bacteria into an iron-starvation mode that ramps up their production of ammonia, the form of nitrogen that is usable for plants.

“This is the first of the 700 peptides in this system for which a really detailed molecular mechanism has been worked out,” says Graham Walker, the American Cancer Society Research Professor of Biology at MIT, a Howard Hughes Medical Institute Professor, and the senior author of the study.

This heme-sequestering peptide could have beneficial uses in treating a variety of human diseases, the researchers say. Removing free heme from the blood could help to treat diseases caused by bacteria or parasites that need heme to survive, such as P. gingivalis (periodontal disease) or toxoplasmosis, or diseases such as sickle cell disease or sepsis that release too much heme into the bloodstream.

“This study demonstrates that basic research in plant-microbe interactions also has potential to be translated to therapeutic applications,” says Siva Sankari, an MIT research scientist and the lead author of the study, which appears today in Nature Microbiology.

Other authors of the paper include Vignesh Babu, an MIT research scientist; Kevin Bian and Mary Andorfer, both MIT postdocs; Areej Alhhazmi, a former KACST-MIT Ibn Khaldun Fellowship for Saudi Arabian Women scholar; Kwan Yoon and Dante Avalos, MIT graduate students; Tyler Smith, an MIT instructor in biology; Catherine Drennan, an MIT professor of chemistry and biology and a Howard Hughes Medical Institute investigator; Michael Yaffe, a David H. Koch Professor of Science and a member of MIT’s Koch Institute for Integrative Cancer Research; and Sebastian Lourido, the Latham Family Career Development Professor of Biology at MIT and a member of the Whitehead Institute for Biomedical Research.

Iron control

For nearly 40 years, Walker’s lab has been studying the symbiosis between legumes and rhizobia, a type of nitrogen-fixing bacteria. These bacteria convert nitrogen gas to ammonia, a critical step of the Earth’s nitrogen cycle that makes the element available to plants (and to animals that eat the plants).

Most of Walker’s work has focused on a clover-like plant called Medicago truncatula. Nitrogen-fixing bacteria elicit the formation of nodules on the roots of these plants and eventually end up inside the plant cells, where they convert to their symbiotic form called bacteroids.

Several years ago, plant biologists discovered that Medicago truncatula produces about 700 peptides that contribute to the formation of these bacteroids. These peptides are generated in waves that help the bacteria make the transition from living freely to becoming embedded into plant cells where they act as nitrogen-fixing machines.

Walker and his students picked one of these peptides, known as NCR247, to dig into more deeply. Initial studies revealed that when nitrogen-fixing bacteria were exposed to this peptide, 15 percent of their genes were affected. Many of the genes that became more active were involved in importing iron.

The researchers then found that when they fused NCR247 to a larger protein, the hybrid protein was unexpectedly reddish in color. This serendipitous observation led to the discovery that NCR247 binds heme, an organic ring-shaped iron-containing molecule that is an important component of hemoglobin, the protein that red blood cells use to carry oxygen.

Further studies revealed that when NCR247 is released into bacterial cells, it sequesters most of the heme in the cell, sending the cells into an iron-starvation mode that triggers them to begin importing more iron from the external environment.

“Usually bacteria fine-tune their iron metabolism, and they don’t take up more iron when there is already enough,” Sankari says. “What’s cool about this peptide is that it overrides that mechanism and indirectly regulates the iron content of the bacteria.”

Nitrogenase, the main enzyme that bacteria use to fix nitrogen, requires 24 to 32 atoms of iron per enzyme molecule, so the influx of extra iron likely helps those enzymes to become more active, the researchers say. This influx is timed to coincide with nitrogen fixation, they found.

“These peptides are produced in a wave in the nodules, and the production of this particular peptide is higher when the bacteria are preparing to fix nitrogen. If this peptide was secreted throughout the whole process, then the cell would have too much iron all the time, which is bad for the cell,” Sankari says.

Without the NCR247 peptide, Medicago truncatula and rhizobium cannot form an effective nitrogen-fixing symbiosis, the researchers showed.

“Many possible directions”

The peptide that the researchers studied in this work may have potential therapeutic uses. When heme is incorporated into hemoglobin, it performs a critical function in the body, but when it’s loose in the bloodstream, it can kill cells and promote inflammation. Free heme can accumulate in stored blood, so having a way to filter out the heme before the blood is transfused into a patient could be potentially useful.

A variety of human diseases lead to free heme circulating in the bloodstream, including sickle cell anemia, sepsis, and malaria. Additionally, some infectious parasites and bacteria depend on heme for their survival but cannot produce it, so they scavenge it from their environment. Treating such infections with a protein that takes up all available heme could help prevent the parasitic or bacterial cells from being able to grow and reproduce.

In this study, Lourido and members of his lab showed that treating the parasite Toxoplasma gondii with NCR427 prevented the parasite from forming plaques on human cells.

The researchers are now pursuing collaborations with other labs at MIT to explore some of these potential applications, with funding from a Professor Amar G. Bose Research Grant.

“There are many possible directions, but they’re all at a very early stage,” Walker says. “The number of potential clinical applications is very broad. You can place more than one bet in this game, which is an intriguing thing.”

Currently, the human protein hemopexin, which also binds to heme, is being explored as a possible treatment for sickle cell anemia. The NCR247 peptide could provide an easier to deploy alternative, the researchers say, because it is much smaller and could be easier to manufacture and deliver into the body.

The research was funded in part by the MIT Center for Environmental Health Sciences, the National Science Foundation, and the National Institutes of Health.

The blueprint of a body
Eva Frederick | Whitehead Institute
July 20, 2022

Multicellular organisms evolved over millennia into a dazzling array of differently adapted NetBet live casinocreatures. With each generation, tiny worms, lavishly plumed birds, and even humans must create themselves anew from a single cell. To do so, they require a plan.

“How that multifunctional body plan is created is one of the deepest questions in developmental biology,” said Zak Swartz, until recently a postdoctoral researcher in the lab of Whitehead Institute Member Iain Cheeseman. “How do you take a single cell and pattern into a body that has different functions and features along it?”

Whitehead Institute researchers are tackling this question through a variety of different lenses. Researchers in Iain Cheeseman’s lab, including Swartz, have delved into the mysterious forces that underlie the polarity of an organism’s first cell. For the lab led by Pulin Li, research comes in at a later stage of development, when multiple cells combine to form a tissue and must communicate with each other to become an organized whole. Work on regeneration in Peter Reddien’s lab shows how some creatures can access their body blueprint throughout their lives to repair nearly any injury, and Yukiko Yamashita’s group studies how organisms pass on their body blueprints to their offspring through germ cells. Jonathan Weissman and his lab have created a “map” which researchers can use to find the function of a given gene, allowing them access to an organism’s most fundamental plans. Read on to learn about these scientists’ work, and more.

Laying out the plan

All multicellular organisms begin with a single cell, the fertilized egg. This cell has an essential role in setting out the body plan for the rest of an organism. It all starts with establishing polarity — in other words, figuring out which side of the cell is the top, and which is the bottom. This polarity establishes an axis of symmetry for the growing organism, and sets the stage for other developmental processes to come.

In a 2021 study, Cheeseman and postdoctoral researcher Zak Swartz investigated how one protein in specific, called Disheveled, localizes in a cell to help create this polarity in sea star embryos. Swartz found that Disheveled started out uniformly distributed in small aggregations throughout the egg cell, or oocyte. As the cell prepared to divide, Disheveled aggregations dissolved and then reformed at what would become the “bottom” of the oocyte.

Once the initial polarity is established, the oocyte can divide, creating a bilaterally symmetric sea star larvae. The burgeoning cluster of cells must then undergo other processes to define the several axes of symmetry that adult sea stars are known for.

Talking through it 

If an organism’s developmental blueprints are to be followed as development progresses, cells must be able to effectively communicate with each other. That cell to cell communication is the area of expertise of Whitehead Institute Member Pulin Li.

During her postdoctoral fellowship at the California Institute of Technology, Li studied tissue patterning — the mechanisms by which an organism’s newly forming tissues are laid out. Specifically, she investigated a developmental mechanism called morphogen gradient formation.

These gradients, composed of chemicals present in developing embryos, function as spatial coordinate systems and help determine how various cell types will be arranged in the organism — for example which groups of cells will form the liver, or the bones, or the brain, and where they will be within the body.

Li was able to recreate these gradients in the lab, in a Petri dish, and then interpret their signals using time lapse imaging and mathematical modeling. Here at Whitehead Institute, she follows a “bottom-up” approach to studying these complex systems. The best way to understand how something works, she says, is to build it yourself.

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 NetBet sportcell “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.”

New findings reveal how neurons build and maintain their capacity to communicate

Nerve cells regulate and routinely refresh the collection of calcium channels that enable them to send messages across circuit connections.

David Orenstein | Picower Institute for Learning and Memory
July 21, 2022

The nervous system works because neurons communicate across connections called synapses. They “talk” when calcium ions flow through channels into “active zones” that are loaded with vesicles carrying molecular messages. The electrically charged calcium causes vesicles to “fuse” to the outer membrane of presynaptic neurons, releasing their communicative chemical cargo to the postsynaptic cell. In a new study, scientists at The Picower Institute for Learning and Memory at MIT provide several revelations about how neurons set up and sustain this vital infrastructure.

“Calcium channels are the major determinant of calcium influx, which then triggers vesicle fusion, so it is a critical component of the engine on the presynaptic side that converts electrical signals to chemical synaptic transmission,” says Troy Littleton, senior author of the new study in eLife and Menicon Professor of Neuroscience in MIT’s departments of Biology and Brain and Cognitive Sciences. “How they accumulate at active zones was really unclear. Our study reveals clues into how active zones accumulate and regulate the abundance of calcium channels.”

Neuroscientists have wanted these clues. One reason is that understanding this process can help reveal how neurons change how they communicate, an ability called “plasticity” that underlies learning and memory and other important brain functions. Another is that drugs such as gabapentin, which treats conditions as diverse as epilepsy, anxiety, and nerve pain, binds a protein called alpha2delta that is closely associated with calcium channels. By revealing more about alpha2delta’s exact function, the study better explains what those treatments affect.

“Modulation of the function of presynaptic calcium channels is known to have very important clinical effects,” Littleton says. “Understanding the baseline of how these channels are regulated is really important.”

MIT postdoc Karen Cunningham led the study, which was her doctoral thesis work in Littleton’s lab. Using the model system of fruit fly motor neurons, she employed a wide variety of techniques and experiments to show for the first time the step-by-step process that accounts for the distribution and upkeep of calcium channels at active zones.

A cap on Cac

Cunningham’s first question was whether calcium channels are necessary for active zones to develop in larvae. The fly calcium channel gene (called “cacophony,” or Cac) is so important, flies literally can’t live without it. So rather than knocking out Cac across the fly, Cunningham used a technique to knock it out in just one population of neurons. By doing so, she was able to show that even without Cac, active zones grow and mature normally.

Using another technique that artificially prolongs the larval stage of the fly she was also able to see that given extra time the active zone will continue to build up its structure with a protein called BRP, but that Cac accumulation ceases after the normal six days. Cunningham also found that moderate increases or decreases in the supply of available Cac in the neuron did not affect how much Cac ended up at each active zone. Even more curious, she found that while Cac amount did scale with each active zone’s size, it barely budged if she took away a lot of the BRP in the active zone. Indeed, for each active zone, the neuron seemed to enforce a consistent cap on the amount of Cac present.

“It was revealing that the neuron had very different rules for the structural proteins at the active zone like BRP that continued to accumulate over time, versus the calcium channel that was tightly regulated and had its abundance capped” Cunningham says.

Regular refresh

The findings showed there must be factors other than Cac supply or changes in BRP that regulate Cac levels so tightly. Cunningham turned to alpha2delta. When she genetically manipulated how much of that was expressed, she found that alpha2delta levels directly determined how much Cac accumulated at active zones.

In further experiments, Cunningham was also able to show that alpha2delta’s ability to maintain Cac levels depended on the neuron’s overall Cac supply. That finding suggested that rather than controlling Cac amount at active zones by stabilizing it, alpha2delta likely functioned upstream, during Cac trafficking, to supply and resupply Cac to active zones.

Cunningham used two different techniques to watch that resupply happen, producing measurements of its extent and its timing. She chose a moment after a few days of development to image active zones and measure Cac abundance to ascertain the landscape. Then she bleached out that Cac fluorescence to erase it. After 24 hours, she visualized Cac fluorescence anew to highlight only the new Cac that was delivered to active zones over that 24 hours. She saw that over that day there was Cac delivery across virtually all active zones, but that one day’s work was indeed only a fraction compared to what had built up over several days before. Moreover, she could see that the larger active zones accrued more Cac than smaller ones. And in flies with mutated alpha2delta, there was very little new Cac delivery at all.

If Cac channels were indeed constantly being resupplied, then Cunningham wanted to know at what pace Cac channels are removed from active zones. To determine that, she used a staining technology with a photoconvertible protein called Maple tagged to the Cac protein that allowed her to change the color with a flash of light at the time of her choosing. That way she could first see how much Cac accumulated by a certain time (shown in green) and then flash the light to turn that Cac red. When she checked back five days later, about 30 percent of the red Cac had been replaced with new green Cac, suggesting 30 percent turnover. When she reduced Cac delivery levels by mutating alpha2 delta or reducing Cac biosynthesis, Cac turnover stopped. That means a significant amount of Cac is turned over each day at active zones and that the turnover is prompted by new Cac delivery.

Littleton says his lab is eager to build on these results. Now that the rules of calcium channel abundance and replenishment are clear, he wants to know how they differ when neurons undergo plasticity — for instance, when new incoming information requires neurons to adjust their communication to scale up or down synaptic communication. He says he is also eager to track individual calcium channels as they are made in the cell body and then move down the neural axon to the active zones, and he wants to determine what other genes may affect Cac abundance.

In addition to Cunningham and Littleton, the paper’s other authors are Chad Sauvola and Sara Tavana.

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

Yiyin Erin Chen and Sam Chunte Peng named as core members of Broad Institute and MIT
Broad Communications
July 12, 2022

Broad Institute of MIT and Harvard has named Erin Chen, a dermatologist and microbiologist, and Sam Peng, a biophysicist and physical chemist with expertise in single-molecule imaging, as core institute members.

Chen will join in January 2023 and will also serve as an assistant professor in the Department of Biology at MIT and an attending dermatologist at Massachusetts General Hospital. Peng joined in July 2022 and will serve as an assistant professor in the Department of Chemistry at MIT.

Chen’s lab will study the communication between the immune system and the diverse microbes that colonize every surface of the human body, with a focus on the human body’s largest organ, the skin.

Peng’s lab will develop novel probes and microscopy techniques to visualize the dynamics of individual molecules in living cells, which will improve the understanding of molecular mechanisms underlying human diseases.

“We are delighted to welcome Sam and Erin to the Broad community,” said Todd Golub, director of the Broad. “These creative scientists are each taking inventive approaches to understand the molecular signals and interactions that underlie biological processes in health and disease. These insights will help further the Broad’s mission of advancing the understanding and treatment of human disease.”

Erin Chen.
Erin Chen

Erin Chen earned her BA in biology from the University of Chicago, her PhD from MIT, and her MD from Harvard Medical School. Prior to joining the Broad, Chen was a Howard Hughes Medical Institute Hanna Gray Postdoctoral Fellow at Stanford University, in the lab of Michael Fischbach. She was also an attending dermatologist at the University of California San Francisco and at the San Francisco VA Medical Center. During her postdoctoral research, Chen developed netbet sports betting appgenetic methods to study harmless commensal skin bacteria. She engineered these bacteria to generate anti-tumor immunity, pioneering a novel approach to vaccination and cancer immunotherapy.

At the Broad, members of the Chen lab will continue to employ microbial genetics, immunologic approaches, and mouse models to dissect the molecular signals used by commensal microbes to educate the immune system. Ultimately, Chen aims to harness these microbe-host interactions to engineer novel therapeutics for human disease.

“I’m excited to join the collaborative scientific community at the Broad and MIT, including those who have pioneered novel tools for examining biological mechanisms at higher spatial resolution,” said Chen. “The biology I study is quite basic, but I’m motivated by the potential impact it could have on patients. Figuring out how commensal skin bacteria are captured by the immune system could unlock a whole new therapeutic toolbox.”

Sam Peng
Sam Peng

Sam Peng earned his BS in chemistry from the University of California, Berkeley, and his PhD from MIT in physical chemistry. He completed his postdoctoral research at Stanford University as an NIH K99 Pathway to Independence scholar in the lab of Steve Chu. During his postdoctoral research, he developed long-term single molecule imaging in live cells using a novel class of nanoprobes. He applied this new technique to study axonal transport in neurons and the molecular dynamics of dynein — a motor protein involved in transporting cargo in cells.

At the Broad, the Peng group will aim to elucidate the molecular mechanisms underlying human diseases. Lab members will develop and integrate a diverse toolbox spanning single-molecule microscopy, super-resolution microscopy, spectroscopy, nanomaterial engineering, biophysics, chemical biology, and quantitative modeling to uncover previously unexplored biological processes. With bright and photostable probes, lab members will have unprecedented capability to record ultra-long-term “molecular movies” in living systems with high spatiotemporal resolutions and to reveal molecular interactions that drive biological functions. Peng’s group will focus on studying molecular dynamics, protein-protein interactions, and cellular heterogeneity involved in neurobiology and cancer biology. Their long-term goal is to translate these mechanistic insights into drug discovery.

“Because my research is so multi-disciplinary, joining the Broad and MIT communities allows us to integrate a range of experimental tools and to collaborate with colleagues and students from diverse backgrounds,” said Peng. “I’m excited to see how our techniques can enable discoveries for a variety of cellular processes, including those underlying complex brain functions and dysfunctions. Many problems that previously seemed inaccessible now appear to be within reach in the foreseeable future.”

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 netbet sports betting applevels 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.”