Research Area: Cell Biology
March 30, 2020
Greta Friar | Whitehead Institute
February 25, 2020
Meet the stars
Zak Swartz, a postdoctoral researcher in Whitehead Institute Member Iain Cheeseman’s lab, gets an unusual delivery a few times a year. It comes in a cardboard box a few feet in length on each side with a “perishable” label on top. When the most recent box arrived, Swartz took it to a small, chilly room across from Cheeseman’s lab. He cut and tore his way through several layers of packaging, insulation, and cold packs to get to his prize: a bunch of plastic bags half-filled with water, the sort of thing that might contain a goldfish at a county fair. Instead of goldfish, these makeshift aquaria each contained two or three bat stars (Patiria miniata), a hardy species of starfish seemingly unphased by their transcontinental trip in a cardboard box.
Bat stars are so named because the thick webbing between their arms—of which they typically have five, though they can have up to nine—gives their short limbs a bat-wing-like appearance. The stars are most often some shade of red or orange, but come in a variety of colors and patterns. Each star that Swartz pulled out of the box had a unique design coating its body.
Swartz untied the plastic bags and took the bat stars out one by one. He gently dropped each star into an aquarium in the corner of the room, where it would sink slowly to the bottom, then crawl to the sides and inch its way up, clinging to the glass. Although their movements are barely perceptible to a human watching, within minutes bat stars coated the walls of the aquarium, the tiny tube feet on the underside of each arm sticking them firmly in place.
As the first round of bat stars settled onto their chosen perches, Swartz returned to the box. He took another bat star out of its bag and held it in his hand for a moment.
“This one’s heavy,” he said, with satisfaction; heavy stars are more likely to be full of eggs, and that’s what Swartz is interested in. He’s researching how cells, such as immature egg cells, that remain dormant or non-dividing for a long time retain their ability to divide. The proteins necessary for cell division degrade over time, like parts of a machine rusting and breaking down, and yet many cells remain able to divide long after their unused cellular machinery should have become useless. In the case of humans, precursor egg cells can spring back into action after decades of dormancy in the ovaries, and can go on to perform that most impressive feat of cell division: the creation of a whole new organism from one cell. But human eggs are not the most accessible or readily available research material, and so Swartz has turned to the bat stars, an excellent source of reproductive cells, to help answer his questions.
Fertile ground for discovery
Bats stars reproduce by spawning. The females release millions of eggs into the ocean through pores in between their arms, while at the same time males release clouds of sperm. The reproductive characteristics of bat stars make them ideal research animals for Swartz. They have a long breeding season during which they can ovulate, they produce millions of eggs at a time, and they release these eggs out into their environment, where they develop externally.
In the lab, Swartz must extract the immature egg cells before they are released, so he can study the processes that take place in the cells during their development. This is much easier than extracting cells from mammalian ovaries; all it requires is a minimally invasive procedure from which the starfish quickly recover. Once Swartz has extracted the eggs in their “hibernating” pre-spawn state, he can control and observe all of the steps of their development, from their re-activation through to fertilization and beyond.
In the wild, fertilized bat star eggs develop into embryos, which quickly become tiny, transparent larvae that swim freely and live as plankton. These larvae have strong regenerative capabilities: if a bat star larva is cut in half, each half can regrow the missing parts of its body. Some species of starfish maintain robust regenerative capacity into their adult stage.
Unlike the adult stage of the bat star, the larvae have bilateral symmetry, with mirroring left and right sides, just like humans. Bat stars develop through several stages as bilateral larva, becoming a bipinnaria and then a brachiolaria. These stages are transparent, their insides easily visible—a good feature for research subjects. Only when the stars metamorphose into their juvenile and then adult forms do they assume their familiar, five-point radially symmetric shape.
Bat stars share a common ancestor with mammals, and bat star and human embryonic development are similar enough for the former to be a useful model for the latter in research like Swartz’s. In fact, bat stars belong to the phylum Echinodermata, a group of animals also including sea urchins, sea cucumbers, sand dollars and others, that have historically proved to be important tools for research into reproduction. The first observation of sperm fertilizing an egg occurred in transparent sea urchin eggs, which helped solve the mystery of how each sex’s gametes contribute to sexual reproduction.
One reason Swartz has chosen bat stars over other starfish species is because of their hardiness. Bat stars fare well in a laboratory aquarium—they even do well being shipped in a cardboard box—whereas other species that Swartz considered using are less adaptable to these conditions. So, while it may have been more convenient to use a species from local Atlantic waters, in the interest of maintaining a healthy lab population of specimens, Swartz has had to procure his research subjects long distance. The geographical range of bat stars is the stretch of Pacific Ocean along the coast of North America.
How are the animals getting from the Pacific coast to Whitehead Institute? They are collected by a contact Swartz made several years ago in California.
Scuba diving for science
Josh Ross runs a research specimen procurement company based in San Pedro, California called South Coast Bio-Marine. Ross has been collecting starfish for Swartz since 2015, and he also collects a variety of marine animals, from sea urchins to limpets to nudibranchs, for researchers at other institutions. Specimens from South Coast Bio-Marine have been used in research on, among other topics, fertilization, memory formation, sleep, and shape changes in oocytes.
Most mornings, Ross and his employees load up a boat with scuba gear and equipment and head to their chosen dive spots, where they collect specimens for the first half of the day.
“I love the fact that I get to dive and work in the ocean every day. When Monday comes, all the weekend boaters and fishermen go back to their jobs, and we have the ocean almost all to ourselves. We are out in the wilderness with truly wild ocean creatures,” Ross says—though sometimes those wild creatures can interfere with their collection plans.
The team then take what they have collected back to Ross’ lab, where the specimens are kept in chilled seawater tanks for a few days to acclimate before Ross ships them to researchers. Ross takes care to harvest specimens with sustainability in mind. When finding starfish for Swartz, he will only take animals that have had time to grow large and spawn several times in their native habitat before being collected.
Ross searches for bat stars at dive sites in 55- to 70-foot deep water, either out in the open, on rocky shelf reefs, or on the sand near the reefs. Bat stars’ habitat ranges from the low intertidal zone, the part of the seashore that’s covered with water except at low tide, into the mild depths of the subtidal zone. They live among kelp and surf grass forest, and use the numerous tube feet lining the underside of each arm to crawl across the sandy ocean floor and cling to rocks. Instead of blood, starfish pump sea water through a water vascular system to circulate nutrients through their bodies and control their limbs. They pump water into and out of their tube feet to make them extend and contract, allowing the stars to move. The tube feet can also release glue-like chemicals that help the stars adhere to rocks even in the strong currents of the ocean—or cling to glass walls in aquariums.
Bat stars are voracious eaters—scavengers as well as predators—and Ross often finds them in the middle of a meal. Bat stars eat by extending their stomachs out of their bodies to dissolve their prey in digestive juices, then drink it up. This system allows them to eat larger prey than their small mouths would otherwise allow. A second stomach that remains inside the star further digests the food.
Ross tries to select starfish for Swartz that feel “ripe,” meaning they are ready or nearly ready to spawn. Indicators that Ross uses include ripe stars having larger “shoulders” and being puffier than unripe stars. After Ross has collected enough bat stars, he ships them from California to Cambridge, Massachusetts, where their role in Swartz’ research begins. Swartz is interested in female specimens, but there is no good way to identify a starfish’s sex on sight, so the bat stars that Ross sends to Swartz tend to be fifty-fifty female and male. Swartz must examine a small biopsy of the gonad to find out which of them contain oocytes, the immediate precursor cells to fertilizable eggs. Once identified, the animals are separated into two aquariums by sex, ready to provide oocytes for experiments. Swartz feeds the starfish a steady diet primarily consisting of raw, peeled shrimp, which keeps them developing new oocytes. When the starfish have completed their time in the lab, Swartz tries to donate them to local aquariums.
Eggs with answers: What we’ve learned from bat stars
Swartz is using the bat stars to investigate how cells divide—specifically, how cells retain the ability to divide after long periods without doing so, and how cell division processes are adapted to the context of animal reproduction and development. Whitehead Member Iain Cheeseman’s lab, where Swartz is a postdoc, investigates the cellular machinery required for cell division. In particular, Cheeseman’s team studies the kinetochore, a complex of proteins involved in orchestrating the precise segregation of chromosomes during cell division, and the centromere, the region in the middle of the chromosome where the kinetochore assembles. The centromere is not defined by its DNA sequence, but by proteins that attach there and signal the kinetochore to assemble at that location. One of the necessary proteins that marks the centromere is called CENP-A. Without CENP-A, the centromere won’t function properly, so chromosomes won’t be correctly distributed into the two new cells created during cell division. However, as with other proteins, there was an open question whether CENP-A degrades over time. Once it is lost at the centromere the cell cannot get it back, and loses the ability to divide. This fact caused Cheeseman and Swartz to wonder how cells that spend long periods of time without dividing can start up again. What sort of maintenance do cells need to do to keep their cell division machinery operational?
Eggs and oocytes, the precursor cells that will develop into eggs, are a great test case because they remain non-dividing for a very long time. Swartz harvested the bat stars’ oocytes and used a fluorescent tag to track the quantity of CENP-A inside of the cells as they progressed through their cell cycle stages. To get a close look at what happens to the CENP-A in oocytes during their dormancy, Swartz maintained the cells in a state of arrested development in petri dishes by putting them in a mixture he calls “starfish juice,” a blend of culture fluids, antibiotics, and some of the bat stars’ own natural fluids.
With the help of the fecund bat stars, Swartz and Cheeseman found the answer to their questions about CENP-A. In research published in Developmental Cell in 2019 [4], the scientists discovered that cells slowly replace their CENP-A over time, swapping out the old protein at risk of breaking down with new functional protein. This finding upended the previous understanding of CENP-A as a static protein that was placed on the centromere once and then remained as long as it could. The researchers also tested a human cell line that can enter dormancy and divide later, and found that, like the sea star oocytes, those cells gradually exchanged CENP-A. In contrast, the researchers discovered that cell types that never need to divide again, like muscle cells or other specialized cells, let most of their CENP-A degrade and so permanently lose the capacity to divide. This finding means that the presence of CENP-A may be a good indicator for use in determining whether any given cell retains the ability to divide in the future. The question of a specialized, or terminally differentiated, cell’s potential for renewed cell division is of great interest in regenerative medicine research. Indeed, this work sparked collaboration between Cheeseman, Swartz and Whitehead Institute Fellow Kristin Knouse, who studies regeneration in mouse and human cells.
The findings could also explain why tissues like muscle rarely develop cancers; the cells cannot replicate and so cannot grow tumors. Furthermore, Swartz thinks that their findings could prove valuable for assisted fertility research.
“Understanding the natural biology that keeps eggs in good shape, able to resume and finish their development after long dormancies, could provide insight into what goes wrong when eggs do not remain viable,” Swartz says.
The possibilities for future research spawning from Swartz’ work are many. The advances that may come, whether in regenerative medicine, assisted fertility, or elsewhere, will all be owed in part to a group of bat stars that travelled across a continent, from ocean to ocean, in a chilled cardboard box to help unravel the mysteries of cell division.
Raleigh McElvery
February 26, 2020
After decades of speculation, researchers have demonstrated that a classical DNA repair enzyme also binds to RNA, affecting blood cell development.
The DNA-dependent protein kinase, otherwise known as DNA-PK, is one of the most important enzymes that binds DNA and repairs double-stranded breaks. This mode of repair is essential for generating receptors that help the immune system fight off intruders. But DNA-PK doesn’t just bind DNA; it also binds RNA. Although researchers have known this for decades, they didn’t fully understand what kinds of RNAs DNA-PK bound in mammalian cells, or the physiological consequences of this binding.
In a new study published on February 26 in Nature, researchers from MIT and Columbia University have uncovered a mechanism whereby DNA-PK binds to the RNA involved in ribosome assembly. Ribosomes — the cell’s protein synthesis machinery — ensure that stem cells give rise to enough red blood cells. The researchers found that mutating DNA-PK prevents the ribosomes from being built properly, which prevents blood cells from doing their job and leads to blood disorders.
“This is the first biochemical evidence of DNA-PK assembly and activation by RNA inside cells,” says Eliezer Calo, a co-senior author and assistant professor in MIT’s Department of Biology. “We’re still trying to determine the mechanisms that regulate protein synthesis in stem cells, and this study reveals one of them.”
Co-senior author, Shan Zha from Columbia University, had previously studied DNA-PK’s role in DNA repair by generating a mouse model that carried enzymatically-dead versions of DNA-PK. While using this model to investigate tumorigenesis, Zha’s lab found these mutant mice developed a form of blood cancer known as myeloid disease. At the same time, another research group showed that mutations in DNA-PK also led to anemia, which occurs when the body does not have enough healthy red blood cells
Neither myeloid disease nor anemia could be easily explained by DNA repair defects alone. However, the two blood disorders did share some similarities to diseases caused by ribosome defects. Because DNA-PK resides in the same organelle where ribosomes are made, the Zha and Calo labs began to wonder whether DNA-PK could bind to the RNA there and control ribosome biogenesis.
In this new study, the Zha lab found that DNA-PK mutations impaired protein translation in red blood cell progenitors, which might contribute to anemia. In parallel, the Calo lab was investigating ribosomal RNA processing and was surprised to find that DNA-PK seemed to be implicated in ribosome assembly. The Calo lab then mapped all the RNAs in cells that bind DNA-PK. The enzyme unexpectedly attached to U3, a small RNA that helps assemble one of the subunits comprising the ribosome. Once it binds U3, DNA-PK can transfer a phosphate group to several specific sites on one of its own subunits. If DNA-PK is defective and cannot transfer the phosphate group, protein synthesis in blood stem cells is impaired, eventually causing anemia.
DNA-PK is essential for cellular viability in nearly all human cell lines, including cancer cell lines, while many other proteins involved in same DNA repair pathway are dispensable. Several studies, including one published by the Zha lab, showed that DNA-PK protein levels are 50-fold higher in common human cell lines than in rodent cell lines. The researchers do not yet know why the enzyme is so critical, but they suspect it might have to do with its ability to bind RNA. “We are interested in exploring whether this new role for DNA-PK could provide clues to this puzzle,” Zha says.
Calo says their findings could also have important implications for cancer treatment, because DNA-PK has emerged as a promising target for cancer therapy. Drugs that inhibit DNA-PK could prevent cancer cells from repairing their DNA and replicating successfully, but he warns these same remedies could also impact stem cell function. The next step is to explore DNA-PK’s other RNA binding targets and the related molecular pathways.
“We’ve demonstrated that DNA-PK has an entirely separate role that has nothing to do with DNA repair,” Calo says. “In the future, we’re excited to learn what additional RNA-related duties it may have beyond stem cell maintenance.”
Top Image: Ribosomes are assembled in the nucleoli (shown here in human cells).
Citation:
“DNA-PKcs has KU-dependent function in rRNA processing and haematopoiesis”
Nature, online February 26, 2020, DOI: 10.1038/s41586-020-2041-2
Zhengping Shao, Ryan A. Flynn, Jennifer L. Crowe, Yimeng Zhu, Jialiang Liang, Wenxia Jiang, Fardin Aryan, Patrick Aoude, Carolyn R. Bertozzi, Verna M. Estes, Brian J. Lee, Govind Bhagat, Shan Zha, and Eliezer Calo
Greta Friar | Whitehead Institute
January 16, 2020
Toxoplasma gondii (T. gondii) is a parasite that chronically infects up to a quarter of the world’s population, causing toxoplasmosis, a disease that can be dangerous, or even deadly, for the immunocompromised and for developing fetuses. One reason that T. gondii is so pervasive is that the parasites are tenacious occupants once they have infected a host. They can transition from an acute infection stage into a quiescent life cycle stage and effectively barricade themselves inside of their host’s cells. In this protected state, they become impossible to eliminate, leading to long term infection. Researchers used to think that a combination of genes were involved in triggering the parasite’s transition into its chronic stage, due to the complexity of the process and because a gene essential for differentiation had not been identified. However, new research from Whitehead Institute Member Sebastian Lourido, who is also an assistant professor of biology at the Massachusetts Institute of Technology (MIT), and graduate student Benjamin Waldman has identified a sole gene whose protein product is the master regulator, which is both necessary and sufficient for the parasites to make the switch. Their findings, which appear online in the journal Cell on January 16, illuminate an important aspect of the parasite’s biology and provide researchers with the tools to control whether and when T. gondii transitions, or undergoes differentiation. These tools may prove valuable for treating toxoplasmosis, since preventing the parasites from assuming their chronic form keeps them susceptible to both treatment and elimination by the immune system.
T. gondii spreads when a potential host, which can be any warm-blooded animal, ingests infected tissue from another animal—in the case of humans, by eating undercooked meat or unwashed vegetables—or when the parasite’s progeny are shed by an infected cat, T. gondii’s target host for sexual reproduction. When T. gondii parasites first invade the body, they are in a quickly replicating part of their life cycle, called the tachyzoite stage. Tachyzoites invade a cell, isolate themselves by forming a sealed compartment from the cell’s membrane, and then replicate inside of it until the cell explodes, at which point they move on to another cell to repeat the process. Although the tachyzoite stage is when the parasites do the most damage, it’s also when they are easily targetable by the immune system and medical therapies. In order for the parasites to make their stay more permanent, they must differentiate into bradyzoites, a slow-growing stage, during which they are less susceptible to drugs and have too little effect on the body to trigger the immune system. Bradyzoites construct an extra thick wall to isolate their compartment in the host cell and encyst themselves inside of it. This reservoir of parasites remains dormant and undetectable, until, under favorable conditions, they can spring back into action, attacking their host or spreading to new ones.
Although the common theory was that multiple genes collectively orchestrate the transition from tachyzoite to bradyzoite, Lourido and Waldman suspected that there was instead a single master regulator.
“Differentiation is not something a parasite wants to do halfway, which could leave them vulnerable,” Waldman says. “Multiple genes means more chances for things to go wrong, so you would want a master regulator to ensure that differentiation happens cleanly.”
To investigate this hypothesis, Waldman used CRISPR-based screens to knock out T. gondii genes, and then tested to see if the parasite could still differentiate from tachyzoite to bradyzoite. Waldman monitored whether the parasites were differentiating by developing a strain of T. gondii that fluoresces in its bradyzoite stage. The researchers also performed a first of its kind single-cell RNA sequencing of T. gondii in collaboration with members of Alex Shalek’s lab in the department of chemistry at MIT. This sequencing allowed the researchers to profile the genes’ activity at each stage in unprecedented detail, shedding light on changes in gene expression during the parasite’s cell-cycle progression and differentiation.
The experiments identified one gene, which the researchers named Bradyzoite-Formation Deficient 1 (BFD1), as the only gene both sufficient and necessary to prevent the transition from tachyzoite to bradyzoite: the master regulator. Not only was T. gondii unable to make the transition without the BFD1 protein, but Waldman found that artificially increasing its production induced the parasites to become bradyzoites, even without the usual stress triggers required to cue the switch. This means that the researchers can now control Toxoplasma differentiation in the lab.
These findings may inform research into potential therapies for toxoplasmosis, or even a vaccine.
“Toxoplasma that can’t differentiate is a good candidate for a live vaccine, because the immune system can eliminate an acute infection very effectively,” Lourido says.
The researchers’ findings also have implications for food production. T. gondii and other cyst-forming parasites that use BFD1 can infect livestock. Further research into the gene could inform the development of vaccines for farm animals as well as humans.
“Chronic infection is a huge hurdle to curing many parasitic diseases,” Lourido says. “We need to study and figure out how to manipulate the transition from the acute to chronic stages in order to eradicate these diseases.”
This study was supported by an NIH Director’s Early Independence Award (1DP5OD017892), a grant from the Mathers Foundation, the Searle Scholars Program, the Beckman Young Investigator Program, a Sloan Fellowship in Chemistry, the NIH (1DP2GM119419, 2U19AI089992, 5U24AI118672), and the Bill and Melinda Gates Foundation.
Written by Greta Friar
***
Sebastian Lourido’s primary affiliation is with Whitehead Institute for Biomedical Research, where his laboratory is located and all his research is conducted. He is also an assistant professor of biology at the Massachusetts Institute of Technology.
***
Citation:
“Identification of a master regulator of differentiation in Toxoplasma”
Cell, online January 16, DOI: 10.1016/j.cell.2019.12.013
Benjamin S. Waldman (1,2), Dominic Schwarz (1,3), Marc H. Wadsworth II (4,5,6), Jeroen P. Saeij (7), Alex K. Shalek (4,5,6), Sebastian Lourido (1,2)
1. Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA
2. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA
3. Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany
4. Institute for Medical Engineering & Science (IMES), Department of Chemistry, and Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
5. Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
6. Ragon Institute of MGH, MIT and Harvard, Cambridge, MA 02319, USA
7. Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, Davis, CA 95616, USA
Biologists devise an efficient method to prepare fluorescently tagged proteins and simulate their native environment.
Raleigh McElvery | Department of Biology
December 18, 2019
All cells have a lipid membrane that encircles their internal components — forming a protective barrier to control what gets in and what stays out. The proteins embedded in these membranes are essential for life; they help facilitate nutrient transport, energy conversion and storage, and cellular communication. They are also important in human disease, and represent around 60 percent of approved drug targets. In order to study these membrane proteins outside the complexity of the cell, researchers must use detergent to strip away the membrane and extract them. However, determining the best detergent for each protein can involve extensive trial and error. And, removing a protein from its natural environment risks destabilizing the folded structure and disrupting function.
In a study published on Dec. 9 in Cell Chemical Biology, scientists from MIT devised a rapid and generalizable way to extract, purify, and label membrane proteins for imaging without any detergent at all — bringing along a portion of the surrounding membrane to protect the protein and simulate its natural environment. Their approach combines well-established chemical and biochemical techniques in a new way, efficiently isolating the protein so it can be fluorescently labeled and examined under a microscope.
“I always joke that it’s not very lifelike to study proteins in soap,” says senior author Barbara Imperiali, a professor of biology and chemistry. “We’ve created a workflow that allows membrane proteins to be imaged while maintaining their native identities and interactions. Hopefully now fewer people will shy away from studying membrane proteins, given their importance in many physiological processes.”
As a member of the Imperiali lab, former postdoc and lead author Jean-Marie Swiecicki investigated membrane proteins from the foodborne pathogen Campylobacter jejuni. In this study, Swiecicki focused on PglC and PglA, two membrane proteins that play a role in enabling the bacteria to infect human cells. His experiments required labeling PglC and PglA with fluorescent tags in order to track them. However, he wasn’t satisfied with existing methods to do so.
In some cases, the fluorescent tags that must be incorporated into the protein in order to visualize it are too large to be placed at defined positions. In other cases, these tags don’t shine brightly enough, or interfere with the structure and function of the protein.
To avoid such issues, Swiecicki decided to use a method known as “unnatural amino-acid mutagenesis.” Amino acids are the units that compose the protein, and unnatural amino-acid mutagenesis involves adding a new amino acid containing an engineered chemical group within the protein sequence. This chemical group can then be labeled with a brightly glowing tag.
Swiecicki inserted the genetic code for the C. jejuni membrane proteins into a different bacterium, Escherichia coli. Inside E. coli, he could incorporate the unnatural amino acid, which could be chemically modified to add the fluorescent label.
When it came time to remove the proteins from the membrane, he substituted a different substance for the detergent: a polymer of styrene-maleic acid (SMA). Unlike detergent, SMA wraps the extracted protein and a small segment of the associated membrane in a protective shell, preserving its native environment. Imperiali explains, “It’s like a scarf protecting your neck from the cold.”
Swiecicki could then monitor the glowing proteins under a microscope to verify his technique was selective enough to isolate individual membrane proteins. The entire process, he says, takes just a few days, and is generally much faster and more reliable than detergent-based extraction methods, which can take months and require the expertise of highly-trained biochemists to optimize.
“I wouldn’t say it’s a magic bullet that’s going to work for every single protein,” he says. “But it’s a highly efficient tool that could make it easier to study many different kinds of membrane proteins.” Eventually, he says, it may even help facilitate high throughput drug screens.
“As someone who works on membrane protein complexes, I can attest to the great need for better methods to study them,” says Suzanne Walker, a professor of microbiology at Harvard Medical School who was not involved in the study. She hopes to extend the approach outlined in the paper to the protein complexes she investigates in her own lab. “I appreciated the extensive detail included in the text about how to apply the strategy successfully,” she adds.
The next steps will be testing the technique on mammalian proteins, and isolating multiple proteins at once in the SMA shell to observe their interactions. And, of course, every new technique deserves a name. “We’re still working on a catchy acronym,” Imperiali says. “Any ideas?”
This research was funded by the Jane Coffin Childs Memorial Fund for Medical Research, Philippe Foundation, and National Institutes of Health.
Biologists uncover an evolutionary trick to control gene expression that reverses the flow of genetic information from RNA splicing back to transcription.
Raleigh McElvery | Department of Biology
December 9, 2019
Sometimes, unexpected research results are simply due to experimental error. Other times, it’s the opposite — the scientists have uncovered a new phenomenon that reveals an even more accurate portrayal of our bodies and our universe, overturning well-established assumptions. Indeed, many great biological discoveries are made when results defy expectation.
A few years ago, researchers in the Burge lab were comparing the genomic evolution of several different mammals when they noticed a strange pattern. Whenever a new nucleotide sequence appeared in the RNA of one lineage, there was generally an increase in the total amount of RNA produced from the gene in that lineage. Now, in a new paper, the Burge lab finally has an explanation, which redefines our understanding of how genes are expressed.
Once DNA is transcribed into RNA, the RNA transcript must be processed before it can be translated into proteins or go on to serve other roles within the cell. One important component of this processing is splicing, during which certain nucleotide sequences (introns) are removed from the newlymade RNA transcript, while others (the exons) remain. Depending on how the RNA is spliced, a single gene can give rise to a diverse array of transcripts.
Given this order of operations, it makes sense that transcription affects splicing. After all, splicing cannot occur without an RNA transcript. But the inverse theory — that splicing can affect transcription — is now gaining traction. In a recent study, the Burge lab showed that splicing in an exon near the beginning of a gene impacts transcription and increases gene expression, offering an explanation for the patterns in their previous findings.
“Rather than Step A impacting Step B, what we found here is that Step B, splicing, actually feeds back to influence Step A, transcription,” says Christopher Burge, senior author and professor of biology. “It seems contradictory, since splicing requires transcription, but there is actually no contradiction if — as in our model — the splicing of one transcript from a gene influences the transcription of subsequent transcripts from the same gene.”
The study, published on Nov. 28 in Cell, was led by Burge lab postdoc Ana Fiszbein.
Promoting gene expression
In order for transcription to begin, molecular machines must be recruited to a special sequence of DNA, known as the promoter. Some promoters are better at recruiting this machinery than others, and therefore initiate transcription more often. However, having different promoters available to produce slightly different transcripts from a gene helps boost expression and generates transcript diversity, even before splicing occurs mere seconds or minutes later.
At first, Fiszbein wasn’t sure how the new exons were enhancing gene expression, but she theorized that new promoters were involved. Based on evolutionary data available and her experiments at the lab bench, she could see that wherever there was a new exon, there was usually a new promoter nearby. When the exon was spliced in, the new promoter became more active.
The researchers named this phenomenon “exon-mediated activation of transcription starts” (EMATS). They propose a model in which the splicing machinery associated with the new exon recruits transcription machinery to the vicinity, activating transcription from nearby promoters. This process, the researchers predict, likely helps to regulate thousands of mammalian genes across species.
A more flexible genome
Fiszbein believes that EMATS has increased genome complexity over the course of evolution, and may have contributed to species-specific differences. For instance, the mouse and rat genomes are quite similar, but EMATS could have helped produce new promoters, leading to regulatory changes that drive differences in structure and function between the two. EMATS may also contribute to differences in expression between tissues in the same organism.
“EMATS adds a new layer of complexity to gene expression regulation,” Fiszbein says. “It gives the genome more flexibility, and introduces the potential to alter the amount of RNA produced.”
Juan Valcárcel, a research professor at the Catalan Institution for Research and Advanced Studies in the Center for Genomic Regulation in Barcelona, Spain, says understanding the mechanisms behind EMATS could also have biotechnological and therapeutic implications. “A number of human conditions, including genetic diseases and cancer, are caused by a defect or an excess of particular genes,” he says. “Reverting these anomalies through modulation of EMATS might provide innovative therapies.”
Researchers have already begun to tinker with splicing to control transcription. According to Burge, pharmaceutical companies like Ionis, Novartis, and Roche are concocting drugs to regulate splicing and treat diseases like spinal muscular atrophy. There are many ways to decrease gene expression, but it’s much harder to increase it in a targeted manner. “Tweaking splicing might be one way to do that,” he says.
“We found a way in which our cells change gene expression,” Fiszbein adds. “And we can use that to manipulate transcript levels as we want. I think that’s the most exciting part.”
This research was funded by the National Institutes of Health and the Pew Latin American Fellows Program in the Biomedical Sciences.
Nicole Giese Rura | Whitehead Institute
November 25, 2019
Salicylic acid, which may be best known as a treatment for skin conditions such as acne and warts and in its modified form as aspirin, is a critical plant hormone involved in growth and development as well as regulating plants’ immune defenses. Unable to move and evade physical damage or attacks by bacteria and other pathogens, plants respond to these assaults through the biosynthesis of salicylic acid, which in turn controls cascades of other defense responses. Consequently, control of salicylic acid production in agricultural plants could boost crops’ resilience to pathogens and insects, thereby reducing the overuse of potentially toxic pesticides that can lead to pathogen resistance. Yet scientists have been missing a key tool necessary for manipulating salicylic acid levels in plants: a full description of the pathway necessary to synthesize the hormone. Now Whitehead Institute Member Jing-Ke Weng, along with Weng lab postdoc Michael Torrens-Spence, have uncovered the last missing steps in the Arabidopsis plant’s salicylic acid pathway and solved a puzzle that has dogged Weng and his field for decades.
The quest to define the salicylic acid biosynthesis pathway started about 50 years ago when researchers determined that salicylic acid is principally formed downstream from a ubiquitous compound called chorismate. In 2001 another step was resolved: Chorismate is converted to isochorismate before eventually becoming salicylic acid. Encouraged by this progress, many in the fields of plant biology and biochemistry thought that the rest of the biosynthesis pathway in plants would be quickly defined by looking for enzymes similar to those that comprise the bacterial version of the pathway, rather an almost two decade-long drought in discoveries followed instead.
Weng and Torrens-Spence tried a different tack using genetic and biochemical methods to break the dry spell in the identification of the pathway’s missing links. Their work is described online this week in the journal Molecular Plant. From previous research, Torrens-Spence knew that the enzymes encoded by two genes – PBS3 and EPS1 – play roles in salicylic acid accumulation after pathogen attacks. In order to determine the role of these enzymes in salicylic acid biosynthesis pathway, Torrens-Spence generated plants lacking in S3H and DMR6, two genes known to breakdown salicylic acid and keep its production in check. With those genes disrupted, plants overproduce salicylic acid to an extreme extent, resulting in a severely stunted growth and other physical traits associated with surplus salicylic acid. Using these transgenic plants, Torrens-Spence had a model in which he could see if a particular gene affects salicylic acid production: If Torrens-Spence mutates genes responsible for salicylic acid biosynthesis, salicylic acid production should be abolished along with the associated visible plant characteristics. Mutations in PBS3 and EPS1 did just that – they rescued the stunted phenotypes associated with salicylic acid overproduction, and the plants accumulated less salicylic acid in their leaves than plants without the PBS3 or EPS1 mutations.
Next Torrens-Spence analyzed and compared the metabolites – the compounds created by cellular processes – in the leaves of plants without mutations and plants with PBS3 or EPS1 mutations. The results identified the probable products of the PBS3 protein’s enzymatic activity and also determined that the EPS1 protein likely acts downstream of PBS3. In order to confirm PBS3 and EPS1’s roles in salicylic acid biosynthesis, Torrens-Spence recreated the pathway in the test tube and in a relative of the tobacco plant. In both models, the reconstructed pathway efficiently converts isochorismate into salicylic acid. Interestingly, Torrens-Spence found that the intermediate produced by PBS3 could be spontaneously converted to salicylic acid in plants, but EPS1 greatly increased this step’s efficiency.
A recent evolutionary study indicates that PBS3 and variations of this gene are found throughout flowering plants, and Torrens-Spence’s work uncovered that PBS3 is an essential enzyme in the production of salicylic acid likely across all flowering plants as well. EPS1 is found only within the mustard family, which includes broccoli, Brussel sprouts, and turnips. According to Torrens-Spence and Weng, other enzymes may fulfill a role similar in plants that lack EPS1. Though the EPS1 aspect of the biosynthesis pathway described by Torrens-Spence and Weng are specific to Arabidopsis, their work provides a roadmap that researchers could follow to explore salicylic acid production in other organisms.
Weng, who has been trying to solve salicylic acid’s biosynthesis pathway in plants since he was in graduate school, says that he’s proud to have finally identified the remaining steps in Arabidopsis. With the complete salicylic acid biosynthesis pathway in Arabidopsis now known, agricultural scientists can use it to try to precisely manipulate salicylic acid’s immunological benefits in crop plants without the stunted growth associated with its excessive production.
This work was supported by the Pew Scholar Program in the Biomedical Sciences, the Searle Scholars Program, and the National Science Foundation (CHE-1709616).
Written by Nicole Giese Rura
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Jing-Ke Weng’s primary affiliation is with Whitehead Institute for Biomedical Research, where his laboratory is located and all his research is conducted. He is also an associate professor of biology at Massachusetts Institute of Technology.
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Citation:
“PBS3 and EPS1 complete salicylic acid biosynthesis from isochorismate in Arabidopsis”
Molecular Plant, online November 21, 2019 [online] DOI:10.1016/j.molp.2019.11.005
Michael P. Torrens-Spence (1), Anastassia Bobokalonova(1,2), Valentina Carballo(1), Christopher M. Glinkerman(1), Tomáš Pluskal(1), Amber Shen(1,2), and Jing-Ke Weng(1,2)
1. Whitehead Institute, Cambridge, MA 02142, USA
2. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Raleigh McElvery
November 6, 2019
An international research collaboration has discovered a new toxin, which bacteria inject into their neighboring cells to hinder growth and compete for limited resources. Their findings were published on November 6 in Nature.
At McMaster University in Ontario, Canada, co-senior author John Whitney and his team were studying a secretion system that allows bacteria to deliver these deleterious molecules, when they came across a new toxin. This toxin was an enzyme, and one they had never seen before. Based on their structural analyses, it looked a lot like the enzymes that synthesize guanosine tetra- and penta-phosphate, collectively known as “(p)ppGpp.” (p)ppGpp is a signaling molecule that helps bacteria safely dial down their growth rate in response to starvation. Suspecting the toxin might produce (p)ppGpp in recipient cells and ultimately impact their growth, the McMaster team shared their findings with Michael Laub, a professor of biology at MIT and a Howard Hughes Medical Institute investigator.
Boyuan Wang, a postdoc in the Laub lab who specializes in (p)ppGpp synthesis, examined the unknown enzyme’s activity to determine its product. He soon realized that, rather than making (p)ppGpp, this enzyme was instead producing related molecules, adenosine tetraphosphate and adenosine pentaphosphate, collectively referred to as (p)ppApp. Somehow, (p)ppApp production was hindering growth.
“Scientists have known about (p)ppApp for decades, but it hadn’t been shown to have a physiological role in organisms until now,” says Wang, a co-first author. Researchers had previously speculated that (p)ppApp was merely a non-specific product generated during (p)ppGpp synthesis, so it was surprising to find an enzyme that made it specifically.
The researchers named their enzyme Tas1, and determined that it uses the cell’s main energy currency, ATP, and its precursor, ADP, to produce (p)ppApp. In fact, one molecule of Tas1 was enough to consume 180,000 molecules of ATP per minute — two orders of magnitude faster than the fastest known (p)ppGpp synthetases work to make (p)ppGpp. Using metabolomic analyses, the MIT group showed that this exceptional rate of (p)ppApp production requires so much energy that there’s not enough left to carry out essential cellular processes, effectively killing the bacterium.
“Bacteria can inject only one Tas1 molecule at a time, and yet the toxin has such a powerful impact on its target, depleting the ATP supply in a matter of minutes,” Wang says. “The secretion system is kind of like a miniaturized intercontinental ballistic missile in terms of its structure and impact, except it functions ‘intercompartmentally’ between two bacteria.”
“It’s amazing that the first (p)ppApp synthase ever discovered actually serves as a novel, and quite clever, means of killing another cell,” says Laub, a co-senior author. “Findings like these really highlight the diversity of mechanisms that bacteria use to inhibit each other’s growth.”
Tas1, the researchers believe, may augment other known toxins that bacteria inject into one another to hinder cell growth, including those that work in the cytoplasm or target the cell envelope.
As a biochemist, Wang is excited by the prospect of using Tas1 as a tool in future experiments to deplete ATP, and probe the networks of metabolic regulation within bacteria and higher organisms.
“It’s fascinating to uncover the strategies nature uses to repurpose proteins,” Wang says. “Before this study, we wouldn’t have considered the possibility that a member of this protein family could be used as a deadly toxin.”
Image: Tas1, a newly discovered enzyme, has a similar structure to the widespread bacterial Rel proteins that produce (p)ppGpp to promote survival during starvation. Tas1 alters its specificity to quickly produce large amounts of (p)ppApp, serving as a toxin in Pseudomonas aeruginosa and killing competing bacteria. Credit: Boyuan Wang.
Citation:
“An interbacterial toxin inhibits target cell growth by synthesizing (p)ppApp”
Nature, online November, 6, DOI: 10.1038/s41586-019-1735-9
Shehryar Ahmad, Boyuan Wang, Matthew D. Walker, Hiu-Ki R. Tran, Peter J. Stogios, Alexei Savchenko, Robert A. Grant, Andrew G. McArthur, Michael T. Laub, and John C. Whitney.
Engineered signaling pathways could offer a new way to build synthetic biology circuits.
Anne Trafton | MIT News Office
October 23, 2019
Inside a living cell, many important messages are communicated via interactions between proteins. For these signals to be accurately relayed, each protein must interact only with its specific partner, avoiding unwanted crosstalk with any similar proteins.
A new MIT study sheds light on how cells are able to prevent crosstalk between these proteins, and also shows that there remains a huge number of possible protein interactions that cells have not used for signaling. This means that synthetic biologists could generate new pairs of proteins that can act as artificial circuits for applications such as diagnosing disease, without interfering with cells’ existing signaling pathways.
“Using our high-throughput approach, you can generate many orthogonal versions of a particular interaction, allowing you to see how many different insulated versions of that protein complex can be built,” says Conor McClune, an MIT graduate student and the lead author of the study.
In the new paper, which appears today in Nature, the researchers produced novel pairs of signaling proteins and demonstrated how they can be used to link new signals to new outputs by engineering E. coli cells that produce yellow fluorescence after encountering a specific plant hormone.
Michael Laub, an MIT professor of biology, is the senior author of the study. Other authors are recent MIT graduate Aurora Alvarez-Buylla and Christopher Voigt, the Daniel I.C. Wang Professor of Advanced Biotechnology.
New combinations
In this study, the researchers focused on a type of signaling pathway called two-component signaling, which is found in bacteria and some other organisms. A wide variety of two-component pathways has evolved through a process in which cells duplicate genes for signaling proteins they already have, and then mutate them, creating families of similar proteins.
“It’s intrinsically advantageous for organisms to be able to expand this small number of signaling families quite dramatically, but it runs the risk that you’re going to have crosstalk between these systems that are all very similar,” Laub says. “It then becomes an interesting challenge for cells: How do you maintain the fidelity of information flow, and how do you couple specific inputs to specific outputs?”
Most of these signaling pairs consist of an enzyme called a kinase and its substrate, which is activated by the kinase. Bacteria can have dozens or even hundreds of these protein pairs relaying different signals.
About 10 years ago, Laub showed that the specificity between bacterial kinases and their substrates is determined by only five amino acids in each of the partner proteins. This raised the question of whether cells have already used up, or are coming close to using up, all of the possible unique combinations that won’t interfere with existing pathways.
Some previous studies from other labs had suggested that the possible number of interactions that would not interfere with each other might be running out, but the evidence was not definitive. The MIT researchers decided to take a systematic approach in which they began with one pair of existing E. coli signaling proteins, known as PhoQ and PhoP, and then introduced mutations in the regions that determine their specificity.
This yielded more than 10,000 pairs of proteins. The researchers tested each kinase to see if they would activate any of the substrates, and identified about 200 pairs that interact with each other but not the parent proteins, the other novel pairs, or any other type of kinase-substrate family found in E. coli.
“What we found is that it’s pretty easy to find combinations that will work, where two proteins interact to transduce a signal and they don’t talk to anything else inside the cell,” Laub says.
He now plans to try to reconstruct the evolutionary history that has led to certain protein pairs being used by cells while many other possible combinations have not naturally evolved.
Synthetic circuits
This study also offers a new strategy for creating new synthetic biology circuits based on protein pairs that don’t crosstalk with other cellular proteins, the researchers say. To demonstrate that possibility, they took one of their new protein pairs and modified the kinase so that it would be activated by a plant hormone called trans-zeatin, and engineered the substrate so that it would glow yellow when the kinase activated it.
“This shows that we can overcome one of the challenges of putting a synthetic circuit in a cell, which is that the cell is already filled with signaling proteins,” Voigt says. “When we try to move a sensor or circuit between species, one of the biggest problems is that it interferes with the pathways already there.”
One possible application for this new approach is designing circuits that detect the presence of other microbes. Such circuits could be useful for creating probiotic bacteria that could help diagnose infectious diseases.
“Bacteria can be engineered to sense and respond to their environment, with widespread applications such as ‘smart’ gut bacteria that could diagnose and treat inflammation, diabetes, or cancer, or soil microbes that maintain proper nitrogen levels and eliminate the need for fertilizer. To build such bacteria, synthetic biologists require genetically encoded ‘sensors,’” says Jeffrey Tabor, an associate professor of bioengineering and biosciences at Rice University.
“One of the major limitations of synthetic biology has been our genetic parts failing in new organisms for reasons that we don’t understand (like cross-talk). What this paper shows is that there is a lot of space available to re-engineer circuits so that this doesn’t happen,” says Tabor, who was not involved in the research.
If adapted for use in human cells, this approach could also help researchers design new ways to program human T cells to destroy cancer cells. This type of therapy, known as CAR-T cell therapy, has been approved to treat some blood cancers and is being developed for other cancers as well.
Although the signaling proteins involved would be different from those in this study, “the same principle applies in that the therapeutic relies on our ability to take sets of engineered proteins and put them into a novel genomic context, and hope that they don’t interfere with pathways already in the cells,” McClune says.
The research was funded by the Howard Hughes Medical Institute, the Office of Naval Research, and the National Institutes of Health Pre-Doctoral Training Grant.