Placement: Promote to Homepage

Multi “-omics” approach uncovers the riches of traditional global medicine
Greta Friar | Whitehead Institute
July 22, 2019

Cambridge, MA — Kava (Piper methysticum) is a plant native to the Polynesian islands that people there have used in a calming drink of the same name in religious and cultural rituals for thousands of years. The tradition of cultivating kava and drinking it during important gatherings is a cultural cornerstone shared throughout much of Polynesia, though the specific customs — and the strains of kava — vary from island to island. Over the last few decades, kava has been gaining interest outside of the islands for its pain relief and anti-anxiety properties as a potentially attractive alternative to drugs like opioids and benzodiazepines because kavalactones, the molecules of medicinal interest in kava, use slightly different mechanisms to affect the central nervous system and appear to be non-addictive. Kava bars have been springing up around the United States, kava supplements and teas lining the shelves at stores like Walmart, and sports figures including former and current NFL players in need of safe pain relief are touting its benefits.

This growing usage suggests that there would be a sizeable market for kavalactone based medical therapies, but there are roadblocks to development: for one, kava is hard to cultivate, especially outside of the tropics. Kava takes years to reach maturity, and as a domesticated species that no longer produces seeds it can only be propagated using cuttings. This can make it difficult for researchers to get a large enough quantity of kavalactones for investigations or clinical trials. New research from Whitehead Institute Member and associate professor of biology at MIT Jing-Ke Weng and postdoctoral researcher Tomáš Pluskal, published online in Nature Plants on July 22, describes a way to solve that problem, as well as to create kavalactone variants not found in nature that may be more effective or safe as therapeutics.

“We’re combining historical knowledge of this plant’s medicinal properties, established through centuries of traditional usage, with modern research tools in order to potentially develop new drugs,” Pluskal says.

Weng’s lab has shown that if researchers figure out the genes behind a desirable natural molecule—in this case, kavalactones—they can clone those genes, insert them into species like yeast or bacteria that grow quickly and are easier to maintain in a variety of environments than a temperamental tropical plant, and then get these microbial bio-factories to mass produce the molecule. In order to achieve this, first Weng and Pluskal had to solve a complicated puzzle: how does kava produce kavalactones? There is no direct kavalactone gene; complex metabolites like kavalactones are created through a series of steps using intermediate molecules. Cells can combine these intermediates, snip out parts of them, and add bits onto them to create the final molecule—most of which is done with the help of enzymes, cells’ chemical reaction catalysts. So, in order to recreate kavalactone production, the researchers had to identify the complete pathway plants use to synthesize it, including the genes for all of the enzymes involved.

The researchers could not use genetic sequencing or common gene editing tools to identify the enzymes because the kava genome is huge; it has 130 chromosomes compared to humans’ 46. Instead they turned to other methods, including sequencing the plant’s RNA to survey the genes expressed, to identify the biosynthetic pathway for kavalactones.

“It’s like you have a lot of LEGO pieces scattered on the floor,” Weng says, “and you have to find the ones that fit together to build a certain object.”

Weng and Pluskal had a good starting point: they recognized that kavalactones had a similar structural backbone to chalcones, metabolites shared by all land plants. They hypothesized that one of the enzymes involved in producing kavalactones must be related to the one involved in producing chalcones, chalcone synthase (CHS). They looked for genes encoding similar enzymes and found two synthases that had evolved from an older CHS gene. These synthases, which they call PmSPS1 and PmSPS2, help to shape the basic scaffolding of kavalactones molecules.

Then, with some trial and error, Pluskal found the genes encoding a number of the tailoring enzymes that modify and add to the molecules’ backbone to create a variety of specific kavalactones. In order to test that he had identified the right enzymes, Pluskal cloned the relevant genes and confirmed that the enzymes they encode produced the expected molecules. The team also identified key enzymes in the biosynthetic pathway of flavokavains, molecules in kava that are structurally related to kavalactones and have been shown in studies to have anti-cancer properties.

Once the researchers had their kavalactone genes, they inserted them into bacteria and yeast to begin producing the molecules. This proof of concept for their microbial bio-factory model demonstrated that using microbes could provide a more efficient and scalable production vehicle for kavalactones. The model could also allow for the production of novel molecules engineered by combining kava genes with other genes so the microbes would produce modified kavalactones. This could allow researchers to optimize the molecules for efficiency and safety as therapeutics.

“There’s a very urgent need for therapies to treat mental disorders, and for safer pain relief options,” Weng says. “Our model eliminates several of the bottlenecks in drug development from plants by increasing access to natural medicinal molecules and allowing for the creation of new-to-nature molecules.”

Kava is only one of many plants around the world containing unique molecules that could be of great medicinal value. Weng and Pluskal hope that their model—combining the use of drug discovery from plants used in traditional medicine, genomics, synthetic biology, and microbial mass production—will be used to better harness the great diversity of plant chemistry around the world in order to help patients in need.

 

This work was supported by grants from the Smith Family Foundation, Edward N. and Della L. Thome Memorial Foundation, the Family Larsson-Rosenquist Foundation, and the National Science Foundation (CHE-1709616). T.P. is a Simons Foundation Fellow of the Helen Hay Whitney Foundation. J.K.W is supported by the Beckman Young Investigator Program, Pew Scholars Program in the Biomedical Sciences (grant number 27345), and the Searle Scholars Program (grant number 15-SSP-162).

 

Written by Greta Friar

 

***

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.

***

 

Full citation:

“The biosynthetic origin of psychoactive kavalactones in kava”

Nature Plants, online July 22, 2019, doi: 10.1038/s41477-019-0474-0

Tomáš Pluskal (1), Michael P. Torrens-Spence (1), Timothy R. Fallon (1,2), Andrea De Abreu (1,2), Cindy H. Shi (1,2), and Jing-Ke Weng (1,2)

1. Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142 USA.

2. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139 USA.

Genetic study takes research on sex differences to new heights

Differences in male and female gene expression, including those contributing to height differences, found throughout the body in humans and other mammals.

Greta Friar | Whitehead Institute
July 19, 2019

Throughout the animal kingdom, males and females frequently exhibit sexual dimorphism: differences in characteristic traits that often make it easy to tell them apart. In mammals, one of the most common sex-biased traits is size, with males typically being larger than females. This is true in humans: Men are, on average, taller than women. However, biological differences among males and females aren’t limited to physical traits like height. They’re also common in disease. For example, women are much more likely to develop autoimmune diseases, while men are more likely to develop cardiovascular diseases.

In spite of the widespread nature of these sex biases, and their significant implications for medical research and treatment, little is known about the underlying biology that causes sex differences in characteristic traits or disease. In order to address this gap in understanding, Whitehead Institute Director David Page has transformed the focus of his lab in recent years from studying the X and Y sex chromosomes to working to understand the broader biology of sex differences throughout the body. In a paper published in Science, Page, a professor of biology at MIT and a Howard Hughes Medical Institute investigator; Sahin Naqvi, first author and former MIT graduate student (now a postdoc at Stanford University); and colleagues present the results of a wide-ranging investigation into sex biases in gene expression, revealing differences in the levels at which particular genes are expressed in males versus females.

The researchers’ findings span 12 tissue types in five species of mammals, including humans, and led to the discovery that a combination of sex-biased genes accounts for approximately 12 percent of the average height difference between men and women. This finding demonstrates a functional role for sex-biased gene expression in contributing to sex differences. The researchers also found that the majority of sex biases in gene expression are not shared between mammalian species, suggesting that — in some cases — sex-biased gene expression that can contribute to disease may differ between humans and the animals used as models in medical research.

Having the same gene expressed at different levels in each sex is one way to perpetuate sex differences in traits in spite of the genetic similarity of males and females within a species — since with the exception of the 46th chromosome (the Y in males or the second X in females), the sexes share the same pool of genes. For example, if a tall parent passes on a gene associated with an increase in height to both a son and a daughter, but the gene has male-biased expression, then that gene will be more highly expressed in the son, and so may contribute more height to the son than the daughter.

The researchers searched for sex-biased genes in tissues across the body in humans, macaques, mice, rats, and dogs, and they found hundreds of examples in every tissue. They used height for their first demonstration of the contribution of sex-biased gene expression to sex differences in traits because height is an easy-to-measure and heavily studied trait in quantitative genetics.

“Discovering contributions of sex-biased gene expression to height is exciting because identifying the determinants of height is a classic, century-old problem, and yet by looking at sex differences in this new way we were able to provide new insights,” Page says. “My hope is that we and other researchers can repeat this model to similarly gain new insights into diseases that show sex bias.”

Because height is so well studied, the researchers had access to public data on the identity of hundreds of genes that affect height. Naqvi decided to see how many of those height genes appeared in the researchers’ new dataset of sex-biased genes, and whether the genes’ sex biases corresponded to the expected effects on height. He found that sex-biased gene expression contributed approximately 1.6 centimeters to the average height difference between men and women, or 12 percent of the overall observed difference.

The scope of the researchers’ findings goes beyond height, however. Their database contains thousands of sex-biased genes. Slightly less than a quarter of the sex-biased genes that they catalogued appear to have evolved that sex bias in an early mammalian ancestor, and to have maintained that sex bias today in at least four of the five species studied. The majority of the genes appear to have evolved their sex biases more recently, and are specific to either one species or a certain lineage, such as rodents or primates.

Whether or not a sex-biased gene is shared across species is a particularly important consideration for medical and pharmaceutical research using animal models. For example, previous research identified certain genetic variants that increase the risk of Type 2 diabetes specifically in women; however, the same variants increase the risk of Type 2 diabetes indiscriminately in male and female mice. Therefore, mice would not be a good model to study the genetic basis of this sex difference in humans. Even when the animal appears to have the same sex difference in disease as humans, the specific sex-biased genes involved might be different. Based on their finding that most sex bias is not shared between species, Page and colleagues urge researchers to use caution when picking an animal model to study sex differences at the level of gene expression.

“We’re not saying to avoid animal models in sex-differences research, only not to take for granted that the sex-biased gene expression behind a trait or disease observed in an animal will be the same as that in humans. Now that researchers have species and tissue-specific data available to them, we hope they will use it to inform their interpretation of results from animal models,” Naqvi says.

The researchers have also begun to explore what exactly causes sex-biased expression of genes not found on the sex chromosomes. Naqvi discovered a mechanism by which sex-biased expression may be enabled: through sex-biased transcription factors, proteins that help to regulate gene expression. Transcription factors bind to specific DNA sequences called motifs, and he found that certain sex-biased genes had the motif for a sex-biased transcription factor in their promoter regions, the sections of DNA that turn on gene expression. This means that, for example, a male-biased transcription factor was selectively binding to the promoter region for, and so increasing the expression of, male-biased genes — and likewise for female-biased transcription factors and female-biased genes. The question of what regulates the transcription factors remains for further study — but all sex differences are ultimately controlled by either the sex chromosomes or sex hormones.

The researchers see the collective findings of this paper as a foundation for future sex-differences research.

“We’re beginning to build the infrastructure for a systematic understanding of sex biases throughout the body,” Page says. “We hope these datasets are used for further research, and we hope this work gives people a greater appreciation of the need for, and value of, research into the molecular differences in male and female biology.”

This work was supported by Biogen, Whitehead Institute, National Institutes of Health, Howard Hughes Medical Institute, and generous gifts from Brit and Alexander d’Arbeloff and Arthur W. and Carol Tobin Brill.

Bacteria use “spare part” proteins to repair damage and survive inhospitable conditions
Saima Sidik
July 17, 2019

Millennia ago, before the evolution of multicellular life, bacteria developed a group of proteins called glycyl radical enzymes to help them turn food into cellular energy. Glycyl radical enzymes functioned efficiently for millions of years until bacteria encountered a new hurdle: oxygen build-up in the atmosphere. Glycyl radicals are easily damaged by oxygen, and bacteria needed a way to continue to process nutrients under these new conditions.

In a recent study published in Journal of Biological Inorganic Chemistry, Sarah Bowman et al. from MIT provide structural evidence for how bacteria use “spare part” proteins to repair glycyl radical enzymes by replacing their damaged portions.

“Instead of degrading the whole protein and building a new one, bacteria make a small spare part protein that can bind to the glycyl radical enzyme and restore its function,” says Lindsey Backman, a graduate student in the MIT Department of Chemistry and co-author on the study.

Senior author Catherine Drennan, an MIT professor of biology and chemistry and a Howard Hughes Medical Institute Investigator, is leading the effort to characterize a glycyl radical enzyme called pyruvate formate lyase, or PFL, and its corresponding spare part protein, YfiD. Using a technique called nuclear magnetic resonance spectroscopy, her lab examined the shape of YfiD. Previous work revealed that upon oxygen exposure, part of PFL is cleaved off, leaving the enzyme unable to perform its chemistry. Then, using in silico modeling, the Drennan lab showed that YfiD fits neatly into the hole in oxygen-damaged PFL, replacing the missing piece.

“If you have a car and you get a flat tire, you don’t buy a new car, you just change the tire,” Backman says.

Although YfiD is one of only two spare part proteins that have been identified, Drennan suspects there are many more. It’s easy for researchers to mistake spare parts for the portions of proteins that they replace, and Drennan thinks that scientists have probably overlooked spare parts when analyzing the complex milieu of proteins found in cells.

Although identifying spare parts is technically challenging, Drennan thinks the applications for synthetic biology make it well worth the effort. Specifically, she’s interested in bacteria that live in the deep sea where there’s very little oxygen and use other glycyl radical enzymes to degrade hydrocarbons. Because they can break down hydrocarbons, these bacteria could be valuable tools for cleaning up oil spills. Oil spills occur on the ocean’s surface where there’s a lot of oxygen, so spare parts might be necessary to stabilize oil-degrading proteins under these conditions.

“The question always becomes, ‘what about the oxygen sensitivity?’” Drennan says, referring to these oil-degrading proteins. “What if we expressed a spare part to make them more stable?”

Backman began working on spare part proteins as an undergraduate participating in the MIT Summer Research Program, or MSRP, as did several of the study’s other authors, and Drennan says that this project’s success highlights the valuable role these students play in MIT labs. Now a full-time graduate student, Backman has teamed up with Drennan lab postdoc and co-author Mary Andorfer, and together they plan to continue characterizing the interaction between YfiD and PFL. They think their findings may be the first step in showing that spare parts are a common protein repair mechanism, and that characterizing them will add a new tool to the synthetic biology toolbox.

Citation:
“Solution structure and biochemical characterization of a spare part protein that restores activity to an oxygen-damaged glycyl radical enzyme”
JBIC Journal of Biological Inorganic Chemistry, online June 26, 2019, DOI: 10.1007/s00775-019-01681-2.
Sarah E. J. Bowman, Lindsey R. F. Backman, Rebekah E. Bjork, Mary C. Andorfer, Santiago Yori, Alessio Caruso, Collin M. Stultz, and Catherine L. Drennan.

Unmasking mutant cancer cells

A new dosing regimen for an old cancer drug shows new promise as an immunotherapy.

Bendta Schroeder | Koch Institute
July 16, 2019

As cancer cells progress, they accumulate hundreds and even thousands of genetic and epigenetic changes, resulting in protein expression profiles that are radically different from that of healthy cells. But despite their heavily mutated proteome, cancer cells can evade recognition and attack by the immune system.

Immunotherapies, particularly checkpoint inhibitors that reinvigorate exhausted T cells, have revolutionized the treatment of certain forms of cancer. These breakthrough therapies have resulted in unprecedented response rates for some patients. Unfortunately, most cancers fail to respond to immunotherapies and new strategies are therefore needed to realize their full potential.

A team of cancer biologists including members of the laboratories of David H. Koch Professor of Biology Tyler Jacks, director of the Koch Institute for Integrative Cancer Research at MIT, and fellow Koch Institute member Forest White, the Ned C. and Janet Bemis Rice Professor and member of the MIT Center for Precision Cancer Medicine, took a complementary approach to boosting the immune system.

Although cancer cells are rife with mutant proteins, few of those proteins appear on a cell’s surface, where they can be recognized by immune cells. The researchers repurposed a well-studied class of anti-cancer drugs, heat shock protein 90 (HSP90) inhibitors, that make cancer cells easier to recognize by revealing their mutant proteomes.

Many HSP90 inhibitors have been studied extensively for the past several decades as potential cancer treatments. HSP90 protects the folded structure of a number of proteins when cells undergo stress, and in cancer cells plays an important role in stabilizing protein structure undermined by pervasive mutations. However, despite promising preclinical evidence, HSP90 inhibitors have produced discouraging outcomes in clinical trials, and none have achieved FDA approval.

In a study appearing in Clinical Cancer Research, the researchers identified a potential reason behind those disappointing results. HSP90 inhibitors have only been clinically tested at bolus doses — intermittent, large doses — that often result in unwanted side effects in patients.

RNA profiling of human clinical samples and cultured cancer cell lines revealed that this bolus-dosing schedule results in the profound suppression of immune activity as well as the activation of heat shock factor 1 protein (HSF1). Not only does HSF1 activate the cell’s heat shock response, which counteracts the effect of the HSP90 inhibitor, but it is known to be a powerful enabler of cancer cell malignancy.

In striking contrast, the researchers used cancer mouse models with intact immune systems to show that sustained, low-level dosing of HSP90 inhibitors avoids triggering both the heat shock response and the immunosuppression associated with high doses.

Using a method devised by the White lab that combines mass spectrometry-based proteomics and computational modeling, the researchers discovered that the new dosing regimen increased the number and diversity of peptides (protein fragments) on the cell surface. These peptides, which the team found to be released by HSP90 during sustained low-level inhibition, were then free to be taken up by the cell’s antigen-presenting machinery and used to flag patrolling immune cells.

“These results connect a fundamental aspect of cell biology — protein folding — to anti-tumor immune responses” says lead author Alex Jaeger, a postdoctoral fellow in the Jacks lab and a former member of the laboratory of the late MIT biologist and Professor Susan Lindquist, whose work inspired the study’s HSP90 dosing scheule. “Hopefully, our findings can reinvigorate interest in HSP90 inhibition as a complementary approach for immunotherapy.”

Using the new dosing regimen, the researchers were able to clear tumors in mouse models at drug concentrations that are 25-50 times lower than those used in clinical trials, significantly reducing the risk for toxic side effects in patients. Importantly, because several forms of HSP90 inhibitors have already undergone extensive clinical testing, the new dosing regimen can be tested in patients quickly.

This work was supported in part by the Damon Runyon Cancer Research Foundation, the Takeda Pharmaceuticals Immune Oncology Research Fund, and an MIT Training Grant in Environmental Science; foundational work on HSF1 was supported by the Koch Institute Frontier Research Program.

Meet the 2019 tenured professors in the School of Science

Eight faculty members are granted tenure in five science departments.

School of Science
July 10, 2019

MIT granted tenure to eight School of Science faculty members in the departments of Biology; Chemistry; Earth, Atmospheric and Planetary Sciences; Mathematics; and Physics.

William Detmold’s research within the area of theoretical particle and nuclear physics incorporates analytical methods, as well as the power of the world’s largest supercomputers, to understand the structure, dynamics, and interactions of particles like protons and to look for evidence of new physical laws at the sub-femtometer scale probed in experiments such as those at the Large Hadron Collider. He joined the Department of Physics in 2012 from the College of William and Mary, where he was an assistant professor. Prior to that, he was a research assistant professor at the University of Washington. He received his BS and PhD from the University of Adelaide in Australia in 1996 and 2002, respectively. Detmold is a researcher in the Center for Theoretical Physics in the Laboratory for Nuclear Science.

Semyon Dyatlov explores scattering theory, quantum chaos, and general relativity by employing microlocal analytical and dynamical system methods. He came to the Department of Mathematics as a research fellow in 2013 and became an assistant professor in 2015. He completed his doctorate in mathematics at the University of California at Berkeley in 2013 after receiving a BS in mathematics at Novosibirsk State University in Russia in 2008. Dyatlov spent time after finishing his PhD as a postdoc at the Mathematical Sciences Research Institute before moving to MIT.

Mary Gehring studies plant epigenetics. By using a combination of genetic, genomic, and molecular biology, she explores how plants inherit and interpret information that is not encoded in their DNA to better understand plant growth and development. Her lab focuses primarily on Arabidopsis thaliana, a small flowering plant that is a model species for plant research. Gehring joined the Department of Biology in 2010 after performing postdoctoral research at the Fred Hutchinson Cancer Research Center. She received her BA in biology from Williams College in 1998 and her doctorate from the University of California at Berkeley in 2005. She is also a member of the Whitehead Institute for Biomedical Research.

David McGee performs research in the field of paleoclimate, merging information from stalagmites, lake deposits, and marine sediments with insights from models and theory to understand how precipitation patterns and atmospheric circulation varied in the past. He came to MIT in 2012, joining the Department of Earth, Atmospheric and Planetary Sciences after completing a NOAA Climate and Global Change Postdoctoral Fellowship at the University of Minnesota. Before that, he attended Carleton College for his BA in geology in 1993-97, Chatham College for an MA in teaching from 1999 to 2003, Tulane University for his MS from 2004 to 2006, and Columbia University for his PhD from 2006 to 2009. McGee is the director of the MIT Terrascope First-Year Learning Community, a role he has held for the past four years.

Ankur Moitra works at the interface between theoretical computer science and machine learning by developing algorithms with provable guarantees and foundations for reasoning about their behavior. He joined the Department of Mathematics in 2013. Prior to that, he received his BS in electrical and computer engineering from Cornell University in 2007, and his MS and PhD in computer science from MIT in 2009 and 2011, respectively. He was a National Science Foundation postdoc at the Institute for Advanced Study until 2013. Moitra was a 2018 recipient of a School of Science Teaching Prize. He is also a principal investigator in the Computer Science and Artificial Intelligence Laboratory (CSAIL) and a core member of the Center for Statistics.

Matthew Shoulders focuses on integrating biology and chemistry to understand how proteins function in the cellular setting, including proteins’ shape, quantity, and location within the body. This research area has important implications for genetic disorders and neurodegenerative diseases such as Alzheimer’s, diabetes, cancer, and viral infections. Shoulders’ lab works to elucidate, at the molecular level, how cells solve the protein-folding problem, and then uses that information to identify how diseases can develop and to provide insight into new targets for drug development. Shoulders joined the Department of Chemistry in 2012 after earning a BS in chemistry and minor in biochemistry from Virginia Tech in 2004 and a PhD in chemistry from the University of Wisconsin at Madison in 2009. He is also an associate member of the Broad Institute of MIT and Harvard, and a member of the MIT Center for Environmental Health Sciences.

Tracy Slatyer researches fundamental aspects of theoretical physics, answering questions about both visible and dark matter by searching for potential indications of new physics in astrophysical and cosmological data. She has developed and adapted novel techniques for data analysis, modeling, and calculations in quantum field theory; her work has also inspired a range of experimental investigations. The Department of Physics welcomed Slatyer in 2013 after she completed a three-year postdoctoral fellowship at the Institute for Advanced Study. She majored in theoretical physics as an undergraduate at the Australian National University, receiving a BS in 2005, and completed her PhD in physics at Harvard University in 2010. In 2017, Slatyer received the School of Science Prize in Graduate Teaching and was also named the first recipient of the school’s Future of Science Award. She is a member of the Center for Theoretical Physics in the Laboratory for Nuclear Science.

Michael Williams uses novel experimental methods to improve our knowledge of fundamental particles, including searching for new particles and forces, such as dark matter. He also works on advancing the usage of machine learning within the domain of particle physics research. He joined the Department of Physics in 2012. He previously attended Saint Vincent College as an undergraduate, where he double majored in mathematics and physics. Graduating in 2001, Williams then pursued a doctorate at Carnegie Mellon University, which he completed in 2007. From 2008 to 2012 he was a postdoc at Imperial College London. He is a member of the Laboratory for Nuclear Science.

Cancer biologists identify new drug combo

Two drugs that block cell division synergize to kill tumor cells.

Anne Trafton | MIT News Office
July 10, 2019

When it comes to killing cancer cells, two drugs are often better than one. Some drug combinations offer a one-two punch that kills cells more effectively, requires lower doses of each drug, and can help to prevent drug resistance.

MIT biologists have now found that by combining two existing classes of drugs, both of which target cancer cells’ ability to divide, they can dramatically boost the drugs’ killing power. This drug combination also appears to largely spare normal cells, because cancer cells divide differently than healthy cells, the researchers say. They hope a clinical trial of this combination can be started within a year or two.

“This is a combination of one class of drugs that a lot of people are already using, with another type of drug that multiple companies have been developing,” says Michael Yaffe, a David H. Koch Professor of Science and the director of the MIT Center for Precision Cancer Medicine. “I think this opens up the possibility of rapid translation of these findings in patients.”

The discovery was enabled by a new software program the researchers developed, which revealed that one of the drugs had a previously unknown mechanism of action that strongly enhances the effect of the other drug.

Yaffe, who is also a member of the Koch Institute for Integrative Cancer Research, is the senior author of the study, which appears in the July 10 issue of Cell Systems. Koch Institute research scientists Jesse Patterson and Brian Joughin are the first authors of the paper.

Unexpected synergy

Yaffe’s lab has a longstanding interest in analyzing cellular pathways that are active in cancer cells, to find how these pathways work together in signaling networks to create disease-specific vulnerabilities that can be targeted with multiple drugs. When the researchers began this study, they were looking for a drug that would amplify the effects of a type of drug known as a PLK1 inhibitor. Several PLK1 inhibitors, which interfere with cell division, have been developed, and some are now in phase 2 clinical trials.

Based on their previous work, the researchers knew that PLK1 inhibitors also produce a type of DNA and protein damage known as oxidation. They hypothesized that pairing PLK1 inhibitors with a drug that prevents cells from repairing oxidative damage could make them work even better.

To explore that possibility, the researchers tested a PLK1 inhibitor along with a drug called TH588, which blocks MTH1, an enzyme that helps cells counteract oxidative damage. This combination worked extremely well against many types of human cancer cells. In some cases, the researchers could use one-tenth of the original doses of each drug, given together, and achieve the same rates of cell death of either drug given on its own.

“It’s really striking,” Joughin says. “It’s more synergy than you generally see from a rationally designed combination.”

However, they soon realized that this synergy had nothing to do with oxidative damage. When the researchers treated cancer cells missing the gene for MTH1, which they thought was TH588’s target, they found that the drug combination still killed cancer cells at the same high rates.

“Then we were really stuck, because we had a good combination, but we didn’t know why it worked,” Yaffe says.

To solve the mystery, they developed a new software program that allowed them to identify the cellular networks most affected by the drugs. The researchers tested the drug combination in 29 different types of human cancer cells, then fed the data into the software, which compared the results to gene expression data for those cell lines. This allowed them to discover patterns of gene expression that were linked with higher or lower levels of synergy between the two drugs.

This analysis suggested that both drugs were targeting the mitotic spindle, a structure that forms when chromosomes align in the center of a cell as it prepares to divide. Experiments in the lab confirmed that this was correct. The researchers had already known that PLK1 inhibitors target the mitotic spindle, but they were surprised to see that TH588 affected the same structure.

“This combination that we found was very nonobvious,” Yaffe says. “I would never have given two drugs that both targeted the same process and expected anything better than just additive effects.”

“This is an exciting paper for two reasons,” says David Pellman, associate director for basic science at Dana-Farber/Harvard Cancer Center, who was not involved in the study. “First, Yaffe and colleagues make an important advance for the rational design of drug therapy combinations. Second, if you like scientific mysteries, this is a riveting example of molecular sleuthing. A drug that was thought to act in one way is unmasked to work through an entirely different mechanism.”

Disrupting mitosis

The researchers found that while both of the drugs they tested disrupt mitosis, they appear to do so in different ways. TH588 binds to microtubules, which form the mitotic spindle, and slows their assembly. Many similar microtubule inhibitors are already used clinically to treat cancer. The researchers showed that some of those microtubule inhibitors also synergize with PLK1 inhibitors, and they believe those would likely be more readily available for rapid use in patients than TH588, the drug they originally tested.

While the PLK1 protein is involved in multiple aspects of cell division and spindle formation, it’s not known exactly how PLK1 inhibitors interfere with the mitotic spindle to produce this synergy. Yaffe said he suspects they may block a motor protein that is necessary for chromosomes to travel along the spindle.

One potential benefit of this drug combination is that the synergistic effects appear to specifically target cancer cell division and not normal cell division. The researchers believe this could be because cancer cells are forced to rely on alternative strategies for cell division because they often have too many or too few chromosomes, a state known as aneuploidy.

“Based on the work we have done, we propose that this drug combination targets something fundamentally different about the way cancer cells divide, such as altered cell division checkpoints, chromosome number and structure, or other structural differences in cancer cells,” Patterson says.

The researchers are now working on identifying biomarkers that could help them to predict which patients would respond best to this drug combination. They are also trying to determine the exact function of PLK1 that is responsible for this synergy, in hopes of finding additional drugs that would block that interaction.

The research was funded by the National Institutes of Health, the Charles and Marjorie Holloway Foundation, the Ovarian Cancer Research Fund, the MIT Center for Precision Cancer Medicine, the Koch Institute Dana Farber/Harvard Cancer Center Bridge Project, an American Cancer Society Postdoctoral Fellowship, the Koch Institute Support (core) Grant from the National Cancer Institute, and the Center for Environmental Health Support Grant.

Researchers determine what makes some proteins “slippery” enough to evade destruction
Raleigh McElvery
July 10, 2019

All cells must balance generating new proteins with eliminating excess or damaged ones by way of powerful degradation machines — which, much like wood chippers, chew up proteins and spit them out. But, these proteins are often folded into intricate structures, and must be unfurled before they can be fed into these degradation machines, broken into tiny bits, and ultimately recycled. In bacteria, a molecular motor known as ClpX must grip the end of the ill-fated protein and apply force to straighten it. However, until now, researchers weren’t sure precisely how ClpX gripped its target tightly enough to accomplish this task.

There had been evidence to suggest that some amino acids — the chemical building blocks that comprise proteins — are “slippery” and thus more difficult to grip. In a new study published in eLife, researchers at the MIT Department of Biology examined each amino acid’s individual contribution to grip. By parsing the physical basis for this molecular interaction, they hope to better understand how some proteins evade destruction.

“Previous studies had shown that small amino acids were notoriously hard to grip, but no one really understood why,” says Tristan Bell, graduate student and first author on the paper. “It’s like watching a game of tug-of-war and knowing that a person’s hands are important for pulling on the rope, but having no idea what allows the hands to get a good grip on the rope.”

ClpX, he explains, is roughly shaped like donut with loops protruding into the center hole. These loops grip the target protein, jamming it against the surface of ClpX, and unfolding it so it can be threaded through the hole and shredded.

The researchers engineered proteins with tails comprised of various amino acid combinations, and measured how well ClpX could grip them, both in bacteria and in test tubes. They determined that ClpX can only grip between six and eight amino acids at a time, and that only a handful of the 20 possible amino acids could actually be “well-gripped.” When ClpX was able to grasp multiple amino acids simultaneously, its grip strength increased.

“We think that somehow the charge is preventing ClpX from making strong contacts with the target protein, preventing it from achieving a stable grip state,” Bell says.Just like in previous experiments, large amino acids appeared easier to grip than small ones, “similar to the way a knotted rope is easier to grasp than a smooth, slippery one,” Bell says. But, regardless of size, amino acids that carried electric charge seemed to be more slippery.

The team thinks that proteins with slippery tails might have an evolutionary advantage, because they are harder to grip and therefore less likely to be degraded.

Invaders like viruses have been known to insert a slippery sequence into certain proteins to prevent the host cell from destroying them and thus promoting replication. Even healthy cells produce proteins with strategically placed slippery sequences, which allow a portion of the protein to break away from the degradation machinery unscathed. In the bacteria Caulobacter crescentus, this planned breakage actually produces a version of one protein that’s needed for DNA replication.

“Next,” Bell says, “we’re hoping to look across entire proteomes in different organisms to find more proteins that escape destruction.”

“Tristan’s experiments and results reveal some of the molecular determinants of grip in the bacterial degradation machines we study,” says Bob Sauer, the Salvador E. Luria Professor of Biology and senior author on the study. “Many of the rules he discovered apply to related machines that function in all biological organisms, including humans, emphasizing the common evolution of these machines.”

Citation:
“Interactions between a subset of substrate side chains and AAA+ motor pore loops determine grip during protein unfolding”
eLife, online June 28, 2019, DOI: 10.7554/eLife.46808
Tristan A. Bell, Tania A. Baker, and Robert T. Sauer.

Researchers identify important proteins hijacked by pathogens during cell-to-cell spread
Raleigh McElvery
July 9, 2019

Listeria monocytogenes, the food-borne bacterium responsible for listeriosis, can creep from one cell to the next, stealthily evading the immune system. This strategy of cell-to-cell spread allows them to infect many different cell types, and can spur complications like meningitis. Yet the molecular details of this spread remain a mystery.

In a paper recently published in Molecular Biology of the Cell, researchers from the MIT Department of Biology, University of California, Berkeley, and Chan Zuckerberg Biohub are beginning to piece together the elusive means by which Listeria moves from one cell to the next. This mode of transport, the scientists suggest, looks a lot like trans-endocytosis, a process that healthy, uninfected cells use to exchange organelles and various cytoplasmic components. In fact, the two processes are so similar that Listeria may be co-opting the host cell’s trans-endocytosis machinery for its own devices.

Although the particulars of trans-endocytosis are poorly understood, the process permits neighboring cells to exchange materials via membrane-bound compartments called vacuoles, which release their cargo upon reaching their final destination.

Much like trans-endocytosis, cell-to-cell spread relies on vacuoles to ferry Listeria. First, the pathogen commandeers the host cell’s own machinery to assemble a tail of proteins that allows it to rocket around inside the cell and ram against both the membrane of the host and that of the adjacent cell. The resulting protrusion is then somehow engulfed into a double-membrane vacuole, and the bacteria burst through their containment to begin the process anew in the recipient cell.

“There’s been a lot of work looking at Listeria cell-to-cell spread,” says Rebecca Lamason, the Robert A. Swanson (1969) Career Development Assistant Professor in the MIT Department of Biology and senior author on the study. “But we still don’t really understand the molecular mechanisms that allow the bacteria to manipulate the membrane to promote engulfment. Depending on what we uncover, we might also be able to apply that information to better grasp how an uninfected cell regulates trans-endocytosis.”

Lamason and her team anticipated that the same proteins implicated in trans-endocytosis would also be involved in Listeria cell-to-cell spread, which would indicate that the pathogen was appropriating these proteins for its own purposes. The researchers made a list of 115 host genes of interest, and then used an RNAi screen to identify just 22 that are critical for cell-to-cell spread.

They were excited to find that, of those 22 genes, several are also implicated in endocytosis, which suggests Listeria is using a similar strategy. These include genes encoding caveolin proteins that control membrane trafficking and remodeling, as well as another protein called PACSIN2 that interacts with caveolins to regulate protrusion engulfment.

Now that the researchers have pinpointed these key proteins, the next step is to determine how they work together in order to promote cell-to-cell spread — especially since the protrusions created by Listeria are much larger than those required for trans-endocytosis.

“As we drill down even deeper into the molecular mechanisms, it will be interesting to see where trans-endocytosis and cell-to-cell spread differ, and where they are similar,” Lamason says. “Our hope is that investigating the mechanisms of bacterial spread will reveal fundamental insights into host intercellular communication.”

Citation:
“RNAi screen reveals a role for PACSIN2 and caveolins during bacterial cell-to-cell spread”
Molecular Biology of the Cell, online June 26, 2019, DOI: 10.1091/mbc.E19-04-0197
Allen G. Sanderlin, Cassandra Vondrak, Arianna J. Scricco, Indro Fedrigo, Vida Ahyong, and Rebecca L. Lamason

Unusual labmates: Lighting up the lab
July 7, 2019

Unusual Labmates is a series that explores some of the more unusual models used for research at Whitehead Institute. From rare plants to luminescent beetles to regenerative starfish and worms, these organisms and their unusual traits provide insights into the underlying biology and incredible diversity of living things.

Massachusetts Institute of Technology (MIT) graduate student Tim Fallon is standing in a field in New Jersey, holding a net and waiting for the last glimmers of sunlight to disappear. As the trees surrounding the field fade into shadow, Fallon watches the ground intently. The air is still and then, hovering above the grass, a small point of light appears. It floats through the air in a concave upwards arc and winks out. Soon, more lights follow suit. These are fireflies, unassuming beetles by day, but at night they put on a dazzling luminescent display in the hope of attracting a mate. Fallon sweeps his net through the air, capturing some of them. He has traveled to New Jersey with other members of Whitehead Institute Member and associate professor of biology at MIT Jing-Ke Weng’s lab to collect specimens of Photinus pyralis, the big dipper firefly, in order to sequence a firefly genome for the very first time.

Fireflies have been around for more than one hundred million years, and in that time have diverged into more than 2,000 species and spread to every continent except Antarctica. The beetles (despite their name, fireflies are actually beetles) are widely known, and often beloved, for their enchanting courtship rituals, but they have also piqued the interest of scientists, who have harnessed the gene behind their light emitting capability for use in research. However, for all of fireflies’ appeal, they are a difficult animal to work with in the lab, and much of their biology remains shrouded in mystery. In the hopes of improving that situation, Weng, Fallon, and collaborators—including Sarah Lower, assistant professor of biology at Bucknell University, and Yuichi Oba, professor at Chubu University in Japan—have been investigating fireflies, primarily by sequencing and analyzing their genomes. This research is providing insights into the evolution of fireflies’ light-producing ability, the biomolecular pathways the fireflies use to luminesce, and could perhaps even inform how the use of firefly-based luminescence in research can be improved.

The chemistry of light

Fireflies produce light using two main ingredients: an enzyme called luciferase and the small molecule luciferin. Luciferase facilitates a chemical reaction that oxidizes the luciferin, and one product of the reaction is light. In the 1980s, researchers recreated the process in plants and plant cells by cloning the firefly gene responsible for the luciferase enzyme from big dipper fireflies and then inserting it into the genomes of their specimens in the lab.[1] When they injected luciferin into the specimens, they began to glow – just like fireflies. Researchers now use this approach in both plant and animal models to track various aspects of biology. They can link the luminescence to a trait or process, and then measure the level of light emitted. For example, researchers can fuse the luciferase gene to a gene of interest such that the two genes will be expressed as one. Then they introduce luciferin to the system and measure the light output using very sensitive equipment that can sense minute changes. The more light that is emitted, the higher the activity level of both luciferase and the gene of interest. This is a widely used assay that, Fallon says, every biologist uses or at least learns about during their training. Luciferase-luciferin has many applications, and along with tracking gene expression it has also been used to track cancer metastasis, monitor medical treatment efficacy, and check for microbial contamination—including on space vehicles such as the Mars Curiosity Rover.[2] The extreme sensitivity of luciferin-luciferase tests makes them an attractive choice in many experiments.

In spite of the widespread use of firefly luminescence, a lot about it still isn’t known, especially when it comes to luciferin. While the gene encoding luciferase has been identified, luciferin is thought to be created through a process involving multiple genes, and the complete set of those genes is unknown, as are the steps involved in the production of luciferin: the intermediate molecules produced and how they are modified to reach the final product. Although Weng’s lab generally studies plants and focuses on understanding how plants evolved biochemical pathways to produce unique small molecules with traits of interest, particularly those with medicinal value, when Fallon joined the lab, he convinced Weng that investigating the unknowns of the small molecule luciferin was a similar and suitable project.

Fireflies in the wild

When Fallon joined the Weng lab, there were not a lot of research tools available for investigating fireflies, starting with the lack of a sequenced genome. Only a handful of firefly genes had even ever been identified. Weng and his collaborators crowdfunded the money to sequence the first firefly genome, and then received funding to sequence a second firefly species and a bioluminescent click beetle, providing a wealth of new data to explore.

The lack of tools for firefly research was due in part to how difficult it is to rear fireflies in the lab, which makes them tricky animals to study. Fireflies are very sensitive to changes in their environments, and in the lab it’s difficult to mimic the right vegetation, climate, seasonal shifts, and other factors that the beetles rely on to time their metamorphoses and thrive.

Unpredictable environmental changes are becoming a common challenge for fireflies beyond the lab as well, due to the impact of humans on their habitats. Fireflies’ sensitivity to these changes may be causing their disappearance. Anecdotally, many people have fond memories of seeing fireflies every summer when they were younger and can attest to the absence of fireflies in those same places now. A large citizen science research project called Firefly Watch is currently underway to figure out the extent to which firefly populations are declining across the United States (U.S.).[3] And in a case indicative of a larger problem, conservation groups recently submitted an urgent petition to recognize the Bethany Beach firefly, whose key habitat in Delaware is at risk due to human development, as the first endangered firefly species in the U.S.[4]

A significant manmade disruption to the fireflies’ habitats is light pollution. Fireflies find each other by signaling with light, but their lanterns are no match for electricity; a firefly trying to outshine a streetlamp might as well be facing off with the sun. For species that evolved faint glows to flash in the dead of night, in the dark of forests, finding a place where their light can be seen is getting harder and harder. Big dipper fireflies have fared better than many others species. They’re large, with bright lanterns, and they tend to flash at twilight, so they are used to competing with some ambient light. Unsurprisingly, big dipper fireflies remain abundant in the wild, including in and near cities. They can be spotted all over the eastern and midwestern U.S., where they are easily identifiable thanks to the distinctive J-shape—resembling the curve of a dipper—that they make as they flash.

Big dipper fireflies have been important in biology research—it is from them that the firefly luciferase gene was first cloned—and so they were the first species that Fallon and his collaborators chose for their genome sequencing project. However, big dipper fireflies fare no better than other firefly species in a lab setting. No one has ever successfully reared big dipper fireflies through a full lifecycle (from egg to egg) in the lab, Fallon says. So, in spite of the ease of collecting big dipper fireflies in the eastern U.S., in order to get a population of fireflies going in the lab, Fallon had to look elsewhere: to Japan.

In Japan, fireflies are beloved. Watching them is a popular summer pastime, and they are celebrated in myths, songs, and other media. The two dominant species are both aquatic in their larval stages: the heike-botaru (Aquatica lateralis), which mostly lives in flooded rice paddy fields, and the larger and brighter genji-botaru (Luciola cruciata), which lives in streams and rivers. Both species need clean bodies of water to survive, and so their numbers have diminished in cities—but unlike in America, Japan has not allowed fireflies to fade away quietly into the night. As fireflies have grown scarcer, breeding centers have begun rearing the insects in large numbers, using big facilities that can recreate the fireflies’ natural habitat on a scale not possible in a U.S. lab. Fireflies are sometimes bred for conservation purposes, such as an effort to bolster the heike population in the water by the Imperial Palace in Tokyo. The fireflies are also bred for spectacle, released during summer festivals and firefly-watching events in the city to recreate the lost experience of glow-filled nights. The Fussa firefly festival has drawn large crowds for more than fifty years.

Rearing insects is also a relatively common pastime in Japan. When Fallon was looking for a type of firefly that would be a good addition to the genome sequencing project and could survive in lab conditions, he learned of the heike, which are kept in captivity and used in research in Japan. Although heike are still very sensitive to small changes in their environments, they have been demonstrated to survive for multiple generations indoors, unlike the big dipper firefly. The larvae are aquatic, and maintaining a controlled aquarium is also easier than a terrarium, Fallon says. Fallon received his fireflies from a collaborator in Japan, firefly expert Dr. Yuichi Oba, a professor at Chubu University. Oba works with Haruyoshi Ikeya, a high school teacher in Yokohama, Japan who cracked the code of rearing heike fireflies indoors several decades ago; the population from which Fallon received his specimens has been lab-bred since its original capture in 1990. Fallon received tips from Oba on how to successfully rear the fireflies—though not all of these could be followed, due to stricter regulations for how to keep the fireflies contained in the U.S., where the United States Department of Agriculture considers them a potential plant pest.

Fireflies in the lab

Fallon rears the fireflies in a small room separate from the main space of the Weng lab. The room is tightly packed with supplies and various aquariums: Fallon must move the firefly specimens between environments as they go through their lifecycle. There is tinfoil over the windows so when Fallon switches the lights off the room becomes a darkroom, where he can observe the fireflies flashing.

Maintaining a lab population serves a number of purposes. First of all, collecting fireflies is time-consuming, limited by season, and in the case of foreign species, involves further transportation and regulatory roadblocks. When collecting specimens in the wild, it’s easy to find adults, but harder to get access to every stage of the lifecycle, especially eggs and pupae, the way you can in the lab. Having a lab population also ensures that you are working with one species, whereas when collecting in the wild it can be easy to get a mixed-up batch—one field may contain a dozen similar-looking species—and molecular biology research requires species-specific material. Overall, having a lab population means access to live specimens whenever you need them: whenever the researchers have a new research question to answer or a new experiment to run. However, maintaining this beneficial resource is not a simple feat.

Rearing fireflies in captivity is difficult in part because each stage of their life cycle has different requirements. Fireflies hatch from eggs, which the heike lay in moss near water. Heike larvae live in water, where they spend most of their lifespan, usually around one year, though in the lab this stage can be as short as six months. During this time, the larvae typically go through five or six instars, or stages of growth between molting. Insects must molt in order to grow, as their size is restricted by their exoskeletons. The heike’s first instar is only a few millimeters long, while the final instar may grow to be closer to two centimeters.

For most of their time as larvae, the specimens live in shoebox sized aquariums inside a metal cabinet. Fallon feeds them bladder snails and waits for them to grow. When the larvae are in their later instars, around the right size and age to pupate, Fallon moves them into another aquarium with a mock riverbank inside, with a soil mixture devised by Haruyoshi Ikeya. The riverbank is necessary because the firefly larvae move to land to pupate. First, they begin to exhibit what is called climbing or landing behavior, during which they will flash—fireflies are capable of emitting light at all stages of their lifecycle. The flashes may be a signal to help coordinate pupation. If a larva doesn’t synchronize with the others when it is ready to pupate, there won’t be any mates around when it hatches. Fireflies don’t live very long as adults, so they must find a mate quickly. In the lab, once the adults hatch, Fallon moves them into another container to prevent them from drowning in the water of the mock river, and hopes that they mate. If they are successful, then the female lays her eggs in the moss Fallon provides, starting the cycle over again.

The fireflies are not easy to keep alive in lab conditions, so the researchers have been experimenting with different conditions for rearing them, like keeping them in boxes of different sizes, changing the aeration of their water, and providing different spaces for the adults to rest when they first hatch.

“We’ve been iterating through a lot of different ways to rear them,” Fallon says. “It’s not like with fruit flies, where you could leave two alone in a room with a banana, and soon you’ve got more fruit flies than you know what to do with. The fireflies are really quite finicky. Over time we’re learning what works and what doesn’t.”

One thing that Fallon has learned from rearing fireflies is how often they glow, during every part of their life cycle. The heike lay their eggs in clumps, which are cumulatively visible to the naked eye. Big dipper firefly eggs also luminesce, but faintly enough that it’s only visible using a sensitive camera. If there’s an evolutionary advantage to having the eggs luminesce, it’s not known. The larvae’s ability to glow is typically believed to be, at least in part, an aposematic signal: a warning to potential predators that the larvae are toxic so the predators won’t try to eat them. Other species use bright colors for the same purpose—think of brilliantly colored poison dart frogs or the popular mnemonic “red touches yellow kills a fellow” used to identify venomous coral snakes—and what’s brighter than something that glows in the dark?

Fireflies’ toxicity comes from chemicals called lucibufagins. Only some firefly species produce these, though the rest may benefit by association if predators associate glowing beetles with a bitter meal. Fallon has noticed that the heike larvae will glow anytime they appear threatened or are faced with the unfamiliar, like if he jostles the box they live in. This threat response is consistent with the expectations for an aposematic signal, but the heike larvae also glow in other conditions, such as during their climbing behavior right before they pupate—and the pupae glow as well. The adults, meanwhile, can also warn off predators with their flashes but primarily luminesce to communicate and find a mate. Different firefly species have distinct flash patterns, as do males and females, allowing adults to identify a suitable partner—and allowing anyone with enough firefly knowledge to identify different species just by watching their flashes.

In every stage of life, fireflies have adapted to make the most of their light-emitting ability. Once they had the genome sequences and firefly specimens in hand, Weng and Fallon wanted to find out how.

What can we learn from fireflies?

The researchers used the firefly genomes to delve into the biomolecular pathways that fireflies use to create light. They identified a luciferin-derived molecule in fireflies that may be a storage form for luciferin, as well as the gene encoding the enzyme that converts luciferin into this molecule. They have also been looking into fireflies’ mechanism for recycling luciferin. The one significant handicap of using luciferase-luciferin in research is that, since the genes encoding luciferin are unknown, the chemical must be fed or injected into specimens manually. This limits the duration of experiments based on when the luciferin is used up, or requires disrupting specimens to reinject them. In the wild, fireflies are able to reuse their luciferin molecules after they have been oxidized to produce light; if researchers could figure out how they do this, it could potentially be applied in the lab.

One major mystery surrounding fireflies that the researchers wanted to solve was whether beetles evolved the ability to luminesce once, or multiple times. Fireflies are one of at least four beetle families that can luminesce—the others are click beetles (Elateridae), glowworm beetles or railroad worms (Phengodidae), and starworms (Rhagophthalmidae). These different beetles all use similar chemistry to luminesce: similar luciferase enzymes and structurally identical luciferins. This commonality suggests that luminescence evolved in a shared ancestor of the four families, and was lost in other related beetles that do not luminesce. However, as Charles Darwin noted, the families of luminescent beetles are very different morphologically, including having distinct light organs—the click beetle emits light from lanterns by its head, whereas the fireflies emit light from their rears. These dissimilarities in shape suggested that the beetle families each evolved luminescence separately; if the light organs evolved from a common ancestor, researchers would expect them to resemble each other. Complicating the matter is that not every species in these families can luminesce; while all known fireflies can emit light, only some click beetles can. This suggests that even within the bioluminescent beetle families, luminescence has evolved multiple times, or been lost multiple times, or some combination thereof.

In order to solve this puzzle, the researchers sequenced the genome of three beetles and compared them: the big dipper firefly, the heike firefly, and the cucubano click beetle (Ignelater luminosus), which is also capable of luminescence. The last common ancestor of heike and big dipper fireflies lived over 100 million years ago, making them good candidates for evolutionary comparison. The researchers found evidence that fireflies and click beetles evolved luminescence independently. They pinpointed where the luciferase gene was in each species’ genome, and what its neighbors were—in the fireflies, the gene was surrounded by genes involved in fatty acid metabolism, suggesting that it evolved from one of these. Meanwhile, the researchers found the click beetle’s luciferase gene in a completely different genetic neighborhood, suggesting that it evolved separately and from a different ancestral gene.

It may seem unusual for such an extraordinary trait as luminescence to have evolved multiple times, but in fact it is a trait that evolution constantly stumbles upon, Fallon says. Beetles are far from the only creatures capable of emitting light; bioluminescence has also evolved in many niches, including in species of fish, coral, jellyfish, squid, snails, fungi, and bacteria.

Fireflies’ luminescence is not a unique trait, but it’s one worth preserving. From fireflies lighting up the night sky at summer festivals and in backyards, to children chasing them through the grass trying to capture a little magic in their hands, to researchers exploring biology with the help of the ultra-sensitive luciferase gene, people benefit from sharing our world with these dazzling little beetles. With the new data coming out of labs like Weng’s, further research benefits from fireflies’ light-making machinery may be on the horizon.

Fallon has learned a lot about the difficulties of rearing fireflies as he tries to maintain a sustainable population in the lab; meanwhile conservationists are struggling to protect populations of fireflies out in the wild. Even the wild population from which Fallon’s fireflies were originally captured no longer exists. Though the species survives, that particular population’s habitat disappeared, leaving the lab-bred beetles as their only legacy. The more that researchers learn about fireflies, the better equipped we may be to protect them from the sort of environmental vulnerabilities that killed off the Weng lab fireflies’ ancestors—both for the sake of the fireflies themselves, and for own sake as spectators and researchers.

Credits

Written by Greta Friar

Video by Conor Gearin

Audio production by Conor Gearin

Cover video by Radim Schreiber / FireflyExperience.org

“The chemistry of light” title card photo by Tim Fallon

“Fireflies in the wild” title card photo by Radim Schreiber / FireflyExperience.org

“Fireflies in the lab” title card photo by Conor Gearin

“What can we learn from fireflies?” title card photo by Conor Gearin

Special thanks to Radim Schreiber, Tim Fallon and Jing-Ke Weng

Works cited:

[1] https://science.sciencemag.org/content/234/4778/856

[2] https://www.science.gov/topicpages/p/planetary+protection+protocols

[3] https://www.massaudubon.org/get-involved/citizen-science/firefly-watch

[4] https://xerces.org/2019/05/15/bethany-beach-firefly/

Junk DNA makes a comeback

Third-year graduate student Emma Kowal is searching DNA for sequences that regulate gene expression.

Saima Sidik
July 8, 2019

“I went into science because of a certain obsession with the romance of it,” says Emma Kowal, a third-year graduate student in Chris Burge’s lab in the MIT Department of Biology. “I loved the idea of the scientist as an adventurer exploring the frontiers of knowledge and the universe. And I haven’t let go of that yet.”

Kowal has always been an avid science fiction reader, and now she’s living out a real-life scientific odyssey. The quest she’s taken on for her PhD research involves an understudied type of DNA sequence called an intron, and the roles that introns might play in regulating gene expression.

Introns lie between the DNA sequences that cells use for protein production, and are initially incorporated into the messenger RNA, or mRNA, that cells produce as an intermediate step in synthesizing proteins from DNA. But before they complete protein synthesis, cells remove introns from mRNA through a process called splicing, which has led many people to view introns as junk DNA with splicing acting like a garbage disposal.

“Introns appeal to me as the underdog genomic region,” Kowal says. Although they’re often seen as unimportant, introns are ubiquitous and plentiful, collectively making up 24% of the human genome. All eukaryotes have them, and, on average, each human gene encodes eight. Many researchers, including Kowal, think that introns have been underestimated, and that they may play an important role in regulating gene expression.

Introns are only the latest chapter in Kowal’s RNA story. She began her research career as a Harvard University undergraduate student working in the Szostak Lab at Massachusetts General Hospital, where she studied how RNA catalyzed the evolution of cells on the early earth. Although studying primordial life was intellectually stimulating, Kowal wanted to work on something more applied, and so she joined the Church Lab in the Harvard Department of Genetics. There she developed methods for purifying and imaging enigmatic RNA-containing lipid compartments called extracellular vesicles, which cells release into their surrounding environments possibly to communicate with one another.

For the sequel to her bachelor’s degree, Kowal chose to attend MIT Biology because she’d heard that, “at MIT, everyone is one standard deviation nerdier, on average, than they are at other schools.” In this sense, she has not been disappointed. Kowal calls the energy at MIT “unparalleled,” and she says, “people are jazzed about what they’re doing, and the whole campus reflects that.”

In some ways, these reflections are physical. Much of the artwork around MIT pays homage to major scientific discoveries, and Kowal says this reverence for science is one factor that attracted her to MIT. From the mural of DNA in the Biology Department to the golden neurons that descend alongside the staircase in the McGovern Institute for Brain Research, it’s as if the community is saying, “look at how awesome the universe is!” as Kowal puts it.

In other ways, this energy is reflected in the people she converses with daily. “I really like the students here,” Kowal says. “Everyone is enthusiastic, but also down to earth.” When she’s not exploring the realms of science, Kowal sometimes has more fanciful adventures with the Dungeons and Dragons group that she’s formed with some of her classmates.

Kowal didn’t necessarily intend to continue working on RNA at MIT Biology, but when she heard about Chris Burge’s lab, which focuses on RNA and the proteins that mediate its production and stability, she felt a call to action.

The Burge Lab combines high throughput experimental techniques with bioinformatics, and Kowal wants to develop expertise in both these fields. “If you’re skilled in generating and analyzing big data sets, you can ask questions that other people can’t,” she says. The Burge lab seemed like the perfect setting for her PhD.

Over and over, scientists have noticed that cells produce more protein from genes that contain introns than when those same introns are removed. Intron mediated enhancement (IME), as this effect is called, is a “stunningly broad phenomenon,” Kowal says, and scientists have observed it in a wide range of organisms, from yeast to plants to humans.Burge asks his students to begin their degrees with a month-long reading period during which they sift through the literature to find a topic that they want to study. “You’re not allowed to pick up a pipette or do any analysis during your reading period,” Kowal says. “You just read and discuss your ideas and let things percolate.” As she read, Kowal came across a number of studies that discussed the influence that introns have on gene expression levels.

Splicing machinery, which removes introns from mRNA, likely plays a role in IME. This machinery binds mRNA as it’s being produced from DNA, then interacts with, and influences, the RNA production machinery. However, researchers have created mutant introns that can’t be recognized by splicing machinery, and sometimes these introns still enhance gene expression, so splicing isn’t the only factor that drives IME. Moreover, replacing one intron with another of the same size containing a different DNA sequence can change its effect, implying that the exact DNA sequences within introns may dictate their effects on gene expression. Kowal is intrigued by this last point, and wants to find these intronic sequences and figure out which have the largest effects on gene expression and why.

“This is an old mystery that’s ripe for new tools,” Kowal says. Over the last decade, researchers have begun using a technique called RNAseq to count the copies of mRNA that are made from each gene in a population of cells. Instead of replacing an intron with a single alternative DNA sequence, Kowal plans to replace an intron with a myriad of random DNA sequences, then use RNAseq to count how many copies of mRNA cells make when they encode each of these random introns.

Preparing to test these random sequences has been an odyssey in and of itself, and Kowal has spent the last year building the system that she’ll use. First, she needed to decide which intron to replace. She chose one from a gene called UbC. Removing this intron reduces expression of UbC by ten-fold.

Besides contributing strongly to IME, the UbC intron is a great candidate for Kowal’s experiment because it lies in a regulatory region of the UbC mRNA that precedes the portion that’s translated into protein. This let her replace the UbC protein coding region with a fluorescent protein that she’ll use to visualize how much protein cells make when they encode each random intron sequence.

Kowal has spent the last year meticulously incorporating a library of random introns into this synthetic version of the UbC gene. She anticipates being able to introduce them into cells soon, to see which random introns result in the highest levels of mRNA and protein production. Thanks to RNAseq, she’ll be able to monitor how much each random intron contributes to mRNA expression. Because she can measure how brightly the fluorescent protein glows, she can correlate these mRNA levels with protein levels. From this, she’ll learn which intron sequences enhance gene expression most strongly, and she’ll also know whether these introns lead to higher levels of mRNA production, or if the same amount of mRNA is made into more protein. This distinction will offer her clues about the mechanism that introns use to enhance gene expression.

Once Kowal knows which intron sequences promote gene expression most effectively, she’ll take advantage of the Burge lab’s bioinformatics expertise to analyze the distribution of these sequences throughout genomes and predict how they affect global gene expression. Kowal suspects certain intron sequences are bound by proteins that mediate mRNA production and stability, and she thinks her work will identify these protein-intron pairs.

Kowal balances her scientific adventures with outdoor adventures. Specifically, she’s recently fallen in love with rock climbing. “Climbing is a great counterpart to science because it’s something you can chip away at, and then there’s this huge satisfaction when you finally achieve a climb,” she says. “And also, between climbing and pipetting, I have really strong fingers.”

As for her love of science fiction, Kowal hopes to one day pen a science-based adventure of her own, but not before she’s made her mark as a scientist, either as a professor or in industry. ”It makes sense for me to focus most of my energy on science right now,” she says. “But after I’ve led a spectacular, adventurous life in science, maybe I’ll use my reflections to write a novel.”

Posted 7.8.19