Caltech Receives Gift from Sackler Foundation to Advance Biomedical Science Research

PASADENA, Calif.—The California Institute of Technology (Caltech) and UCLA have launched highly productive collaborations in cancer research and other areas of biomedicine in recent years, frequently through the Caltech lab of Nobel Laureate and President Emeritus David Baltimore. Now, an endowment established by the Raymond and Beverly Sackler Foundation will strengthen the Caltech-UCLA partnership and advance the Baltimore lab's interdisciplinary research into areas where mathematics and engineering converge with biology.

The Sackler endowment will support three primary areas: postdoctoral researchers working in areas that have a convergent theme; students pursuing a joint MD/PhD degree through the Caltech-UCLA Medical Scientist Training Program; and a joint seminar series between Caltech and UCLA emphasizing the interface of medicine, engineering, and the physical sciences.

"Caltech has a strong interdisciplinary focus that is fueling its outstanding achievements in biomedical research," says Caltech's president Jean-Lou Chameau. "The support from the Raymond and Beverly Sackler Foundation further enhances this research, which can lead to therapies for some of the world's most critical diseases."

The research supported by the endowment will encompass many of the numerous disciplines that have become intertwined with biology in recent years, such as computational mathematics and physics. There is also an important educational component, as the Sackler endowment will help train MD/PhD students and strengthen the link between Caltech's basic science expertise and UCLA's translational medicine focus.

"The Sackler gift is very important because it allows us to expand our research in a direction in which I very much want it to go, and at the same time it will support our very significant MD/PhD program," says Baltimore, the Robert Andrews Millikan Professor of Biology. "We are grateful to Raymond and Beverly Sackler for their generosity and for their foresight in recognizing the importance of our work."

Dr. Raymond Sackler is a physician, entrepreneur, and philanthropist who has supported numerous scientific and cultural initiatives throughout the world. With his late brothers, Arthur and Mortimer, he sponsored the Sackler Faculty of Medicine, and with his wife, Beverly, has sponsored the Raymond and Beverly Sackler Faculty of Exact Sciences, both at Tel Aviv University. David Baltimore and Caltech are one of 10 recipients of endowments from the foundation as part of a recent global program in support of convergent research.

"The purpose of this gift is to catalyze new convergent science investigations and to honor the important research that has been carried out by David Baltimore," says Raymond Sackler. "He made groundbreaking scientific accomplishments at a relatively early age, and now—working at the crossroads of several disciplines—he continues to make major discoveries in biomedical research. We hope that this new endowment will foster his research and Caltech's dynamic collaborations with UCLA."

Baltimore is best known for his work with viruses and the immune system, and he shared the Nobel Prize in Physiology or Medicine in 1975 for his discoveries concerning cancer-inducing viruses. Several years ago, he decided that his research should move toward translational applications, and in 2009 he became the director of a new effort with UCLA—the Joint Center for Translational Medicine—which supports numerous research projects with the potential for clinical applications.

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Caltech Biologist Stephen Mayo Named Inaugural Bowes Division Chair

Bowes Foundation gift will seed innovative projects in the biological sciences

PASADENA, Calif.—Stephen L. Mayo, chair of the Division of Biology and Bren Professor of Biology and Chemistry at the California Institute of Technology (Caltech), has been named the William K. Bowes Jr. Foundation Division Chair. The William K. Bowes, Jr. Foundation, based in San Francisco, endowed the new division leadership chair with a $5 million gift, supplemented by an additional $2.5 million provided by the Gordon and Betty Moore Matching Program.

The new chair is named after the philanthropic foundation created by William K. Bowes, Jr., founding partner of U.S. Venture Partners in Silicon Valley.  For nearly 50 years, Bowes has helped launch numerous biotechnology and other high-tech companies, including Amgen, Applied Biosystems, and Sun Microsystems.

Unlike typical professorships, which fund salaries, the Bowes Foundation Chair—also called a leadership chair—is a permanent endowment that will provide funds that Mayo and future biology division chairs will use to support innovative research projects with potential for scientific and societal impact. It will also be used to support Caltech's education mission and outreach programs.

"The Bowes Chair is unprecedented at Caltech and will be a tremendous asset to advance our science and engineering research and teaching," says Caltech president Jean-Lou Chameau. "This is the first in what will be a series of leadership chairs providing high-leverage, unrestricted support that the faculty who head our divisions will use to seed the Institute's most ambitious educational and research ideas. We are extremely grateful for Bill Bowes's inspirational and generous support."

Bowes has been connected with Caltech for more than 30 years, most notably through the founding of Amgen, which included Caltech biologists Norman Davidson and Leroy Hood as two of its scientific founders; they were also original members of the company's scientific advisory board.  "I have known firsthand for years that Caltech faculty have the unique intellectual skills, imagination, and track record to propel promising entrepreneurial ventures," says Bowes. "With the Bowes Foundation Division Chair, I hope to give Caltech's extremely talented biological scientists the freedom to turn more of their ideas into reality. This is really a carefully considered investment in the future."

 "Bill Bowes's important gift will give Caltech's biology division critical unrestricted funding to foster valuable research, teaching, and outreach programs," says Mayo. "It is an honor to be named the first Bowes Foundation Division Chair, not only because this is such a groundbreaking initiative for Caltech, but also because Bill has been so instrumental in launching entrepreneurial ventures that have improved so many people's lives."

Bowes has had both a varied and an influential career. After service in the Army infantry in the South Pacific and Japan during and after World War II, Bowes received a BA in economics from Stanford and then an MBA from Harvard. He was an investment banker in San Francisco for nearly three decades before founding U.S. Venture Partners in 1981. He founded and served as the first chairman and treasurer of Amgen, which would become the world's largest biotech company.

U.S. Venture Partners has played a leading role in the development of the software, health-care, and e-commerce industries, building companies such as Sun Microsystems and Applied Biosystems. It has also been one of the leading investors in Caltech start-ups, including Axiom Microdevices, Cleave Biosciences, Contour Energy Systems, and Proteolix. (For more on these companies, see "A Snapshot of Start-ups.")

In recent years, Bowes has focused his energies on philanthropy, supporting nonprofit organizations in such areas as medical research, higher education, and the environment. He has also served on numerous committees and boards for a broad range of institutions, including the Environmental Defense Fund, the San Francisco Exploratorium, the Hoover Institution, and the San Francisco Conservatory of Music.

"Bill Bowes has not only been one of the nation's pioneers in venture capital, but he has also demonstrated the effectiveness of prudent investing in philanthropy," says Caltech senior trustee William Davidow, founding partner of Mohr Davidow Ventures, a venture-capital firm based in Menlo Park. "The early experimental research that Bill is supporting today at Caltech will become the innovative entrepreneurial enterprises of tomorrow."

Steve Mayo is a pioneer in protein-design technology, having been the first to design a protein on a computer and then build it in a lab. Working at the interface of theory, computation, and wet-laboratory experimentation, Mayo focuses on developing quantitative approaches to protein engineering, aiming to understand the physical and chemical determinants of protein structure, stability, and function.

His design approach has been incorporated in a suite of software programs called Comprehensive Protein Design Software, and has been applied to a variety of problems ranging from protein fold stabilization to enzyme design. The end goal, he says, is to create protein-based therapies against diseases, new ways of improving agricultural production, and other applications. Mayo has founded or cofounded several companies, including Molecular Simulations, Xencor, and Protabit.

Caltech has a distinguished history of discovery in the biological sciences, recognized by numerous Nobel Prizes. Over its more than 75 years, Caltech's Division of Biology has made many of the research advances that led to the biotechnology and genetic revolutions. In the past, Caltech's biologists made fundamental insights in cellular and molecular biology; today our investigators are both continuing to blaze that path of discovery and applying their knowledge to finding innovative solutions to cancer, AIDS, and other diseases.

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The Physics of Going Viral

Caltech researchers measure the rate of DNA transfer from viruses to bacteria

PASADENA, Calif.—Researchers at the California Institute of Technology (Caltech) have been able, for the first time, to watch viruses infecting individual bacteria by transferring their DNA, and to measure the rate at which that transfer occurs. Shedding light on the early stages of infection by this type of virus—a bacteriophage—the scientists have determined that it is the cells targeted for infection, rather than the amount of genetic material within the viruses themselves, that dictate how quickly the bacteriophage's DNA is transferred.

"The beauty of our experiment is we were able to watch individual viruses infecting individual bacteria,"says Rob Phillips, the Fred and Nancy Morris Professor of Biophysics and Biology at Caltech and the principal investigator on the new study. "Other studies of the rate of infection have involved bulk measurements. With our methods, you can actually watch as a virus shoots out its DNA."

The new methods and results are described in a paper titled "A Single-Molecule Hershey–Chase Experiment," which will appear in the July 24 issue of the journal Current Biology and currently appears online. The lead authors of that paper, David Van Valen and David Wu, completed the work while graduate students in Phillips's group.

In the well-known 1952 Hershey-Chase experiment, Alfred Hershey and Martha Chase of the Carnegie Institution of Washington in Cold Spring Harbor convincingly confirmed earlier claims that DNA—and not protein—was the genetic material in cells. To prove this, the researchers used bacteriophages, which are able to infect bacteria using heads of tightly bundled DNA coated in a protein shell. Hershey and Chase radiolabeled sulfur, contained in the protein shell but not in the DNA, and phosphorous, found in the DNA but not in the protein shell. Then they let the bacteriophages infect the bacterial cells. When they isolated the cells and analyzed their contents, they found that only the radioactive phosphorous had made its way into the bacteria, proving that DNA is indeed the genetic material. The results also showed that, unlike the viruses that infect humans, bacteriophages transmit only their genetic information into their bacterial targets, leaving their "bodies" behind.

"This led, right from the get-go, to people wondering about the mechanism—about how the DNA gets out of the virus and into the infected cell," Phillips says. Several hypotheses have focused on the fact that the DNA in the virus is under a tremendous amount of pressure. Indeed, previous work has shown that the genetic material is under more pressure within its protein shell than champagne experiences in a corked bottle. After all, as Phillips says, "There are 16 microns of DNA inside of a tiny 0.05 micron-sized shell. It's like taking 500 meters of cable from the Golden Gate Bridge and putting it in the back of a FedEx truck." 

Phillips's group wanted to find out whether that pressure plays a dominant role in transferring the DNA. Instead, he says, "What we discovered is that the thing that mattered most was not the pressure in the bacteriophage, but how much DNA was in the bacterial cell."

The researchers used a fluorescent dye to stain the DNA of two mutants of a bacteriophage known as lambda bacteriophage—one with a short genome and one with a longer genome—while that DNA was still inside the phage. Using a fluorescence microscope, they traced the glowing dye to see when and over what time period the viral DNA transferred from each phage into an E. coli bacterium. The mean ejection time was about five minutes, though that time varied considerably.

This was markedly different from what the group had seen previously when they ran a similar experiment in a test tube. In that earlier setup, they had essentially tricked the bacteriophages into ejecting their DNA into solution—a task that the phages completed in less than 10 seconds. In that case, once the phage with the longer genome had released enough DNA to make what remained inside the phage equal in length to the shorter genome, the two phages ejected DNA at the same rate. Therefore, Phillips's team reasoned, it was the amount of DNA in the phage that determined how quickly the DNA was transferred.

But Phillips says, "What was true in the test tube is not true in the cell." E. coli cells contain roughly 3 million proteins within a box that is roughly one micron on each side. Less than a hundredth of a micron separates each protein from its neighbors. "There's no room for anything else," Phillips says. "These cells are really crowded." 

And so, when the bacteriophages try to inject their DNA into the cells, the factor that limits the rate of transfer is how jam-packed those cells are. "In this case," Phillips says, "it had more to do with the recipient, and less to do with the pressure that had built up inside the phage."

Looking toward the future, Phillips is interested in using the methods he and his team have developed to study different types of bacteriophages. He also wants to investigate various molecules that could be helping to actively pull the viral DNA into the cells. In the case of a bacteriophage called T7, for instance, previous work has shown that the host cell actually grabs onto the DNA and begins making copies of its genes before the virus has even delivered all the DNA into the cell. "We're curious whether that kind of mechanism is in play with the lambda bacteriophage," Phillips says.

The current findings have implications for the larger question of how biomolecules like DNA and proteins cross membranes in general, and not just into bacteria. Cells are full of membranes that divide them into separate compartments and that separate entire cells from the rest of the world. Much of the business of cellular life involves getting molecules across those barriers. "This process that we've been studying is one of the most elementary examples of what you could call polymer translocation or getting macromolecules across membranes," Phillips says. "We are starting to figure out the physics behind that process."

In addition to Phillips, Van Valen, and Wu, the other authors on the Current Biology paper are graduate student Yi-Ju Chen; Hannah Tuson of the University of Wisconsin at Madison; and Paul Wiggins of the University of Washington. Van Valen is currently a medical student at UCLA's David Geffen School of Medicine, and Wu is an intern at the University of Chicago. The work was supported by funding from the National Science Foundation, a National Institutes of Health Medical Scientist Training Program fellowship, a Fannie and John Hertz Yaser Abu-Mostafa Graduate Fellowship, and an NIH Director's Pioneer Award.

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Sarkis Mazmanian Discusses Benevolent Bacteria in Scientific American

There are trillions of bacteria living in our bodies, making up complex communities of microbes regulating processes like digestion and immunity. For Caltech biologist Sarkis Mazmanian, they also make up the focus of his research: understanding how the "good" bacteria promote human health. Featured in the cover story for the June issue of Scientific American, he makes a case for devoting more attention to the helpful bugs after years of scientific dedication to pathogens. "It goes against dogma to think that bacteria would make our immune systems function better," he says, in the article. "But the picture is getting very clear: the driving force behind the features of the immune system are commensals."

The magazine is available now on newsstands and the article, "The Ultimate Social Network," can be read online with a subscription.

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Decoding Worm Lingo

Caltech biologists discover that many nematode species make the same types of small-molecule pheromones

PASADENA, Calif.—All animals seem to have ways of exchanging information—monkeys vocalize complex messages, ants create scent trails to food, and fireflies light up their bellies to attract mates. Yet, despite the fact that nematodes, or roundworms, are among the most abundant animals on the planet, little is known about the way they network. Now, research led by California Institute of Technology (Caltech) biologists has shown that a wide range of nematodes communicate using a recently discovered class of chemical cues.

A paper outlining their studies—which were a collaborative effort with the laboratory of Frank C. Schroeder, assistant scientist at the Boyce Thompson Institute for Plant Research (BTI) of Cornell University—was published online April 12 in the journal Current Biology

Previous research by several members of this team had recently shown that a much-studied nematode, Caenorhabditis elegans, uses certain chemical signals to trade data. What was unknown was whether other worms of the same phylum "talk" to one another in similar ways.

But when the researchers looked at a variety of nematodes, they found the very same types of chemicals being combined and used for communication, says Paul Sternberg, the Thomas Hunt Morgan Professor of Biology at Caltech and senior author on the study. "It really does look like we've stumbled upon the letters or words of a universal nematode language, the syntax of which we don't yet fully understand," he says.

Nematodes are wide-ranging creatures; they have been found in hot springs, arctic ice, and deep-sea sediments. Many types of nematodes are harmless, or even beneficial, but others cause damage to plants and harm to humans and animals. Decoding the language of these worms could allow us to develop strategies to prevent the spread of unwanted nematode species, saving time and money for the agricultural and health-care industries.

"We can now say that many—maybe all—nematodes are communicating by secreting small molecules to build chemical structures called ascarosides," says Sternberg, whose past research in C. elegans found that those worms secrete ascarosides both as a sexual attractant and as a way to control the social behavior of aggregation. "It's really exciting and a big breakthrough that tells us what to look for and how we, too, might be able to communicate with this entire phylum of animals." 

Building upon Sternberg's previous findings, he and Andrea Choe, then a graduate student and now a postdoctoral scholar in biology at Caltech, decided to look for evidence of ascarosides in other species of nematodes. These included some parasitic organisms as well as some benign roundworm samples.

"I turned a section of Paul's lab into a parasite zoo, and people were both intrigued by it and terrified to come back there," says Choe. "One day they would see me cutting carrots to culture plant parasites, and the next I would be infecting mosquitoes or harvesting hookworms from rat intestines. We really tried to get as many different samples as we could."

Once they had cultured a sufficient number of different nematode species, the creatures were bathed in a liquid solution dubbed "worm water." This worm water collected the chemicals given off by the nematodes. The worms were then filtered out and sent to Schroeder's lab at BTI to be analyzed using a mass spectrometer—a tool used to deduce the chemical structure of molecules.

"When the results came back from BTI, showing that the same ascarosides were present in all the worm-water samples, I thought that they had made a mistake," says Choe. "It was a very surprising finding."

Using technology developed by Dima Kogan, a former graduate student at Caltech and coauthor of the paper, the researchers were also able to test the responses of various worms to particular ascarosides. Worms were placed on an agar plate, along with an experimental cue—a blend of ascarosides. Any action that might occur on the plates was then recorded; Kogan's software analyzed those recordings frame by frame, counting the number of worms that were either attracted or repelled by the given chemicals.

When asked about the development of the software, Choe explains that it all began when Kogan noticed that the current method involved counting worms by eye. "He was stunned that we would spend our time doing this," says Choe, "and he came up with this software in less than a week. It removed user bias, sped up our research 10-fold, and allowed us to study more chemicals and more species."

Next, the researchers will work to learn more about how the worms actually sense the ascarosides.

"Now that we know these chemicals are broadly present in nematodes, we want to find the genes that are responsible for the ability to respond to these chemicals," says Sternberg, who is also an investigator with the Howard Hughes Medical Institute. "That knowledge could open up a whole other angle, not just for dealing with the chemicals, but for actually interfering with those communication systems a little downstream by hitting the receivers."

The team also plans to continue deconstructing the language they have found among nematodes. For example, Sternberg wonders, how many different combinations of chemicals mean "food," or "mate," or "attack"? If the scientists can crack the code in terms of what different blends mean to different species, they can begin to interfere with the actions of the nematodes that wreak havoc across the world—leading to better eradication of plant pests, as well as human and animal parasites.

"There is only one known worm pheromone used in agriculture," says Choe. "It is time for us change that. This research could be a very big breakthrough on that front."

The Current Biology study, "Ascaroside Signaling is Widely Conserved Among Nematodes," was funded by a grant from the National Institutes of Health and was supported by the Howard Hughes Medical Institute. Additional authors on the study are Stephan H. von Reuss, from Schroeder's lab at BTI; Robin B. Gasser, from the University of Melbourne; and Edward G. Platzer, from UC Riverside.

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Determining a Stem Cell's Fate

Caltech biologists scour mouse genome for genes and markers that lead to T cells

PASADENA, Calif.—What happens to a stem cell at the molecular level that causes it to become one type of cell rather than another? At what point is it committed to that cell fate, and how does it become committed? The answers to these questions have been largely unknown. But now, in studies that mark a major step forward in our understanding of stem cells' fates, a team of researchers from the California Institute of Technology (Caltech) has traced the stepwise developmental process that ensures certain stem cells will become T cells—cells of the immune system that help destroy invading pathogens.

"This is the first time that a natural developmental process has been dissected in such detail, going from step to step to step, looking at activities of all the genes in the genome," says Ellen Rothenberg, the principal investigator on the study and Albert Billings Ruddock Professor of Biology at Caltech. "It means that in genetic terms, there is virtually nothing left hidden in this system."

The study was led by Jingli A. Zhang, a graduate student in Rothenberg's lab, who is now a postdoctoral scholar at Caltech. The group's findings appear in the April 13 issue of the journal Cell.

The researchers studied multipotent hematopoietic precursor cells—stem-cell-like cells that express a wide variety of genes and have the capability to differentiate into a number of different blood-cell types, including those of the immune system. Taking into consideration the entire mouse genome, the researchers pinpointed all the genes that play a role in transforming such precursor cells into committed T cells and identified when in the developmental process they each turn on.  At the same time, the researchers tracked genes that could guide the precursor cells to various alternative pathways.  The results showed not only when but also how the T-cell-development process turned off the genes promoting alternative fates.

"We were able to ask, 'Do T-cell genes turn on before the genes that promote some specific alternative to T cells turn off, or does it go in the other order? Which genes turn on first? Which genes turn off first?'" Rothenberg explains. "In most genome-wide studies, you rarely have the ability to see what comes first, second, third, and so on, in a developmental progression. And establishing those before-after relationships is absolutely critical if you want to understand such a complicated process."

The researchers studied five stages in the cascade of molecular events that yields a T cell—two before commitment, a commitment stage, and then two following commitment. They identified the genes that are expressed throughout those stages, including many that code for regulatory proteins, called transcription factors, which turn particular genes on or off. They found that a major regulatory shift occurs between the second and third stages, when T-cell commitment sets in. At that point, a large number of the transcription factors that activate genes associated with uncommitted stem cells turn off, while others that activate genes needed for future steps in T-cell development turn on.

The researchers looked not only at which genes are expressed during the various stages but also at what makes it possible for those genes to be expressed at that particular time. One critical component of regulation is the expression of transcription-factor genes themselves. Beyond that, the researchers were interested in identifying control sequences—the parts of genes that serve as docking sites for transcription factors. These sequences are often very difficult to identify in mice and humans using classical molecular-biology techniques; scientists have spent as many as 10 years trying to create a comprehensive map of the control sequences for a single gene. 

To create a map of likely control sequences, Zhang studied epigenetic markers. These are chemical modifications, such as those that change the way the DNA is bundled. They become associated with particular regions of DNA as a result of the action of transcription factors and can thereby affect how easy or hard it is for a neighboring gene to be turned on or off. By identifying DNA regions where epigenetic markers are added or removed, Rothenberg's group has paved the way for researchers to identify control sequences for many of the genes that turn on or off during T-cell development.

In some ways, Rothenberg says, her team is taking a backward approach to the problem of locating these control sequences. "What we're saying is, if we can tell that a gene is turned on at a certain point in terms of producing RNA, then we should also be able to look at the DNA sequences right around it and ask, 'Is there any stretch of DNA sequence that adds or loses epigenetic markers at the same time?'" Rothenberg says. "If we find it, that can be a really hot candidate for the control sequences that were used to turn that gene on." 

Two methodologies have made it possible to complete this work. First, ultra-high-throughput DNA sequencing was used to identify when major changes in gene expression occur along the developmental pathway. This technique amplifies DNA sequences taken throughout millions of cell samples, puts all of the bits in order, compares them to the known genome sequence (for mice, in this case), and identifies which of the various genes are enriched, or found in greater numbers. Those that are enriched are the ones most likely to be expressed. The team also used a modified version of this sequencing technique to identify the parts of the genome that are associated with particular epigenetic markers. Coauthor Barbara Wold, Caltech's Bren Professor of Molecular Biology, is an expert in these so-called "next-generation" deep-sequencing technologies and provided critical inspiration for the study.

A second important methodology involved an in vitro tissue-culture system developed in the lab of Juan Carlos Zúñiga-Pflücker of the University of Toronto, which enabled the Caltech researchers to mass-produce synchronized early T-cell precursors and to see the effect of altered conditions on individual cells in terms of producing T cells or other cells.

Zhang is lead author of the paper in Cell, titled "Dynamic transformations of genome-wide epigenetic marking and transcriptional control establish T cell identity." In addition to Zhang, Rothenberg, and Wold, Brian Williams, a senior scientific researcher at Caltech, was also a coauthor. Another coauthor, developmental biologist Ali Mortazavi, was part of Wold's lab and was also associated with the lab of Paul Sternberg when the work was completed; he is now an assistant professor at the University of California, Irvine.

The work was supported by the Beckman Institute, the Millard and Muriel Jacobs Genetics and Genomics Laboratory, the L.A. Garfinkle Memorial Laboratory Fund, the Al Sherman Foundation, the Bren Professorship, the A.B. Ruddock Professorship, and grants from the National Institutes of Health.

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Alexander Varshavsky Awarded Otto Warburg Medal

Alexander Varshavsky, Caltech's Howard and Gwen Laurie Smits Professor of Cell Biology, has been awarded the Otto Warburg Medal of the German Society for Biochemistry and Molecular Biology (GBM). The medal is considered to be the highest German award for biochemists and molecular biologists.

Varshavsky's main recognized contribution was the fundamental discovery, in the 1980s, of biological regulation by intracellular protein degradation and its central importance in cellular physiology. This discovery by the Varshavsky laboratory involved the understanding, through genetic and biochemical insights, of the biological functions of the ubiquitin system, a major proteolytic circuit in living cells. Ubiquitin is a small protein that is present in cells either as a free protein or as a part of complexes with many other proteins. The association of ubiquitin with cellular proteins marks them for degradation or other metabolic fates. Through its ability to destroy specific proteins, the ubiquitin system plays a major role in cell growth and differentiation, DNA repair, regulation of gene expression, and many other biological processes.

"Alexander Varshavsky's research on ubiquitin-dependent intracellular protein degradation has revolutionised that area of research," Professor Irmgard Sinning, president of GBM, said in the citation. "We owe to him a series of discoveries with a tremendous impact on cell biology and helping us to better understand and study numerous diseases."

The recipient of numerous awards, including most recently the 2012 King Faisal International Prize for Science, Varshavsky earned his BS from Moscow State University in 1970 and his PhD from the Institute of Molecular Biology in 1973. He has been Smits Professor at Caltech since 1992.

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Do You Hear What I Hear?

Caltech biologists locate brain's processing point for acoustic signals essential to human communication

PASADENA, Calif.—In both animals and humans, vocal signals used for communication contain a wide array of different sounds that are determined by the vibrational frequencies of vocal cords. For example, the pitch of someone's voice, and how it changes as they are speaking, depends on a complex series of varying frequencies. Knowing how the brain sorts out these different frequencies—which are called frequency-modulated (FM) sweeps—is believed to be essential to understanding many hearing-related behaviors, like speech. Now, a pair of biologists at the California Institute of Technology (Caltech) has identified how and where the brain processes this type of sound signal.

Their findings are outlined in a paper published in the March 8 issue of the journal Neuron.

Knowing the direction of an FM sweep—if it is rising or falling, for example—and decoding its meaning, is important in every language. The significance of the direction of an FM sweep is most evident in tone languages such as Mandarin Chinese, in which rising or dipping frequencies within a single syllable can change the meaning of a word.

In their paper, the researchers pinpointed the brain region in rats where the task of sorting FM sweeps begins.

"This type of processing is very important for understanding language and speech in humans," says Guangying Wu, principal investigator of the study and a Broad Senior Research Fellow in Brain Circuitry at Caltech. "There are some people who have deficits in processing this kind of changing frequency; they experience difficulty in reading and learning language, and in perceiving the emotional states of speakers. Our research might help us understand these types of disorders, and may give some clues for future therapeutic designs or designs for prostheses like hearing implants."

The researchers—including co-author Richard I. Kuo, a research technician in Wu's laboratory at the time of the study (now a graduate student at the University of Edinburg)—found that the processing of FM sweeps begins in the midbrain, an area located below the cerebral cortex near the center of the brain—which, Wu says, was actually a surprise.

"Some people thought this type of sorting happened in a different region, for example in the auditory nerve or in the brain stem," says Wu. "Others argued that it might happen in the cortex or thalamus. "

To acquire high-quality in-vivo measurements in the midbrain, which is located deep within the brain, the team designed a novel technique using two paired—or co-axial—electrodes. Previously, it had been very difficult for scientists to acquire recordings in hard-to-access brain regions such as the midbrain, thalamus, and brain stem, says Wu, who believes the new method will be applicable to a wide range of deep-brain research studies.

In addition to finding the site where FM sweep selectivity begins, the researchers discovered how auditory neurons in the midbrain respond to these frequency changes. Combining physical measurements with computational models confirmed that the recorded neurons were able to selectively respond to FM sweeps based on their directions. For example, some neurons were more sensitive to upward sweeps, while others responded more to downward sweeps.

"Our findings suggest that neural networks in the midbrain can convert from non-selective neurons that process all sounds to direction-selective neurons that help us give meanings to words based on how they are spoken. That's a very fundamental process," says Wu.  

Wu says he plans to continue this line of research, with an eye—or ear—toward helping people with hearing-related disorders. "We might be able to target this area of the midbrain for treatment in the near future," he says.

The Neuron study, "The Generation of Direction Selectivity in the Auditory System," was funded by grants from the Broad Fellows Program in Brain Circuitry of the Broad Foundation and Caltech.

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Understanding Bacterial Sensors

Researchers Piece Together Model of Chemoreceptor Arrays

Nearly all motile bacteria can sense and respond to their surroundings—finding food, avoiding poisons, and targeting cells to infect, for example—through a process called chemotaxis. This allows the bacteria to move towards chemicals they are attracted to, and away from ones that repel them. Because chemotaxis plays a critical role in the first steps of bacterial infection, a better understanding of the process could pave the way for the development of new, more effective antibiotics. Researchers at Caltech are helping to reveal just how chemotaxis works.

In bacteria, the sensing process begins with chemoreceptors—proteins that extend, like tiny antennae, from the cell body to the exterior of the cell. Chemoreceptors bind to attractants, like sugars and amino acids, and to repellents, like metals; they then send signals to motors controlling the whiplike flagella that steer the swimming bacterium in a particular direction.

In an effort to better understand chemotaxis, Grant Jensen, a professor of biology at Caltech, has been working with research specialist Ariane Briegel to determine the exact arrangement of these exquisitely sensitive receptors. Using advanced electron microscopy techniques and new crystallography results, Jensen and Briegel, working with researchers from Cornell University, have built the first model that depicts precisely how chemoreceptors and the proteins around them are structured at the sensing tip of bacteria. Their results appeared recently in the Proceedings of the National Academy of Science (PNAS).

The entire chemotaxis system functions with about 11 proteins, making it one of the simplest examples of a signal transduction pathway (a system in which the activation of a receptor leads to any number of chemical steps that produce a specific response—in this case, a bacterium swimming in a particular direction). In humans, signaling pathways control everything from development and tissue repair to immunity and aspects of brain function; defects in such pathways produce diseases such as diabetes and cancer.  In animal cells, a signal transduction pathway might include 500 proteins. The relatively simple pathway producing the chemotaxis system, therefore, “is the best starting point to understand a full signal transduction pathway," Briegel says.

Brian Crane of Cornell University solved a new crystal structure (top) featuring a double ring of chemoreceptor fragments (pink and purple) and parts of CheA (black and blue) and CheW (green). Split into two (bottom), the receptor fragments in the crystal structure lined up perfectly with Briegel's ECT images.
Credit: Briegel et al./Caltech

In 2009, Jensen's group was able to get the first glimpse of the chemoreceptor architecture. To see it, the researchers used a state-of-the-art electron microscope, purchased by Caltech using a gift from the Gordon and Betty Moore Foundation, that enabled them to observe bacterial cell samples in a near-native state. Unlike traditional electron microscopy—for which samples must be fixed, embedded in plastic, sectioned, and stained—the new imaging technique, called electron cryotomography (ECT), involves freezing samples so quickly that they become trapped within a layer of transparent, glasslike ice. The microscope can then capture many high-resolution images as the sample is rotated.

With that first look, three years ago, Jensen and Briegel discovered that chemoreceptors are arranged in a regular, repeating lattice of hexagons that are 12 nanometers apart, center-to-center.

By rotating the model with two CheA proteins about part of the CheA pair,the team wound up with two connected rings (top) with the proteins and receptors in the same plane. The spacing between those rings explains the hexagonal lattice Jensen's group saw with ECT (middle and bottom).
Credit: Briegel et al./Caltech

But Jensen and Briegel knew they were not seeing the whole picture. Arriving at the complete model required a multistep effort. First, Briegel used improved sample preparation and data-processing procedures to generate even higher resolution images of the honeycomblike chemoreceptor arrays. These new higher-resolution pictures allowed her to determine the precise arrangement of the receptors in these arrays: she discovered that six chemoreceptors are located at each corner of each hexagon. The chemoreceptors are arranged in a pattern that scientists call "trimers of dimers"—that is, groups of three sets of two pairs of receptors. The trimers are arranged such that each pair points toward a center of a hexagon.

Biologists have long known that two additional proteins, called CheA and CheW, are also found within groups of chemoreceptors. These proteins were thought to hold the receptors together and to activate a protein that then binds to the flagellar motors and causes a change in its spinning direction. But no one knew exactly how CheA, CheW, and the receptors were linked.

Understanding that, Jensen says, is "a huge step forward."

For help with the next piece, Caltech researchers teamed up with Brian Crane of Cornell University, who then solved a crystal structure featuring a double ring of chemoreceptor fragments and parts of CheA and CheW. While viewing a computer model of the structure, the researchers realized that splitting the double ring into two and then lining up the receptor fragments in each ring with the receptors in Briegel's ECT images produced a perfect match.

CheA never works alone: it forms in pairs. The ring of Crane's crystal structure, however, only contained part of one CheA. So Crane's group used data from electron spin resonance (ESR) and crystallography experiments to build a model with two CheA proteins. The team discovered that simple rotations of part of the CheA pair brought all of the proteins and receptors into the same plane and produced two connected rings.

"The spacing between those rings explains the hexagonal lattice we see with ECT," says Briegel. "For the first time we have a very convincing model of how this whole receptor array is put together."

The group's next step is to determine what structural changes take place when an attractant binds to a chemoreceptor to send a signal to the flagella motors. Having a model for the whole receptor array, Jensen says, makes that task easier. "Seeing the arrays was one thing," he says. "Now, seeing the receptors with all the helper molecules and how they're arranged and linked together, we have a chance of understanding what happens when one of them gets activated."

Along with Briegel, Jensen, and Crane, additional authors on the PNAS paper, "Bacterial chemoreceptor arrays are hexagonally packed trimers of receptor dimers networked by rings of kinase and coupling proteins," are Xiaoxiao Li and Alexandrine Bilwes of Cornell, and Kelly Hughes of the University of Utah. The work was supported by the Howard Hughes Medical Institute and the National Institutes of Health.

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Kimm Fesenmaier
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Some Bacteria Attack Using Spring-loaded Poison Daggers

PASADENA, Calif.—Bacteria have evolved different systems for secreting proteins into the fluid around them or into other cells. Some, for example, have syringe-like exterior structures that can pierce other cells and inject proteins. Another system, called a type VI secretion system, is found in about a quarter of all bacteria with two membranes. Despite being common, researchers have not understood how it works. Now a team, co-led by researchers at the California Institute of Technology (Caltech), has figured out the structure of the type VI secretion system apparatus and proposed how it might work—by shooting spring-loaded poison molecular daggers.

"People aren't surprised that animals have really interesting ways to hurt each other—snakes have venom, bears have claws," says Grant Jensen, professor of biology at Caltech and coleader of the study. "But they might be surprised that a single cell within one of those animals' bodies is still 100 times larger than the bacterial cells we're talking about, and yet the bacterial cells contain weapons that are so sophisticated. That's the marvel."

The nano-weapon—which spans a distance no longer than about 80 atoms lined up end-to-end—is a tube that contracts very quickly, firing an inner dagger through the cell's membranes, into the surrounding medium and, possibly, into another cell. The tube then disassembles and can reassemble elsewhere in the cell, ready to fire another molecular dagger.

The findings, made in collaboration with researchers at Harvard Medical School, appear as an advance online publication of the journal Nature.

The work began with an accidental discovery. Researchers in the Jensen lab were using an electron cryomicroscope—an electron microscope that enables researchers to observe samples in a near-native state—to image an environmental strain of Vibrio cholerae cells. Unlike traditional electron microscopy—for which samples must be fixed, dehydrated, embedded in plastic, sectioned, and stained—electron cryotomography (ECT) involves freezing samples so quickly that they become trapped within a layer of transparent, glasslike ice. The microscope can then capture high-resolution images as the sample is rotated, and those images can be stitched together to make 3D videos—so-called tomograms.

Jensen and his team wanted to use the technique to observe how V. cholerae cells segregate two duplicate copies of their genetic material before dividing. Instead, they noticed relatively large tubelike structures spanning the entire width of the cells. And they had no idea what the structures were. 

Jensen started sharing preliminary images of the mysterious structures in lectures around the country, asking if anyone knew what they might be. Finally, someone suggested that he talk to John Mekalanos of Harvard Medical School, who was involved in the original discovery of the type VI secretion system. 

The Mekalanos lab made a version of V. cholerae lacking one of the proteins that makes up the tube structure. With that protein knocked out, the type VI secretion system disappeared. In another experiment, they attached fluorescent tags to the proteins and were actually able to watch the structures form and contract within living cells.

"When the tube contracts, that's when it shoots," says Martin Pilhofer, a postdoctoral scholar in Jensen's lab. "That result agrees well with what we had seen using the electron cryomicroscope, where we observed long tubular structures in two different conformations—extended and contracted. Whereas electron cryomicroscopy allowed us to observe the secretion apparatus at high resolution, the fluorescence study gave us more insight into the dynamics of the system."

The firing mechanism is similar to the one used by bacteriophages, viruses that infect bacteria. Phage tails are made up of an outer sheath and an inner tube that gets ejected. Since other researchers had previously established that proteins in the type VI secretion system are similar to those found in various parts of the phage tail and its associated structures, there is even more support for the newly discovered mechanism for the type VI secretion system.

"These amazing tubes inside the cell went undetected for decades of traditional electron microscopy, and they may have stayed that way for many more," says Jensen, who is also an HHMI investigator. "But Caltech made a wise investment a long time ago, with the generous help of the Gordon and Betty Moore Foundation, into our one-of-a-kind electron cryomicroscope, and it is truly what allowed us to see these structures."

In addition to Jensen, Pilhofer, and Mekalanos, other authors on the Nature paper, "Type VI secretion requires a dynamic contractile phage tail-like structure," include Gregory Henderson, a former graduate student in Jensen's lab who is now a resident physician at the Mayo Clinic, and Marek Basler, a postdoctoral scholar at Harvard Medical School. The work was supported by grants from the National Institute of Allergy and Infectious Diseases and the National Institute of General Medical Sciences.

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Kimm Fesenmaier
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