Caltech Researchers Help Unlock the Secrets of Gene Regulatory Networks

PASADENA, Calif.-- A quartet of studies by researchers at the California Institute of Technology (Caltech) highlight a special feature on gene regulatory networks recently published in the Proceedings of the National Academy of Sciences (PNAS).

The collection of papers, "Gene Networks in Development and Evolution Special Feature, Sackler Colloquium," was coedited by Caltech's Eric H. Davidson, the Norman Chandler Professor of Cell Biology. His coeditor was Michael Levine, professor of genetics, genomics and development at the University of California, Berkeley. 

"The control system that determines how development of an animal occurs in each species is encoded in the genome, and the physical location of the sequences where this code is resident is being revealed in a new area of systems biology--the study of gene regulatory networks," says Davidson. Gene regulatory networks are the complex networks of gene interactions that direct the development of any given species.

The papers in the collection focus on the gene regulatory networks of a variety of organisms, including fruit flies, soil-dwelling nematodes, sea urchins, lampreys, and mice.

"These networks lie at the heart of the regulatory apparatus, and they consist of genes that encode proteins that regulate other genes, and the DNA sequences which control when and where they are expressed," says Davidson, who authored a paper in the special feature about a gene regulatory network found in sea urchin embryos. He and Levine also coauthored a perspective in the same issue of the journal on the properties of gene regulatory networks.

In one paper, Ellen V. Rothenberg, one of the two Albert Billings Ruddock Professors of Biology at Caltech, examines, in mice, the intricate developmental pathway that causes blood stem cells to differentiate into T cells, a varied class of immune system cells that help the body fight off infection. 

The paper, Rothenberg says, represents a "codification of everything we know about T cell development. We've found that getting the right balances of the various regulatory signals is absolutely crucial for the T cells to come out right. It gives one a sense of how subtle and sophisticated the regulation can be."

Another study in the special feature by Marianne Bronner-Fraser, the second Albert Billings Ruddock Professor of Biology, focuses on the gene regulatory network underlying neural crest formation in the lamprey, the most primitive living vertebrate. The neural crest is a group of embryonic cells that are pinched off during the formation of the neural tube--the precursor to the spinal cord--and then migrate throughout the developing body to form other nervous system structures. 

The study "reveals order and linkages within the network at early stages," Bronner-Fraser says. "Because the neural crest cell type represents a vertebrate innovation, our work in lampreys shows that this network is ancient and tightly conserved to the base of vertebrates," she says.

The fourth of the Caltech papers, by Paul W. Sternberg, the Thomas Hunt Morgan Professor of Biology at Caltech and an investigator with the Howard Hughes Medical Institute (HHMI), and his colleagues, looks at a postembryonic gene regulatory network in Caenorhabditis elegans, a soil-dwelling worm commonly studied by developmental biologists. The gene regulatory network studied by Sternberg and his colleagues controls the formation of the worm's vulva, which connects the uterus with the outside and allows the passage of sperm and eggs. 

All of the papers in the special feature arise out of presentations at a Sackler Colloquium held at the National Science Foundation's Beckman Center in Irvine, California, in February 2008. 

Davidson's paper, "Gene regulatory network subcircuit controlling a dynamic spatial pattern of signaling in the sea urchin embryo," coauthored with Caltech postdoctoral scholar Joel Smith, was funded by the National Institutes of Health's (NIH) Institute of Child Health and Development and General Medical Sciences Institute and a California Institute of Regenerative Medicine (CIRM) fellowship to Smith.

Rothenberg's paper, "A gene regulatory network armature for T lymphocyte specification," represents a collaboration between Rothenberg and Hamid Bolouri, a visiting associate at Caltech, with support from the NIH, the Albert Billings Ruddock Professorship, the Louis A. Garfinkle Memorial Laboratory Fund, the Al Sherman Foundation, and the DNA Sequencer Royalty Fund. The paper was coauthored by Caltech senior postdoctoral research scholar Constantin Georgescu, and William Longabaugh of the Institute for Systems Biology in Seattle.

Bronner-Fraser's paper, "Gene regulatory networks in neural crest development and evolution," was coauthored by Caltech postdoctoral research scholars Natalya Nikitina and Tatjana Sauka-Spengler.

Sternberg's paper, "The Caenorhabditis elegans vulva: A post-embryonic gene regulatory network controlling organogenesis," was funded by the NIH and the HHMI.

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Three Caltech Scientists Receive Ellison Medical Foundation Awards

The Senior Scholar Awards are aimed at promoting basic research into the underlying processes that control aging

PASADENA, Calif.--The Ellison Medical Foundation (EMF) has awarded Senior Scholar Awards of nearly $1 million each to three California Institute of Technology (Caltech) researchers for exploratory projects in the molecular biology of aging processes and age-related diseases.

The brainchild of Laurence J. Ellison, Oracle cofounder and CEO, and Nobel Prize-winning biologist Joshua Lederberg, the EMF supports basic research that integrates molecular biology and the biomedicine of aging. Its Senior Scholar Awards fund exploratory work by acclaimed researchers, many new to the study of aging. Over the past decade, a board of six distinguished scientists has selected awardees by adhering to Lederberg and Ellison's belief that the way to get positive scientific results was to "look for smart people who had track records of creative, productive work and who had a good idea," according to EMF's website. The foundation would then "give them money and stand back. It would favor basic research that was too risky or speculative to attract mainstream funding."

Caltech's awardees take that mandate seriously. For instance, Jacqueline Barton, the Arthur and Marian Hanisch Memorial Professor and professor of chemistry, plans to use her Senior Scholar Award to explore novel ways the body can defend itself against oxidative damage, a major contributor to aging. Barton is known for her work in understanding charge transport in DNA--examining the way in which electrical charges are moved along a DNA strand, and what role charge transport plays in creating DNA damage. But now she is beginning to consider ways in which charge transport might actually be protecting DNA as well. For instance, Barton believes that DNA charge transport may provide a way for the DNA to send out a long-range signal when it undergoes oxidative damage, alerting DNA-bound proteins such as p53--known as "the guardian of the genome" because of its role in cancer prevention via DNA repair--to set into motion the processes that will eventually lead to the mending of damaged strands. "This would be a paradigm shift with respect to current biological mechanisms for cellular activation," says Barton.

Judith Campbell, professor of chemistry and biology at Caltech, is exploring the ways in which a yeast protein her lab discovered--a DNA-synthesizing enzyme called Dna2--might work to safeguard the bits of DNA at the end of chromosomes, called telomeres. Telomeres are made of repeated sequences of DNA and act to protect the ends of the chromosome from damage, much like the plastic wrapped around the end of a shoelace. Each time a cell divides, however, its telomeres get a little bit shorter; eventually, this aging process leads to the cell's death. But what Campbell has found is that, in yeast at least, Dna2 seems to help maintain the length of the telomeres, slowing down the aging process. She intends to use her Senior Scholar Award to begin studying Dna2 in humans, rather than yeast. "Extending our work to human cells will allow me to contribute to the application of fundamental biology to the improvement of human health," she says. "This has been a burgeoning but frustratingly slow field. We hope this award will allow us to identify new targets--including but not limited to Dna2--whose manipulation can lead to telomere stability. This can, in turn, be expected to have an effect on the life span of the organism as a whole, by keeping at least some of the diseases of aging at bay."

David Baltimore, Caltech President Emeritus and Robert Andrews Millikan Professor of Biology, has been researching the role of tiny bits of RNA--called micro-RNAs or miRs--in the process of aging. First discovered in the 1990s, micro-RNAs appear to control gene expression and seem to play a role in the development and inhibition of cancer, in the development of immune cells, and in the body's response to inflammation. According to Baltimore, miRs can influence a wide variety of behaviors in cells--everything from differentiation to proliferation to functional behavior. Baltimore's group will use the Senior Scholar Award to compare the micro-RNA profiles in the cells of young mice to the profiles found in the cells of old mice. They will focus particular attention on specific miRs they have already discovered, which they have found to play a role in inflammation--a process that seems to increase as we age. "When we find an miR that is affected by aging, we will examine its targets, its cellular specificity, the effects from its overproduction, and the consequences of a knockout," says Baltimore.

"This award spotlights three of Caltech's most prominent researchers in the field," says Caltech president Jean-Lou Chameau. "It recognizes not only the promise of their research efforts, but also the originality of the ideas which they are pursuing. It is from these sorts of programs--programs that are aimed at allowing researchers to venture into new research arenas--that real creativity is nurtured."

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About Caltech: Caltech is recognized for its highly select student body of 900 undergraduates and 1,200 graduate students, and for its outstanding faculty. Since 1923, Caltech faculty and alumni have garnered 32 Nobel Prizes and five Crafoord Prizes.

In addition to its prestigious on-campus research programs, Caltech operates the W. M. Keck Observatory in Mauna Kea, the Palomar Observatory, the Laser Interferometer Gravitational-Wave Observatory (LIGO) and the Jet Propulsion Laboratory. Caltech is a private university in Pasadena, California. For more information, visit http://www.caltech.edu.

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Caltech Researchers Get First Look at How Groups of Cells Coordinate Their Movements

Findings also provide new and detailed insights into what happens during the early stages of embryonic development

PASADENA, Calif.--Using novel imaging, labeling, and data-analysis techniques, scientists from the California Institute of Technology (Caltech) have been able to visualize, for the first time, large numbers of cells moving en masse during some of the earliest stages of embryonic development.

The findings not only provide insight into this stage of development--called gastrulation--but give a more general glimpse at how a living organism choreographs the motions of thousands of cells at one time. Previous research has been generally limited to imaging the movements of single cells.

The work was published in the December 5 issue of the journal Science.

The Caltech team observed the vast reorganization and migration of cells that occurs during gastrulation in Drosophila, the fruit fly. During gastrulation, a single-layered tube of cells collapses and then begins to spread. The scientists focused their attention on the movement of cells from two cross sections of that tube, paying particular attention to the movements of cells in the mesoderm--the inner layer of cells in the early embryo--relative to the cells in the ectoderm, or outer layer of cells. (The endoderm, the innermost of the three primary germ cell layers, has not yet formed at this stage of development.)

The researchers used a technique called two-photon excited fluorescence imaging to get a glimpse at what goes on during gastrulation in a living embryo. "This is the first time we've been able to see mesoderm cells within a developing Drosophila embryo," says Angelike Stathopoulos, assistant professor of biology at Caltech and the paper's principal investigator. "It was important not only because we were able to watch many cells move at one time, but because we were able to take that data and make sense of it."

Making sense of the data meant reducing the movement of each cell to a vector, and then analyzing that vector not just along its x, y, and z axes, but by looking at its cylindrical coordinates. "The spreading has both an angular and a radial component," Stathopoulos notes.

What the data showed was that the mesodermal cells move in a directed manner, traveling down and moving outward--diverging--at the same time. The ectoderm, on the other hand, moves down but converges as it goes. Thus, the mesoderm--which sits atop the ectoderm--passively rides the downward wave of ectodermal movement, but has to actively swim against the tide to spread outward.

Credit: Science; McMahon, Supatto, Fraser, Stathopoulos/Caltech

"It's as if the mesodermal cells are on a moving sidewalk," says Stathopoulos, "but as they're being moved along, they keep taking steps to the side."

The researchers found, in addition, that this choreography is anything but chaotic. The cells stayed in more or less the same order throughout their travels, following a set of "leader" cells and rarely if ever crossing over the midline of the pack.

"If you look at movies of the motion," says Stathopoulos, "you see all this jiggling, and you'd think the cells are mixing all around. And yet, they're not."

"We were able to follow the whole process," adds Scott Fraser, the Anna L. Rosen Professor of Biology, director of the Beckman Institute's Biological Imaging Center, and a paper coauthor. "We were able to label and watch the cells doing the motion, and the events that guide the motion."

In addition to looking at normal gastrulation, the scientists also looked at what happens to gastrulation when mutations occur. "We watched normal behavior, and the cues that guided it," says Fraser. "Then we could use the power of genetics to break one of the cues and analyze what was different, to determine how that cue was involved in the process. Being able to visualize groups of cells lets us do this in a more complex and powerful way. Before, you could say, 'It's broken.' Now, we can say how it is broken."

The findings turned up some other surprising tidbits about fruit-fly embryology, as well. For instance, while scientists had known that the cells in the mesoderm tended to divide twice during gastrulation, they didn't know if this was applicable only to a particular subset of cells, or if it was a hard-and-fast rule. Turns out, it was a rule. "We saw that each and every cell divides twice," says Stathopoulos. "Even more surprising, we saw that the timing of those divisions is based on the cells' original position in the tube--even though, by the time they divide, they've traveled from where they began. They actually remember where they came from."

While Stathopoulos says she's not yet sure what this timed division means, it's a clear sign that there are many more layers of order in embryonic development than had been previously suspected.

"What's fun about this," says Fraser, "is that all the different parts came together at the same time--genetics, labeling, imaging, analysis. It was like a perfect storm . . . in a good way."

The work detailed in the paper, "Dynamic Analyses of Drosophila Gastrulation Provide Insights into Collective Cell Migration," was supported by funding from the National Institutes of Health, the Searle Scholars Program, the March of Dimes, and Caltech's Beckman Institute.

Caltech graduate student Amy McMahon and postdoctoral scholar Willy Supatto shared primary authorship of this paper.

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Caltech Scientists Show Function of Helical Band in Heart

MRI technique provides evidence that could both settle a 50-plus-year debate and create a roadmap for future cardiac surgical techniques

PASADENA, Calif.--Scientists from the California Institute of Technology (Caltech) have created images of the heart's muscular layer that show, for the first time, the connection between the configuration of those muscles and the way the human heart contracts.

More precisely, they showed that the muscular band--which wraps around the inner chambers of the heart in a helix--is actually a sort of twisting highway along which each contraction of the heart travels.

Their findings were published in the December issue of the American Physiological Society journal, Heart and Circulatory Physiology.

Since the days of Leonardo da Vinci, observers of the human body have known that the heart's beat is not a simple in-and-out movement--that it has more than a little bit of a twist to it. "The heart twists to push blood out the same way you twist a wet towel to wring water out of it," explains Morteza Gharib, the principal investigator on the study, and the Hans W. Liepmann Professor of Aeronautics and professor of bioengineering in the Division of Engineering and Applied Science at Caltech.

Some 50 years ago, anatomist Francisco Torrent-Guasp was the first to show the helical configuration of the heart's myocardium--its muscular middle layer, the one that contracts with each heart beat.

But what he and subsequent generations of scientists were unable to do was to connect that myocardial band to the heart's function--to prove that the helical shape is important to the effective beating of the heart. Without that connection, physicians and scientists have tended to look at the heart as "just a piece of meat," says Gharib.

Until now, that is. Using a technique pioneered by Han Wen and his team at the National Institutes of Health, Gharib and his colleague Abbas Nasiraei Moghaddam, a Caltech graduate and visitor in bioengineering, were able to create some of the first dynamic images of normal myocardium in action at the tissue level. "We tagged and traced small tissue elements in the heart, and looked at them in space, so we could see how they moved when the heart contracts," Gharib explains. "In this way, we were able to see where the maximum physical contraction occurs in the heart and when--and to show that it follows this intriguing helical loop."

With each beat of the heart, a wave of contraction starts at the heart's apex--which, despite its name, is actually at the very bottom of the heart--and then travels up through the myocardium. "The only time the whole helix shows up in the images is at the end of systole, which is when the heart is contracting," says Gharib. "This simple band structure is akin to an engine behind the heart pumping action."

In addition to going a long way toward settling the decades-long structure/function debate surrounding Torrent-Guasp's work, this finding also has major implications for the surgical treatment of heart disease, Gharib says. "It's going to change the way we repair the heart," he explains. Knowing that the contractile wave travels along the helical pathway--instead of occurring throughout the heart all at once--has implications for which parts of the heart will be most vulnerable to a surgeon's scalpel, for instance. "Seventy-five percent of the function of the heart depends on this muscle," Gharib says. "Surgeons now know what to cut and what not to cut. This will help them to come up with new and more effective surgical procedures."

The work detailed in the paper, "Evidence for the existence of a functional helical myocardial band," was supported by funding from the Caltech MRI Center through a Discovery grant.

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Caltech Scientists Develop "Barcode Chip" for Cheap, Fast Blood Tests

PASADENA, Calif.-- A new "barcode chip" developed by researchers at the California Institute of Technology (Caltech) promises to revolutionize diagnostic medical testing. In less than 10 minutes, and using just a pinprick's worth of blood, the chip can measure the concentrations of dozens of proteins, including those that herald the presence of diseases like cancer and heart disease.

The device, known as the Integrated Blood-Barcode Chip, or IBBC, was developed by a group of Caltech researchers led by James R. Heath, the Elizabeth W. Gilloon Professor and professor of chemistry, along with postdoctoral scholar Rong Fan and graduate student Ophir Vermesh, and by Leroy Hood, president of the Institute for Systems Biology in Seattle, Washington.

An IBBC, described in a paper in the advance online edition of Nature Biotechnology, is about the size of a microscope slide and is made out of a glass substrate covered with silicone rubber. The chip's surface is molded to contain a microfluidics circuit--a system of microscopic channels through which the pinprick of blood is introduced, protein-rich blood plasma is separated from whole blood, and a panel of protein biomarkers is measured from the plasma.

The chip offers a significant improvement over the cost and speed of standard laboratory tests to analyze proteins in the blood. In traditional tests, one or more vials of blood are removed from a patient's arm and taken to a laboratory, where the blood is centrifuged to separate whole blood cells from the plasma. The plasma is then assayed for specific proteins. "The process is labor intensive, and even if the person doing the testing hurries, the tests will still take a few hours to complete," says Heath. A kit to test for a single diagnostic protein costs about $50.

"We wanted to dramatically lower the cost of such measurements, by orders of magnitude," he says. "We measure many proteins for the cost of one. Furthermore, if you reduce the time it takes for the test, the test is cheaper, since time is money. With our barcode chip, we can go from pinprick to results in less than 10 minutes."

A single chip can simultaneously test the blood from eight patients, and each test measures many proteins at once. The researchers reported on devices that could measure a dozen proteins from a fingerprick of blood, and their current assays are designed for significantly more proteins. "We are aiming to measure 100 proteins per fingerprick within a year or so. It's a pretty enabling technology," Heath says.

To perform the assay, a drop of blood is added to the IBBC's inlet, and then a slight pressure is applied, which forces the blood through a channel. As the blood flows, plasma is skimmed into narrow channels that branch off from the main channel. This part of the chip is designed as if it were a network of resistors, which optimizes plasma separation.

The plasma then flows across the "barcodes." The barcodes consist of a series of lines, each 20 micrometers across and patterned with a different antibody that allows it to capture a specific protein from the plasma passing over. When the barcode is "developed," the individual bars emit a red fluorescent glow, whose brightness depends upon the amount of protein captured.

In the Nature Biotechnology paper, the researchers used the chip to measure variations in the concentration of human chorionic gonadotropin (hCG), the hormone produced during pregnancy. "The concentration of this protein increases by about 100,000-fold as a woman goes through the pregnancy cycle, and we wanted to show that we could capture that whole concentration range through a single test," Heath says.

The scientists also used the barcode chip to analyze the blood of breast and prostate cancer patients for a number of proteins that serve as biomarkers for disease. The types and concentrations of the proteins vary from disease to disease and between different individuals. A woman with breast cancer, for example, will produce a different suite of biomarkers than will a man with prostate cancer, while a woman with an aggressive form of cancer may produce proteins that are different from a woman with a less-deadly cancer.

Those proteins can also change as a patient receives therapy. Thus, determining these biomarker profiles can allow doctors to create individualized treatment plans for their patients and improve outcomes. The ease and the speed with which results can be obtained using the IBBC also will potentially allow doctors to assess their patients' responses to drugs and to monitor how those responses evolve with time, much as a diabetic patient might use a blood glucose test to monitor insulin delivery.

The barcode chip is now being tested in human clinical trials on patients with glioblastoma, a common and aggressive form of brain tumor. The researchers are also using the chips in studies of healthy individuals, to determine how diet and exercise change the composition of the proteins in the blood.

Currently, the barcoded information is "read" with a common laboratory scanner that is also used for gene and protein expression studies. "But it should be very easy to design something like a supermarket UPC scanner to read the information," making the process even more user-friendly, says Fan, the first author on the paper.

"As personalized medicine develops, measurements of large panels of protein biomarkers are going to become important, but they are also going to have to be done very cheaply," Heath says. "It is our hope that these IBBCs will enable such inexpensive and multiplexed measurements."

The paper, "Integrated barcode chips for rapid, multiplexed analysis of proteins in microliter quantities of blood," will be featured as the cover story in the December print edition of Nature Biotechnology. The work was funded by the National Cancer Institute and by the Institute for Collaborative Biotechnologies through a grant from the United States Army Research Office.

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Caltech's David Baltimore and Fiona Harrison Named among America's Best Leaders for 2008

U.S. News Media Group and Harvard's Center for Public Leadership recognize 24 of the country's foremost professionals

PASADENA, Calif.--Two prominent researchers from the California Institute of Technology (Caltech) have been named among the country's 24 top leaders by U.S. News Media Group in association with the Center for Public Leadership (CPL) at Harvard Kennedy School. The 2008 edition of America's Best Leaders--available online at www.usnews.com/leaders and on newsstands Monday, November 24--includes honors for Caltech's David Baltimore and Fiona Harrison.

According to U.S. News, the Best Leaders issue features "some of the country's most visionary individuals," highlighting those professionals "who continue to offer optimism and hope through their work."

Nobel Laureate David Baltimore, the Robert Andrews Millikan Professor of Biology and former president of Caltech, was lauded by the magazine for the way he has "profoundly influenced national science policy on such issues as recombinant DNA research and the AIDS epidemic. He is an accomplished researcher, educator, administrator and public advocate for science and engineering, and is considered one of the world's most influential biologists."

Baltimore was awarded the Nobel Prize in Physiology or Medicine in 1975, at the age of 37. He served as president of Caltech for nine years and was named president emeritus in 2006. He had previously spent almost 30 years on the faculty of the Massachusetts Institute of Technology, where he contributed widely to the understanding of cancer, AIDS, and the molecular basis of the immune response, and where he served as founding director of the Whitehead Institute for Biomedical Research from 1982 until 1990. He served as president of the American Association for the Advancement of Science from 2007 to 2008, and is currently its chair.

Baltimore says he is "very honored to be named among this exceptional group of leaders."

Leadership, he says, "involves a vision of the future, and a generosity of spirit that allows the leader to take pleasure in the accomplishments of others." Leadership can't be about micromanaging every aspect of a large organization, he notes. "It's about picking the right people, motivating them, and encouraging them. You have to make sure those people are pulling in the same direction. You have to catalyze interaction between people so that they see a commonality of interests--so that they follow a common line."

Fiona Harrison, a professor of physics and astronomy at Caltech, was chosen for her work as principal investigator of the NuSTAR (Nuclear Spectroscopic Telescope Array) Mission, a pathfinder mission that will "open the high energy X-ray sky for sensitive study for the first time," she says. She started developing the technologies necessary to realize NuSTAR more than a decade ago, and assembled and led a team of scientists and engineers to design and implement the mission. NuSTAR was selected by NASA through a competitive process and will launch in mid-2011.

Harrison "has devoted her career to studying energetic phenomena in the Universe, including massive black holes and stellar explosions, and developing advanced instrumentation for focusing and detection of X-rays and gamma-rays," notes U.S. News. She is a recipient of a NASA Graduate Student Research Fellowship and winner of the Robert A. Millikan Prize Fellowship in Experimental Physics, and was awarded a Presidential Early Career Award for Scientists and Engineers by President Clinton. She joined the faculty of Caltech in 1995.

"It is an honor to be recognized among such a diverse and distinguished group of people," says Harrison.

"Most aspiring scientists don't focus on leadership as a quality necessary to accomplish their research," she adds. "As experiments and projects get larger and more complex, however, good leadership is often necessary in order to make progress. For me, creating and motivating an effective team were skills I learned in order to make the science I am passionate about happen."

In a collaborative effort between U.S. News and Harvard's CPL, the leaders were selected by a nonpartisan and independent committee, convened and organized by the center, without the participation of U.S. News editors. The selection criteria used by the committee in choosing the honorees included the ability to set direction, achieve results, and cultivate a culture of growth.

"Even though Americans have lost confidence in current leadership, over the past year they have had unique opportunities to observe and debate the qualities of strong leaders," says Brian Kelly, editor of U.S. News & World Report. "With our Best Leaders issue, we widen the lens to examine people who are showing leadership in unexpected ways across a wide variety of fields."

Other honorees include Lance Armstrong, founder of the Lance Armstrong Foundation; Herbie Hancock, chairman of the Thelonious Monk Institute of Jazz Performance Arts; Marian Wright Edelman, founder and president of the Children's Defense Fund; Anthony Fauci, director of the National Institute for Allergy and Infectious Disease; Robert Gates, U.S. Secretary of Defense; and Steven Spielberg, director and producer, and founder of the Shoah Foundation.

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Caltech Researchers Use Electron Cryotomography to Get First 3-D Glimpse of Bacterial Cell-Wall Architecture

Findings represent important advances in both biology and imaging technology

PASADENA, Calif.--The bacterial cell wall that is the target of potent antibiotics such as penicillin is actually made up of a thin single layer of carbohydrate chains, linked together by peptides, which wrap around the bacterium like a belt around a person, according to research conducted by scientists at the California Institute of Technology (Caltech). This first-ever glimpse of the cell-wall structure in three dimensions was made possible by new high-tech microscopy techniques that enabled the scientists to visualize these biological structures at nanometer scales.

"This is both a technological and biological advance," says Grant Jensen, associate professor of biology at Caltech, a Howard Hughes Medical Institute investigator, and the principal investigator on the study.

Their research appears in the online early edition of the Proceedings of the National Academy of Sciences (PNAS).

"Bacterial cells rely on a cage-like net that surrounds them to maintain their integrity," Jensen explains. "If it weren't for this molecular bag, the bacteria couldn't survive; they would likely rupture."

This bag, called a sacculus, is made out of peptidoglycan, a mesh-like structure of carbohydrates (glycans) and amino-acid peptides. It is the sacculus, Jensen notes, that is targeted by the antibiotic penicillin; penicillin blocks a bacterium's ability to grow and remodel the bag to fit it as the bacterium itself grows. "If the bug can't make this bag," Jensen says, "it can't multiply, and you get better."

Researchers have long been interested in understanding the precise architecture of the sacculus. In particular, Jensen and his colleagues have wondered whether the so-called glycan strands--which are cross-linked by peptides to create peptidoglycan--"wrap around the cell like a belt wraps around a person," or whether they stand up from the surface of the bacterial cell, "like grass."

The answer to this debate has eluded the scientists, however, because trying to image such tiny biological objects has been beyond their technological reach. Until now, that is.

"Six years ago, a gift from the Moore Foundation allowed us to buy what is arguably the world's best electron cryomicroscope," says Jensen. "This allowed us to take a different kind of picture of small biological objects than has ever been possible before. These pictures are 3-D images to molecular resolution--you can actually start to see individual biological molecules. Using it, we were able to see this network of glycan strands. It was just remarkable."

By pairing the electron cryotomography and a purification technique that involved removing the sacculi and flattening them in a very thin layer of water, postdoctoral scholar Lu Gan, the paper's first author and a Damon Runyon Fellow, was able to image the peptidoglycan structure in three dimensions, which allows for a virtual 3-D tour of the bacterial sacculus.

"What we saw were long skinny tubes wrapping around the bag like the ribs of a person or a belt around the waist," says Jensen. "We also saw that the sacculus is just a single layer thick."

"This is a clear answer to this old question," adds Gan. "We now know what the architecture of this most basic shape-determining molecule is. We now know the right answer versus having a family of answers, some of which are wrong."

Understanding how the cell wall is built is important, says Jensen, because scientists have long been in the dark about some of the most basic physical and mechanical aspects of bacterial life, including why they are shaped the way they are. "It's hard to understand how a building is constructed unless you can see the studs," he explains. "Now that we can see the studs--now that we can see the basic architecture of the sacculus--we're closer to understanding how a bacterium could direct its own growth, and how drugs that block that process might work."

Also involved in the research reported in PNAS was Songye Chen, a postdoctoral scholar in biology at Caltech.

The paper, "Molecular organization of gram-negative peptidoglycan," was published in the PNAS Early Edition. This work was supported by grants from the National Institutes of Health, a Searle Scholar Award, Caltech's Beckman Institute, and gifts from the Gordon and Betty Moore Foundation and the Agouron Institute. Lu Gan is supported by a fellowship from the Damon Runyon Cancer Research Foundation.

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Caltech Biologists Spy on the Secret Inner Life of a Cell

PASADENA, Calif.-- The transportation of antibodies from a mother to her newborn child is vital for the development of that child's nascent immune system. Those antibodies, donated by transfer across the placenta before birth or via breast milk after birth, help shape a baby's response to foreign pathogens and may influence the later occurrence of autoimmune diseases. Images from biologists at the California Institute of Technology (Caltech) have revealed for the first time the complicated process by which these antibodies are shuttled from mother's milk, through her baby's gut, and into the bloodstream, and offer new insight into the mammalian immune system.

Newborns pick up the antibodies with the aid of a protein called the neonatal Fc receptor (FcRn), located in the plasma membrane of intestinal cells. FcRn snatches a maternal antibody molecule as it passes through a newborn's gut; the receptor and antibody are enclosed within a sac, called a vesicle, which pinches off from the membrane. The vesicle is then transported to the other side of the cell, and its contents--the helpful antibody--are deposited into the baby's bloodstream.

Pamela Bjorkman, Max Delbrück Professor of Biology at Caltech and an investigator with the Howard Hughes Medical Institute, and her colleagues were able to watch this process in action using gold-labeled antibodies (which made FcRn visible when it picked up an antibody) and a technique called electron tomography. Electron tomography is an offshoot of electron microscopy, a now-common laboratory technique in which a beam of electrons is used to create images of microscopic objects. In electron tomography, multiple images are snapped while a sample is tilted at various angles relative to the electron beam. Those images can then be combined to produce a three-dimensional picture, just as cross-sectional X-ray images are collated in a computerized tomography (CT) scan.

"You can get an idea of movement in a series of static images by taking them at different time points," says Bjorkman, whose laboratory studies how the immune system recognizes its targets, work that is offering insight into the processes by which viruses like HIV and human cytomegalovirus invade cells and cause disease.

The electron tomography images revealed that the FcRn/antibody complexes were collected within cells inside large vesicles, called "multivesicular bodies," that contain other small vesicles. The vesicles previously were believed to be responsible only for the disposal of cellular refuse and were not thought to be involved in the transport of vital proteins.

The images offered more surprises. Many vesicles, including multivesicular bodies and other more tubular vesicles, looped around each other into an unexpected "tangled mess," often forming long tubes that then broke off into the small vesicles that carry antibodies through the cell. When those vesicles arrived at the blood-vessel side of the cell, they fused with the cell membrane and delivered the antibody cargo. The vesicles also appeared to include a coat made from a molecule called clathrin, which helps form the outer shell of the vesicles. Researchers previously believed that a vesicle's clathrin cage was completely shed before the vesicle fused with the cell membrane. The new results suggest that only a small section of that coating is sloughed off, which may allow the vesicle to more quickly drop its load and move on for another.

"We are now studying the same receptor in different types of cells in order to see if our findings can be generalized, and are complementing these studies with fluorescent imaging in live cells," Bjorkman says. "The process of receptor-mediated transport is fundamental to many biological processes, including detection of developmental decisions made in response to the binding of hormones and other proteins, uptake of drugs, signaling in the immune and nervous systems, and more. So understanding how molecules are taken up by and transported within cells is critical for many areas of basic and applied biomedical research," she adds.

The paper, "FcRn-mediated antibody transport across epithelial cells revealed by electron tomography," was published in the September 25 issue of Nature. The work was supported by the National Institutes of Health, a Max Planck Research Award, the Gordon and Betty Moore Foundation, the Agouron Institute, and National University of Singapore AcRF start-up funds.

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Kathy Svitil
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Caltech Scientists Engineer Supersensitive Receptor, Gain Better Understanding of Brain's Dopamine System

Receptor may be good target for treatment of smoking addiction, ADHD, and more

PASADENA, Calif.--Genetically modifying a receptor found on the neurons that produce the neurotransmitter dopamine has given California Institute of Technology (Caltech) researchers a unique glimpse into the workings of the brain's dopamine system--as well as a new target for treating diseases that result from either too much or too little of this critical neurotransmitter.

Caltech scientists Henry Lester, Bren Professor of Biology, and Ryan Drenan, senior postdoctoral scholar in biology, worked with colleagues from Caltech, the University of Colorado at Boulder, the Rockefeller University, the University of Utah, and the pharmaceutical company Targacept. They genetically modified a type of brain receptor known as an "a6-containing nicotinic acetylcholine receptor" to make it more sensitive to both nicotine and acetylcholine. (Acetylcholine is another of the brain's neurotransmitters.)

The receptor in question is found primarily on neurons that produce the neurotransmitter dopamine. When the receptor is kicked into action by the presence of either nicotine or acetylcholine--two of the keys that fit its biochemical lock--the receptor prompts the neurons on which it sits to begin pumping out dopamine.

While previous studies of this same receptor had shown what happens when you block its function--when you put the brakes on dopamine production--this was the first time anyone was able to look at what happens when you make the receptor more sensitive and thus put the dopamine system into overdrive. "We were able to not only isolate this receptor's function, but also to amplify it," says Drenan, "and that allowed us to see exactly what it and it alone is capable of doing in the brain."

As it turns out, it's capable of doing a lot. Revved up by even low doses of nicotine, these receptors prompt the neurons on which they are clustered to let loose with a flood of dopamine. This flooding was obvious from the behavior of mice carrying the genetically modified receptors: because dopamine plays an important role in movement, the mice became quickly and significantly hyperactive. In fact, the researchers note, low doses of nicotine affect mice with these hypersensitive receptors in much the same way that amphetamines affect "normal" mice. Looking more closely at this phenomenon, the researchers write, "could be useful in understanding the causes of human hyperactivity such as that observed in ADHD." "This technique also gives researchers the power to activate dopamine neurons selectively," says Lester. "We plan to exploit this opportunity to obtain new knowledge about dopamine neurons' functions."

While these sensitized receptors appear on dopamine neurons throughout the brain, the researchers note that they seem to play an especially critical role in what is called the mesolimbic pathway--one of four pathways that control dopamine production throughout the brain, and the one implicated in the addictive properties of drugs like nicotine.

To this end, Lester's team and their collaborators have already begun to explore the possibilities of targeting these receptors with specific drugs that might work to reduce their sensitivity to nicotine, potentially providing a new line of attack for treating nicotine addiction. In fact, notes Drenan, these same drugs might also one day prove useful in treating other dopamine-related conditions, such as ADHD, Parkinson's disease, and schizophrenia.

"By uncovering the biological role of these receptors, especially with regard to their role in the midbrain dopamine system, we show that they are excellent drug targets," says Drenan.

The paper, "In Vivo Activation of Midbrain Dopamine Neurons via Sensitized, High-Affinity a6* Nicotinic Acetylcholine Receptors," was published in the October 9 issue of the journal Neuron. This work was supported by grants from the National Institutes of Health, the Moore Foundation, the Croll Research Foundation, California's Tobacco-Related Disease Research Program, a Caltech alumnus, and the Howard Hughes Medical Institute.

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Lori Oliwenstein
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Caltech Scientists Find Cells Coordinate Gene Activity with FM Bursts

PASADENA, Calif.-- How a cell achieves the coordinated control of a number of genes at the same time, a process that's necessary for it to regulate its own behavior and development, has long puzzled scientists. Michael Elowitz, an assistant professor of biology and applied physics at the California Institute of Technology (Caltech), along with Long Cai, a postdoctoral research scholar at Caltech, and graduate student Chiraj Dalal, have discovered a surprising answer. Just as human engineers control devices ranging from dimmer switches to retrorockets using pulsed--or frequency modulated (FM)--signals, cells tune the expression of groups of genes using discrete bursts of activation.

Elowitz, who is also a Bren Scholar and an investigator with the Howard Hughes Medical Institute, and his colleagues discovered this process by combining mathematical and computational modeling with experiments on individual living cells. The scientists looked specifically at the molecular changes within simple baker's yeast (Saccharomyces cerevisiae) cells after exposure to excess calcium, which increases in concentration in cells in response to stressful conditions such as high salt levels, alkaline pH, and cell wall damage.

The scientists tracked that response using a protein called Crz1 labeled with a green fluorescent tag. Crz1 is stimulated in response to high calcium levels and activates genes that help protect the cell. The glowing of the fluorescent marker allowed Elowitz and colleagues to visualize the movement of Crz1 as it travelled within the cell from the cytoplasm into the cell nucleus and out again into the cytoplasm. Using time-lapse microscopy, they created "movies" of that movement.

"This allowed us to discover that the localization of the Crz1 protein was randomly switching between nucleus and cytoplasm," says Elowitz. The researchers were able to see the Crz1 protein moving in a coherent fashion. "What's striking is that most of the Crz1 molecules jump in or out of the nucleus together. The typical length of time they stay in the nucleus is constant, but how often they all jump into the nucleus depends on the signal--in this case, calcium. Thus, you can say that calcium levels are 'encoded' in the frequency of these nuclear localization bursts."

Using mathematical modeling, the researchers were then able to determine that the burst-like movement most likely serves to coordinate gene expression. The process is similar to how a dimmer switch on household lights works. Such knobs control the fraction of time that current, which switches on and off rapidly, goes to the light fixture. Rotating the knob varies the relative amount of time that current is on or off, and the resulting intensity of the light is proportional to the fraction of time the switch is on. "The idea of controlling a system by flipping it between extreme 'on' and 'off' states at different rates, rather than fine-tuning it, is sometimes called 'bang bang' regulation," Elowitz says.

"Similarly, the amount of gene expression in the Crz1 system is proportional to the fraction of time that Crz1 is localized to the nucleus. Unlike the dimmer, it is the frequency--how often there are nuclear localization pulses--not the duration of these pulses, which the cell regulates. But in both cases, it is the fraction of time that the system is 'on' that is being controlled," Elowitz says.

One key point, he adds, "is that as the rate of these jumps changes, all genes are affected in the same way. One way of thinking about it is that each 'jump' activates all of the genes, albeit at different levels. Therefore, the expression of each gene is individually proportional to the number or frequency of these jumps, and they are all proportional to each other as well."

The behavior of Crz1 is believed to control roughly 100 target genes. However, Elowitz and his colleagues suspect that frequency-modulated movement may be a common strategy for gene regulation. "Because the problem of coregulation of genes is very general, we suspect frequency modulation may be widespread across many genes, organisms, and cell types. We're now trying to determine how general this phenomenon is by looking at what other genes and cell types use this type of system," he says.

The paper, "Frequency-modulated nuclear localization bursts coordinate gene regulation," was published in the September 25 issue of the journal Nature. The work was supported by grants from the National Institutes of Health and the Packard Foundation.

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Kathy Svitil
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