Caltech Scientists Show Why Anti-HIV Antibodies are Ineffective at Blocking Infection

Findings provide possible explanation for failure of decades-long AIDS vaccine search

Their findings were published last week in the online early edition of the Proceedings of the National Academy of Sciences (PNAS).

"This study helps to clarify the obstacles that antibodies face in blocking infection," says Pamela Bjorkman, the Max Delbrück Professor of Biology at Caltech and a Howard Hughes Medical Institute Investigator, "and will hopefully shed more light on why developing an effective vaccine for HIV has proven so elusive."

Y-shaped antibodies are best at neutralizing viruses--i.e., blocking their entry into cells and preventing infection--when both arms of the Y are able to reach out and bind to their target proteins at more or less the same time. In the case of HIV, antibodies that can block infection target the proteins that stud the surface of the virus, which stick out like spikes from the viral membrane. But an antibody can only bind to two spikes at the same time if those spikes fall within its span--the distance the antibody's structure allows it to stretch its two arms.

"When both arms of an antibody are able to bind to a virus at the same time," says Joshua Klein, a Caltech graduate student in biochemistry and molecular biophysics and the PNAS paper's first author, "there can be a hundred- to thousandfold increase in the strength of the interaction, which can sometimes translate into an equally dramatic increase in its ability to neutralize a virus. Having antibodies with two arms is nature's way of ensuring a strong binding interaction."

As it turns out, this sort of double-armed binding is easier said than done--at least in the case of HIV.

In their PNAS paper, Bjorkman and Klein looked at the neutralization capabilities of two different monoclonal antibodies isolated from HIV-infected individuals. One, called b12, binds a protein known as gp120, which forms the upper portion of an HIV's protein spike. The other, 4E10, binds to gp41, which is found on a lower portion of the spike known as the stalk.

The researchers broke each of the antibodies down into their component parts and compared their abilities to bind and neutralize the virus. They found, as expected, that one-armed versions of the b12 antibody were less effective at neutralizing HIV than two-armed versions. When they looked at the 4E10 antibody, by comparison, they found that having two arms conferred almost no advantage over having only one arm. In addition, they found that larger versions of 4E10 were less effective than smaller ones. These results highlight potential obstacles that vaccines designed to elicit antibodies similar to 4E10 might face.

But b12 has its own obstacles to overcome as well. In fact, when the researchers looked more closely at their data, they realized that the benefits of having two arms--even for b12--were much smaller than those seen for antibodies against viruses like influenza. In other words, the body's natural anti-HIV antibodies are much less effective at neutralizing HIV than they should be.

But why?

"The story really starts to get interesting when we think about what the human immunodeficiency virus actually looks like," says Klein. Whereas a single influenza virus's surface is studded with approximately 450 spikes, he explains, the similarly sized HIV may have fewer than 15 spikes.

With spikes so few and far between, finding two that both fall within the reach of a b12 or 4E10 antibody--the spans of which generally measure between 12 and 15 nanometers--becomes much more of a challenge.

"HIV may have evolved a way to escape one of the main strategies our immune system uses to defeat infections," says Klein. "Based on these data, it seems that the virus is circumventing the bivalent effect that is so key to the potency of antibodies."

"I consider this a very important paper because it changes the focus of the discussion about why anti-HIV antibodies are so poor," adds virologist David Baltimore, the Robert Andrews Millikan Professor of Biology and a Nobel Prize winner. "It brings attention to a long-recognized but often forgotten aspect of antibody attack--that they attack with two heads. What this paper shows is that anti-HIV antibodies are restricted to using one head at a time and that makes them bind much less well. Responding to this newly recognized challenge will be difficult because it identifies an intrinsic limitation on the effectiveness of almost any natural anti-HIV antibodies."

In addition to Bjorkman and Klein, the authors on the PNAS paper, "Examination of the contributions of size and avidity to the neutralization mechanisms of the anti-HIV antibodies b12 and 4E10," are Caltech research technicians Priyanthi Gnanapragasam, Rachel Galimidi, and Christopher Foglesong, and senior research specialist Anthony West, Jr.

The work described in the paper was supported by a Bill and Melinda Gates Foundation Grant through the Grand Challenges in Global Health Initiative and the Collaboration for AIDS Vaccine Discovery.

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Lori Oliwenstein

Caltech Scientists Control Complex Nucleation Processes using DNA Origami Seeds

PASADENA, Calif.--The construction of complex man-made objects--a car, for example, or even a pizza--almost invariably entails what are known as "top-down" processes, in which the structure and order of the thing being built is imposed from the outside (say, by an automobile assembly line, or the hands of the pizza maker).

"Top-down approaches have been extremely successful," says Erik Winfree of the California Institute of Technology (Caltech). "But as the object being manufactured requires higher and higher precision--such as silicon chips with smaller and smaller transistors--they require enormously expensive factories to be built."

The alternative to top-down manufacturing is a "bottom-up" approach, in which the order is imposed from within the object being made, so that it "grows" according to some built-in design.

"Flowers, dogs, and just about all biological objects are created from the bottom up," says Winfree, an associate professor of computer science, computation and neural systems, and bioengineering at Caltech. Along with his coworkers, Winfree is seeking to integrate bottom-up construction approaches with molecular fabrication processes to construct objects from parts that are just a few billionths of a meter in size that essentially assemble themselves.

In a recent paper in the Proceedings of the National Academy of Sciences (PNAS), Winfree and his colleagues describe the development of an information-containing DNA "seed" that can direct the self-assembled bottom-up growth of tiles of DNA in a precisely controlled fashion. In some ways, the process is similar to how the fertilized seeds of plants or animals contain information that directs the growth and development of those organisms.

"The big potential advantage of bottom-up construction is that it can be cheap"--just as the mold that grows in your kitchen does so for free--"and can be massively parallel, because the objects construct themselves," says Winfree.

But, he adds, while bottom-up approaches have been extremely useful in biology, they haven't played as significant a role in technology, "because we don't have a great grasp on how to design systems that build themselves. Most examples of bottom-up technologies are specific chemical processes that work great for a particular task, but don’t easily generalize for constructing more complex structures."

To understand how complexity can be programmed into bottom-up molecular fabrication processes, Winfree and his colleagues study and understand the processes--or algorithms--that generate organization not just in computers but also in the natural world.

"Tasks can be solved by carrying out well-defined rules, and these rules can be carried out by a mindless mechanism such as a computer," he says. "The same set of rules can perform different tasks when given different inputs, and there exist 'universal programs' that can perform any task required of it, as specified in its input. Your laptop is such a universal computer; it can run any software that you download, and in principle, any feasible task could be programmed."

These principles also have been exploited by natural evolution, Winfree says: "Every cell, it appears, is a kind of universal computer that can be instructed in seemingly limitless ways by a DNA genome that specifies what chemical processes to execute, thus building an active organism. The aim of my lab has been to understand algorithms and information within molecular systems."

Winfree's investigations into algorithmic self-assembly earned him a MacArthur "genius" prize in 2000; his collaborator, Paul W. K. Rothemund, a senior research associate at Caltech and a coauthor of the PNAS paper, was awarded the same no-strings-attached grant in 2007 for his work designing scaffolded "DNA origami" structures that self-assemble into nearly arbitrary shapes (such as a smiley face and a map of the Western Hemisphere).

The structures designed by Rothemund, which could eventually be used in smaller, faster computers, were used as the seeds for the programmed self-assembly of DNA tiles described in the current paper.

In the work, the researchers designed several different versions of a DNA origami rectangle, 95 by 75 nanometers, which served as the seeds for the growth of different types of ribbon-like crystals of DNA. The seeds were combined in a test tube with other bits of DNA, called "tiles," heated, and then cooled slowly.

"As it cools, the first origami seed and the individual tiles form, as their component DNA molecules begin sticking to each other and folding into shape--but the tiles and origami don't stick to each other yet," Winfree explains.

"Then, at a lower temperature, the tiles start to stick to each other and to the origami. The critical concept here is that the DNA tiles will only form crystals if the process gets started by a seed, upon which they can grow," he says.

In this way, the DNA ribbons self-assemble themselves, but only into forms such as ribbons with particular widths and ribbons with stripe patterns prescribed by the original seed.

The work, Winfree says, "exhibits a degree of control over information-directed molecular self-assembly that is unprecedented in accuracy and complexity, which makes me feel that we are finally beginning to understand how to program information into molecules and have that information direct algorithmic processes."

The paper, "An information-bearing seed for nucleating algorithmic self-assembly," was published in the March 24 issue of the Proceedings of the National Academy of Sciences.

The other authors of the paper are undergraduate Robert D. Barish and visiting scholar Rebecca Schulman. The work was supported by grants from the National Aeronautics and Space Administration's astrobiology program, the National Science Foundation, and the Focus Center Research Program, and a gift from Microsoft Research.

Kathy Svitil

Caltech Researchers Train Computers to Analyze Fruit-Fly Behavior

Program will make it possible to link genes to behaviors, scientists say

PASADENA, Calif.--Scientists at the California Institute of Technology (Caltech) have trained computers to automatically analyze aggression and courtship in fruit flies, opening the way for researchers to perform large-scale, high-throughput screens for genes that control these innate behaviors. The program allows computers to examine half an hour of video footage of pairs of interacting flies in what is almost real time; characterizing the behavior of a new line of flies "by hand" might take a biologist more than 100 hours.

This work--led by Pietro Perona, the Allen E. Puckett Professor of Electrical Engineering at Caltech, and David J. Anderson, the Roger W. Sperry Professor of Biology at Caltech, and a Howard Hughes Medical Institute Investigator--is detailed in the April issue of Nature Methods.

"Everyone wants to know how genes control behavior," notes Anderson. "But in order to apply powerful genetic analyses to complicated social behaviors like aggression and courtship, you need accurate ways of measuring--of scoring--those behaviors."

Previously, the only way to do this was to have students "watch video tapes over and over to record one particular type of behavior at a time," says Anderson. Using this method to measure a number of different types of behaviors--like lunging, tussling, chasing, circling, and copulating--or even to determine the way the flies orient their bodies or set their wings when they encounter another fly, requires the student to watch the same bit of video repeatedly, each time looking at the behavior of a single pair of flies. "In order to screen for mutations affecting aggressive behavior, we would have to analyze something like 2,000 pairs of flies," says Anderson. "It's been virtually impossible to do this without a small army of graduate students."

Enter Perona and Heiko Dankert, a postdoctoral scholar in electrical engineering. Using the techniques of machine vision and combining them with other engineering advancements, the two began training computers to see and recognize aggression and courtship behaviors. The result? An automated system that can monitor a wide variety of behaviors in videos of interacting fruit-fly pairs in a matter of minutes.

"This is a coming-of-age moment in this field," says Perona. "By choosing among existing machine vision techniques, we were able to put together a system that is much more capable than anything that had been demonstrated before."

The team fed the computer the characteristic details of what each individual behavior looks like on video. A lunge, for instance, begins with a shortening of the fly's body as the fly rears up; the fly then makes a quick darting movement, closing to within a few centimeters of another fly.

Once the computer had mastered these details, the researchers then compared the computer's analysis of a piece of video to the analysis produced by a human. "We looked at how many instances the computer caught, and how many it missed," says Anderson. "By looking at the errors the computer made, we were able to further refine our descriptions to create an even more accurate system."

In the end, Anderson notes, this back-and-forth resulted in a program that is "actually better than humans at detecting some of the instances of the various behaviors."

"Where previous experiments had been carried out on 100 to 1,000 frames of video, we carried out our experiments on 100,000 frames of video," Perona adds. "And while previous experiments showed numerous errors in tracking, we get very few. We are able to give accurate performance figures."

The next step, says Anderson, is to try to extend this automatic behavior-detection system to mice--a more difficult task when you're dealing with a fuzzy-edged creature like a mammal, but one that is important if we hope to some day link the genes behind fruit-fly behaviors with the genes that may cause similar behaviors in humans.

"Our visual system tells us a lot about what other people are doing--who is eating, who is beating someone else up, who is blushing, who got the guy or girl," Perona notes. "One goal of my field, computational vision, is designing machines that can detect and interpret human intentions, actions and activities. To do that, we need to start with organisms that are simpler and easier to study. David Anderson showed me how interesting and rich fly behaviors are, and so we started collaborating."

"There's a lot of information in these videos that we can now squeeze out in order to understand what controls these social interactions in flies," Anderson adds. "It makes it possible for us to study what we were not capable of studying before."

In addition to Anderson, Perona, and Dankert, other authors on the Nature Methods paper, "Automated Monitoring and Analysis of Social Behavior in Drosophila," include Caltech graduate student Liming Wang, and Eric Hoopfer, a Caltech postdoctoral scholar in biology.

This work was supported by the Howard Hughes Medical Institute, the National Science Foundation, and a Feodor Lynen fellowship awarded to Dankert by the Alexander von Humboldt Foundation.

Lori Oliwenstein

Caltech Researchers Find Tiny Genetic Change Keeps Nicotine from Binding to Muscle Cells

Research provides insight into the way nicotine works in the brain

PASADENA, Calif.--A tiny genetic mutation is the key to understanding why nicotine--which binds to brain receptors with such addictive potency--is virtually powerless in muscle cells that are studded with the same type of receptor. That's according to California Institute of Technology (Caltech) researchers, who report their findings in the March 26 issue of the journal Nature.

By all rights, nicotine ought to paralyze or even kill us, explains Dennis Dougherty, the George Grant Hoag Professor of Chemistry at Caltech and one of the leaders of the research team. After all, the receptor it binds to in the brain's neurons--a type of acetylcholine receptor, which also binds the neurotransmitter acetylcholine--is found in large numbers in muscle cells. Were nicotine to bind with those cells, it would cause muscles to contract with such force that the response would likely prove lethal.

Obviously, considering the data on smoking, that is not what happens. The question has long been: Why not?

"It's a chemical mystery," Dougherty admits. "We knew something subtle had to be going on here, but we didn't know exactly what."

That subtlety, it turns out, lies in the slight tweaking of the structure of the acetylcholine receptor in muscle cells versus its structure in brain cells.

The shape of the acetylcholine receptor, and the way the chemicals that bind with it contort themselves to fit into that receptor, is determined by a number of different weak chemical interactions. Perhaps most important is an interaction that Dougherty calls "underappreciated"--the cation-π interaction, in which a positively charged ion and an electron-rich π system come together.

Back in the late 1990s, Dougherty and colleagues had shown that the cation-π interaction is indeed a key part of acetylcholine's ability to bind to the acetylcholine receptors in muscles. "We assumed that nicotine's charge would cause it to do the same thing, to have the same sort of strong interaction that acetylcholine has," says Dougherty. "But we found that it didn't."

This would explain why smoking doesn't paralyze us; if the nicotine can't get into the muscle's acetylcholine receptors, it can't cause the muscles to contract.

But how, then, does nicotine work its addictive magic on the brain?

It took another decade for the scientists to be able to peek at what happens in brain cells' acetylcholine receptors when nicotine arrives on the scene. Turns out that in brain cells, unlike in muscle cells, nicotine makes the exact same kind of strong cation-π interaction that acetylcholine makes in both brain and muscle cells.

"In addition," Dougherty notes, "we found that nicotine makes a strong hydrogen bond in the brain's acetylcholine receptors. This same hydrogen bond, in the receptors in muscle cells, is weak."

The cause of this difference in binding potency, says Dougherty, is a single point mutation that occurs in the receptor near the key tryptophan amino acid that makes the cation-π interaction. "This one mutation means that, in the brain, nicotine can cozy up to this one particular tryptophan much more closely than it can in muscle cells," he explains. "And that is what allows the nicotine to make the strong cation-π interaction."

Dougherty says the best way to visualize this change is to think of the receptor as a box with one open side. "In muscle cells, this box is slightly distorted, so that the nicotine can't get to the tryptophan," he says. "But in the brain, the box is subtly reshaped. That's the thing: It's the shape, not the composition, of the box that changes. This allows the nicotine to make strong interactions, to become very potent. In other words, it's what allows nicotine to be addictive in the brain."

"Several projects in our labs are converging on the molecular and cellular mechanisms of the changes that occur when the brain is repeatedly exposed to nicotine," adds study coauthor Henry Lester, the Bren Professor of Biology at Caltech. "We think that the important events begin with the rather tight and selective interaction between nicotine and certain receptors in the brain. This Nature paper teaches us how this interaction occurs, at an unprecedented level of resolution."

Dougherty notes that these findings might one day lead to better drugs to combat nicotine addiction and other neurological disorders. "The receptor we describe in this paper is an important drug target," he says. "It might help pharmaceutical companies develop a better drug than nicotine to do the good things nicotine does--enhance cognition, increase attention--without being addictive and toxic."

The research described in the Nature paper, "Nicotine binding to brain receptors requires a strong cation-π interaction," was supported by the National Institutes of Health and the California Tobacco-Related Disease Research Program of the University of California. In addition to Dougherty and Lester, the paper's coauthors include Xinan Xiu, a former Caltech graduate student, and current graduate students Nyssa Puskar and Jai Shanata. Shanata's work on this research was partially supported by a National Research Service Award training grant.

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Lori Oliwenstein

Caltech Scientists Discover Mechanism for Wind Detection in Fruit Flies

Researchers say flies' antennae use different populations of neurons to detect wind and sound

PASADENA, Calif.--Tiny, lightweight fruit flies need to know when it's windy out so they can steady themselves and avoid being knocked off their feet or blown off course. But how do they figure out that it's time to hunker down? According to a team led by California Institute of Technology (Caltech) scientists reporting in this week's issue of the journal Nature, the flies have evolved a specialized population of neurons in their antennae that let them know not only when the wind is blowing, but also the direction from which it is coming.

The behavior of fruit flies in the face of a stiff breeze is remarkable in and of itself, notes David J. Anderson, the Roger W. Sperry Professor of Biology at Caltech, and a Howard Hughes Medical Institute (HHMI) Investigator. "We discovered that you can stop a fly dead in its tracks by blowing a gentle stream of air over it," he explains, adding that the flies' immobility is so complete, you could pick one up with a pair of chopsticks as long as a steady stream of wind was passing over the insect. Once the wind stops blowing, however, the flies immediately start walking around again. [EDITORS: Video of this behavior in the flies is available on request.]

But the response is also of interest from a scientific point of view, because it represents a fairly simple, innate defensive response that scientists can begin to tease apart in order to understand just how such behaviors are programmed in our genes. "It's more than just stupid pet tricks with fruit flies," Anderson says.

"We quickly realized that it would be interesting to ask just how the wind acts on the flies to make them stop walking. How do they sense the wind? How do they transfer that message to their brain so they know to stop moving while the wind is blowing?"

As it turns out, fruit flies are unusual in how they sense wind. Other insects have sensory hairs that stand up from the cuticle--or outer body wall--and, when blown about by a passing wind, trigger a neural response. The fruit flies, on the other hand, use their antennae to detect a breeze and its general direction, based on how the antenna moves in the breeze.

"This posed a bit of a puzzle for us," Anderson explains. "It's been long assumed that the main function of the neurons in the antennae was hearing."

And that is at least one of the antennae's functions. The flies' antennae detect nearby sounds--like the male's courtship song--that cause vibrations in the air, a bit like ripples in a pond after a rock has been thrown. Those vibrations twist the antennae slightly, exciting the neurons within.

Wind, on the other hand, is not a regularly oscillating wave; instead, it's a steady stream of air particles moving past the fly from various directions. The antennae move in the wind, but they don't twist rapidly back and forth as they do in response to sound.

Says Anderson: "What we wanted to understand was, how can flies tell the difference between sound and wind using the same sensory organ?"

There were two possible answers to this question. The first was that a fly's antennae are equipped with a single, versatile type of neuron that changes its firing pattern depending on whether it's detecting sound or wind, and that the differences in that firing pattern are picked up and somehow decoded by the fly's brain.

The other possibility, says Anderson, was that a fly's antennae contain two distinctly different populations of neurons--one that responds to oscillating air to detect sound, and another that responds to flowing air particles to detect wind.

The right answer? Number two. By selectively knocking out subsets of neurons, Anderson's graduate student Suzuko Yorozu was able to show that Johnston's organ--an area in a fruit fly antenna where sound detection is known to occur--does indeed contain at least two entirely separate groups of neurons. She also showed that each neuron type detects only one type of stimulus (sound for one; wind for the other), and that each sends its message to a distinct and separate area of the brain.

"The sound-sensitive neurons are preferentially activated by small movements of the antenna that are oscillatory in nature, firing only when the antenna twists, and turn off quickly," says Anderson. "The neurons that respond to wind, on the other hand, turn on when the antenna is pushed by air flow, and they stay on until the wind stops blowing." In other words, says Yorozu, "the intrinsic properties of these neurons are very different."

The end result of these separate pathways is that the flies exhibit absolutely distinct types of behaviors, with the sound-detecting neurons leading to behaviors like copulation (in the case of the courtship song), while the wind-detecting neurons prompt flies to come to a dead stop for safety's sake when air is blowing past with any real speed.

In addition to Anderson and Yorozu, other authors on the Nature paper, "Distinct sensory representations of wind and near-field sound in the Drosophila brain," include Caltech and HHMI postdoctoral scholar Allan Wong, Caltech visiting associate Brian Fischer, Caltech postdoctoral scholar Heiko Dankert, Maurice Kernan from SUNY Stony Brook, Azusa Kamikouchi from the University of Tokyo and the University of Cologne, and Kei Ito from the University of Cologne.

This work was supported by a grant from the National Science Foundation.

Lori Oliwenstein

Caltech Biologists Find Optimistic Worms Are Ready for Rapid Recovery

PASADENA, Calif.-- For the tiny soil-dwelling nematode worm Caenorhabditis elegans, life is usually a situation of feast or famine. Researchers at the California Institute of Technology (Caltech) have found that this worm has evolved a surprisingly optimistic genetic strategy to cope with these disparate conditions--one that could eventually point the way to new treatments for a host of human diseases caused by parasitic worms.

As reported in a paper published in the February 26 issue of Science Express, Paul W. Sternberg, the Thomas Hunt Morgan Professor of Biology at Caltech and an investigator with the Howard Hughes Medical Institute, along with postdoctoral scholar L. Ryan Baugh, looked at the worms' genetic response to conditions of scarcity and plenty.

In dozens of batches of the worms, consisting of tens of millions of individuals, Baugh, now an assistant professor at Duke University, synchronized hatching, so that all of the animals in each batch emerged from their eggs at the same time.

Some of the hatched worms were allowed to develop under conditions with scarce nutrients, and others with plentiful nutrients. At precise time intervals (3, 6, 9, 12, and 15 hours after hatching), subsets of both populations were killed en masse and ground up. Their messenger RNA--the genetic material that is produced upon the activation of genes and then translated to produce proteins--was harvested and analyzed at Caltech's Jacobs Genetics and Genomics Laboratory, a specialized facility designed to conduct large-scale genetic analyses.

In this way, the researchers measured the expression of every one of the worms' approximately 20,000 genes, to determine how that expression differed depending on food availability.

"We also did an experiment in which we took the starved worms and refed them, and took the fed worms and starved them, to see how rapid their response was to the changing conditions," Sternberg says.

The researchers found that the worms responded far more rapidly to being fed than being starved. Being fed also caused the activation of a far greater number of genes than did starvation. For example, three hours of feeding worm larvae that had previously been starved caused the activation of 381 genes, while starving formerly fed worm larvae for three hours caused the activation of only 56 genes.

In addition, the research revealed that as many genes are involved in the worms' response to nutrition as are involved in their overall development. Many of the genes that play a role in that nutritional response have to do with energy metabolism, and in changing the way the animals utilize and store energy.

"It looks like C. elegans is primed to respond faster to better conditions. It is optimistic," Sternberg says. "These worms live, most of the time, in scarcity. They are facing bad conditions--that is, no food--most of the time. Probably they've evolved to take advantage when times get better for a brief period. They grow and reproduce."

The worms' quick response to food appears to be controlled by a vital cellular protein called RNA Polymerase II (RNA Pol II), which is responsible for transcribing DNA into mRNA. In a separate experiment, Sternberg and his colleagues found that RNA Pol II accumulates on genes that respond rapidly to being fed, but in advance of that feeding.

"We speculate that this polymerase accumulation is part of the way in which they can respond so quickly. It's already engaged, ready to go, ready to send out the message. It's like having Paul Revere on the North Shore, ready to ride, when the food comes," Sternberg says.

"It is kind of interesting in hard economic times to think whether we can learn anything from this organism, in terms of being optimistic or pessimistic. Maybe the take-home message is that sometimes when you are faced with scarcity, you should still be optimistic."

Sternberg speculates that other nematodes, including the parasitic worms that cause elephantiasis in humans, and other lymphatic filarial diseases, may also go through similar transitions in nutrition as they transition from one host (say, a mosquito) to another (a human). Those transitions may be mediated by a similar accumulation of RNA Pol II on particular genes. Identifying those genes could provide potential targets for new types of therapeutic drugs.

The paper, "RNA Pol II Accumulates at Promoters of Growth Genes During Developmental Arrest," was coauthored by Baugh, Sternberg, and John DeModena, a member of the biology research staff at Caltech. The work was supported by the Howard Hughes Medical Institute.


Kathy Svitil

Caltech Scientists Find Evidence for Precise Communication Across Brain Areas During Sleep

PASADENA, Calif.--By listening in on the chatter between neurons in various parts of the brain, researchers from the California Institute of Technology (Caltech) have taken steps toward fully understanding just how memories are formed, transferred, and ultimately stored in the brain--and how that process varies throughout the various stages of sleep.

Their findings, published in the February 26 issue of the journal Neuron, may someday even help scientists understand why dreams are so difficult to remember.

Scientists have long known that memories are formed in the brain's hippocampus, but are stored elsewhere--most likely in the neocortex, the outer layer of the brain. Transferring memories from one part of the brain to the other requires changing the strength of the connections between neurons and is thought to depend on the precise timing of the firing of brain cells.

"We know that if neuron A in the hippocampus fires consistently right before neuron B in the neocortex, and if there is a connection from A to B, then that connection will be strengthened," explains Casimir Wierzynski, a Caltech graduate student in computation and neural systems, and first author on the Neuron paper. "And so we wanted to understand the timing relationships between neurons in the hippocampus and the prefrontal cortex, which is the front portion of the neocortex."

The research team--led by Athanassios Siapas, a Bren Scholar in the Caltech Division of Biology and an associate professor of computation and neural systems--used high-tech recording and computational techniques to listen in on the firing of neurons in the brains of rats. These techniques helped them pinpoint a number of neuron pairs that had precisely the kind of synchronous relationship they were looking for--one in which a hippocampal neuron's firing was followed within milliseconds by the firing of a neuron in the prefrontal cortex.

"This is exactly the kind of relationship that would be needed for the hippocampus to effect changes in the neocortex--such as the consolidation, or laying down, of memories," adds Wierzynski.

Once these spike-timing relationships between the hippocampal and prefrontal cortex neurons had been established, the team used their high-tech eavesdropping techniques to hear what goes on in the brains of sleeping rats--since sleep, as Siapas points out, has long been thought to be the optimal time for the memory consolidation.

As it turns out, those thoughts were right--but only part of the time.

The team did indeed hear "bursts" of neuronal chatter during sleep--but only during a phase of sleep known as slow-wave sleep (SWS), the deep, dreamless periods of sleep. "It turns out that during slow-wave sleep there are these episodes where a lot of the cells in the hippocampus will all fire very close to the same time," says Wierzynski. In response, some cells in the prefrontal cortex will fire in near unison as well, just milliseconds later. "What's interesting is that the bulk of the precise spike timing happens during these bursts, and not outside of these bursts," he adds.

On the other hand, during rapid-eye-movement (REM) sleep, the previously chatty neuron pairs seemed to talk right past each other, firing at the same rates as before but no longer in concert.

"It was surprising," says Wierzynski, "to find that the timing relationship almost completely went away during REM sleep."

Since REM sleep is the phase during which dreaming occurs, the scientists speculate that this absence of memory-consolidating chatter may eventually help to explain why dreams can be so difficult to remember.

As intriguing as that idea may be, the researchers caution that these findings only raise possibilities, providing avenues for further research in the field.

"Now that we've shown this link," says Siapas, "we have a framework we can use to study these questions further. This is just a step toward our goal of some day fully understanding the relationship between memory and sleep."

Other coauthors on the paper, entitled "State-dependent spike timing relationships between hippocampal and prefrontal circuits during sleep," included Evgueniy Lubenov, a postdoctoral scholar in biology at Caltech, and Caltech graduate student Ming Gu.

This work was supported by a National Defense Science and Engineering Graduate Fellowship, the Caltech Information Science and Technology Center for Biological Circuits Design, the James S. McDonnell Foundation, the Bren Foundation, the McKnight Foundation, the Whitehall Foundation, and the National Institutes of Health.

Lori Oliwenstein
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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.



Kathy Svitil

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."


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

Jon Weiner
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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.

Lori Oliwenstein


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