Capturing Snapshots in Time to Pick Apart Synaptic Activity

As we take in the world around us, learn, and form memories, the synapses between neurons in our brains are constantly being modified. Some get stronger, while others are allowed to shrink or get weaker. The network of enzyme-regulated chemical reactions that control these modifications is complex, to say the least. Now Mary Kennedy, the Allen and Lenabelle Davis Professor of Biology at Caltech, has come up with a way to tease apart the elusive details of that network. 

Beyond the basic scientific importance of understanding the ins and outs of our brains, the work could have significant implications for the mental health field. "It's becoming increasingly clear that slight mutations in some of these pathways make people more vulnerable to many disorders, including schizophrenia, bipolar disorder, and autism," Kennedy says.

Over the past 30 years, researchers have pieced together an understanding of the regulatory pathways and enzymes involved in controlling and modifying synaptic activity, and they've created "cartoons"—maps showing how the various components of the pathways interact. Looking at the cartoons, with their tangles of crisscrossing arrows connecting proteins and enzymes, the complexity of the network becomes apparent. 

Researchers have worked out many of the players involved and how they can interact to modify synapse strength. But they still know little about the dynamics of the network and how the processes are activated over time and under different circumstances. "We know how little strings of enzymatic processes can get activated," Kennedy says. "But we don't have very good ways of asking what happens when, say, 20 of these processes are interacting and you tweak one or two."

Now Kennedy believes she's found a way to ask such questions. The key is to stop the action in a series of samples and to see what changes from one second to the next. 

Kennedy is putting together an experimental method that will enable her to capture such "snapshots in time." She recently acquired a plunge-freeze device that she plans to modify so that it can freeze brain tissue samples in liquid propane/ethane as quickly as one second after electrical stimulation. Previously, Kennedy says, “we had no way of putting recording-electrodes into a brain slice, stimulating, and then freezing it solid or stopping it within a second.” 

There really was no reason to do so until the science and technology had progressed to a level that would enable a full analysis of the frozen samples. Kennedy says now is the time to move forward with these studies. Once she has the frozen tissue samples in hand, she'll process them with a new proteomic technique. Then, with the help of the Proteome Exploration Laboratory in the Beckman Institute, she'll be able to inject the sample into a mass spectrometer for analysis.

Many of the enzyme-driven reactions that take place in the synapses involve adding a highly energetic phosphate group to an amino acid, a process called phosphorylation, which alters the function of a protein. Since scientists have already determined the sequences of amino acids surrounding many of the critical sites of regulation in these pathways, Kennedy believes she will be able to use a mass spectrometer to measure the concentration of as many as 20 to 40 known phosphorylated sites in a single small tissue sample.

"That will let us map out the changes that happen in this large network immediately after a synchronized synaptic input," Kennedy says. "That means we'll be able to measure much more globally how these complex pathways interact with each other—which ones are more important at early stages, and which ones come in later—all of which has been very difficult to understand."

She hopes that she'll be able to use her new method to identify synaptic pathways that may be relevant to mental illnesses and Alzheimer's disease. "In order to screen in a more effective way for drugs or anything that could bring a particular set of processes into range, such global measurements are really critical," she says.

Kennedy's new Leica plunge-freeze apparatus was a gift from the Allen and Lenabelle Davis Foundation. The work is also supported by a grant from the National Institute of Mental Health. 

Kimm Fesenmaier

Captivated by Critters: Humans Are Wired to Respond to Animals

PASADENA, Calif.—Some people feel compelled to pet every furry animal they see on the street, while others jump at the mere sight of a shark or snake on the television screen. No matter what your response is to animals, it may be thanks to a specific part of your brain that is hardwired to rapidly detect creatures of the nonhuman kind. In fact, researchers from the California Institute of Technology (Caltech) and UCLA report that neurons throughout the amygdala—a center in the brain known for processing emotional reactions—respond preferentially to images of animals.

Their findings were described in a study published online in the journal Nature Neuroscience.

The collaborative research team was responsible for recruiting 41 epilepsy patients at the Ronald Reagan UCLA Medical Center; these patients were already being monitored for brain activity related to seizures. Using electrodes already in place, the team recorded single-neuron responses in the amygdala as study participants viewed images of people, animals, landmarks, or objects. The amygdalae are two almond-shaped clusters of neurons—cells that are core components of the nervous system—located deep in the medial temporal lobe of the brain.  

"Our study shows that neurons in the human amygdala respond preferentially to pictures of animals, meaning that we saw the most amount of activity in cells when the patients looked at cats or snakes versus buildings or people," says Florian Mormann, lead author on the paper and a former postdoctoral scholar in the Division of Biology at Caltech. "This preference extends to cute as well as ugly or dangerous animals and appears to be independent of the emotional contents of the pictures. Remarkably, we find this response behavior only in the right and not in the left amygdala."

Mormann says this striking hemispheric asymmetry helps strengthen previous findings supporting the idea that, early on in vertebrate evolution, the right hemisphere became specialized in dealing with unexpected and biologically relevant stimuli, or with changes in the environment. "In terms of brain evolution, the amygdala is a very old structure, and throughout our biological history, animals—which could represent either predators or prey—were a highly relevant class of stimuli," he says. 

"This is a pretty novel finding, since most amygdala research in the past was usually about faces of people and emotions related to fear rather than pictures of animals," adds Ralph Adolphs, a coauthor on the paper and Bren Professor of Psychology and Neuroscience and professor of biology at Caltech. "Nobody would have guessed that cells in the amygdala respond more to animals than they do to human faces, and in particular that they respond to all kinds of animals, not just dangerous ones. I think this will stimulate more research and has the potential to help us better understand phobias of animals."

The study is also a clear illustration of how scientists doing basic research can benefit from working with collaborators in a clinical setting and vice versa.

"This is a good example of how special situations in neurosurgery—in this case, patients who are treated in order to cure their epilepsy—can provide a unique window into the workings of the human mind," says Itzhak Fried, a UCLA neurosurgeon and a coauthor of the study.

"A category-specific response to animals in the right human amygdala" was featured online on August 28 as an advance online publication of Nature Neuroscience. The Caltech team was led by Christof Koch, Troendle Professor of Cognitive and Behavioral Biology, and included Julien Dubois, Simon Kornblith, Milica Milosavljevic, Moran Cerf, Naotsugu Tsuchiya, and Alexander Kraskov. Rodrigo Quian Quiroga and Matias Ison from the University of Leicester also contributed to the study.

The research was supported by the European Commission, the National Research Foundation of Korea, the National Institute of Neurological Disorders and Stroke, the G. Harold and Leila Y. Mathers Foundation, the Gimbel Discovery Fund, and the Dana Foundation.  

Katie Neith
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Caltech Group Applies New Techniques and Sees Surprises in Cell Division

PASADENA, Calif.—Researchers at the California Institute of Technology (Caltech) have obtained the first high-resolution, three-dimensional images of a cell with a nucleus undergoing cell division. The observations, made using a powerful imaging technique in combination with a new method for slicing cell samples, indicate that one of the characteristic steps of mitosis is significantly different in some cells.

During mitosis, two sets of chromosomes get paired up at the center of the cell's nucleus. Then hollow rods of proteins called microtubules, which make up a cellular structure called the spindle apparatus, grab on to the chromosomes and essentially pull each set away from the center in opposite directions, so that both daughter cells end up with a full copy of the genetic material. Typically, in the cells of plants, fungi, and many animals, one or more microtubules attach to each chromosome before the spindle will separate the sets of chromosomes from each another.

But when the Caltech researchers observed this step using their new technique, what they saw was not business as usual for a dividing cell. "We've found the first clear example of a cell where there are fewer microtubules used than chromosomes," says Grant Jensen, professor of biology at Caltech and a Howard Hughes Medical Institute investigator. The group's findings appear online in Current Biology and will be published in the September 27 issue of the journal.

Jensen's group is one of just a few in the world that uses electron cryotomography (ECT) to image biological samples. Unlike traditional electron microscopy—for which samples must be dehydrated, embedded in plastic, sectioned, and stained—ECT involves plunge-freezing samples so quickly that they become trapped in a near-native state within a layer of transparent, glasslike ice. A microscope can then capture high-resolution images of the sample as it is rotated, usually one degree at a time. 

One limitation of ECT is that samples cannot be thicker than 500 nanometers—otherwise the electron beam cannot penetrate the sample sufficiently. Therefore, ECT studies have focused on small bacteria and viruses. But Jensen's group wanted to extend the technique to observe eukaryotic cells, which are typically much bigger. So they located the smallest known eukaryote, Ostreococcus tauri, and imaged it with ECT.

The next step was to observe the important process of cell division in a eukaryote. But even tiny O. tauri exceeds the 500-nanometer limit when it is undergoing mitosis, since cells are essentially twice as big as usual when they're dividing. So the researchers needed a way to cut the frozen sample into slices, a process called cryosectioning.

"In the past when people have tried this, the sections have come off sort of like snowflakes—the material has gotten crushed," Jensen says. But Caltech electron microscopy scientist Mark Ladinsky has developed a highly successful new technique for cryosectioning samples. He slices them at about -150°C, using a special machine and a diamond knife. Then he carefully removes the slices from the knife using a micromanipulator. The technique has enabled the researchers to look at slices through dividing cells in a near-native, hydrated state.

With the new imaging and sectioning techniques working together, a former postdoctoral scholar in Jensen's lab, Lu Gan, was able to make detailed observations of mitosis in O. tauri, a cell with 20 chromosomes. Contrary to expectations, Gan observed nowhere near 40 microtubules attached to the two sets of chromosomes during mitosis; instead, he found only about 10 small, incomplete microtubules. This suggests that the chromosomes may link together to form some kind of a bundle that can then be segregated all at once by a smaller number of microtubules.

Previous studies, dating back to the 1970s, claimed to have found unicellular eukaryotic cells with fewer microtubules than chromosomes. However, those cells were chemically fixed and stained—a process that can easily damage the cells—casting doubt on the claims. But with their new imaging and sectioning techniques, the Caltech researchers feel confident that they have indeed imaged such a cell.

Their success bodes well for future studies. "Our work with O. tauri shows that we might be able to get high-resolution, three-dimensional images of other eukaryotic cells, which are much larger than bacteria," Jensen says. "We've even moved on to try to image some human cells using the same process."

The group's report, "Organization of the Smallest Eukaryotic Spindle," was supported in part by a grant from the National Institutes of Health and a fellowship from the Damon Runyon Cancer Research Foundation. The ECT imaging was made possible by a gift from the Gordon and Betty Moore Foundation. 


Kimm Fesenmaier

Caltech Team Says Sporulation May Have Given Rise to the Bacterial Outer Membrane

PASADENA, Calif.—Bacteria can generally be divided into two classes: those with just one membrane and those with two. Now researchers at the California Institute of Technology (Caltech) have used a powerful imaging technique to find what they believe may be the missing link between the two classes, as well as a plausible explanation for how the outer membrane may have arisen.

The "missing link" is a bacterium called Acetonema longum—a member of a little-known family of bacteria that have two membranes and respond to extreme or challenging situations by forming protective spores, a process known as sporulation. Outside of this small family, only bacteria with a single membrane have been found to sporulate.

"When we started looking at Acetonema, we had no idea how unique and interesting it would turn out to be," says Grant Jensen, professor of biology at Caltech and a Howard Hughes Medical Institute investigator. "But when we imaged the sporulation process in this bacterium at high resolution, we saw that a piece of the inner membrane actually becomes the new cell's outer membrane."

The finding shows that because sporulation results in a second membrane covering a cell, the common ancestor of bacteria with a single or double membrane could have been a spore-forming bacterium similar to A. longum.

Jensen's group at Caltech is one of just a few in the world that uses electron cryotomography (ECT) to image biological samples. Unlike traditional electron microscopy—for which samples must be dehydrated, embedded in plastic, sectioned, and stained—ECT involves plunge-freezing samples so quickly that they become trapped in a near-native state within a layer of transparent, glasslike ice. A microscope can then capture high-resolution images of the sample as it is rotated, usually one degree at a time. Finally, those images can be stitched together to create three-dimensional representations of a specimen such as a single bacterial cell or a virus. 

Postdoctoral scholar Elitza Tocheva, a member of Jensen's lab, was exploring possible projects when Jared Leadbetter, professor of environmental microbiology at Caltech, suggested the group collaborate to study a sporulating bacterium isolated from a termite gut. Leadbetter knew that the model for sporulating bacteria, Bacillus subtilis, was too thick for ECT, and this other bacterium, A. longum, happened to be relatively skinny.

Tocheva quickly realized that a unique subject had fallen into her hands—not only was A. longum thin enough to image using ECT, but it also turned out to be one of the rare spore-forming bacteria with two membranes. The images she captured revealed the sporulation process in exquisite detail, and within the images, she noticed something interesting: in order to end up with an outer membrane after sporulation, an inner membrane had to be inverted and converted into an outer membrane. That's no small feat, given that inner and outer membranes are very different both structurally and functionally.

In the case of a double-membraned bacterium, such as A. longum, the sporulation process begins with the inner membrane pinching together asymmetrically, creating a mother cell and a smaller daughter cell, all within the outer membrane. Next, the mother cell engulfs the daughter, giving the daughter an extra layer of membrane derived from the original inner membrane. The product is a spore surrounded by two membranes within the mother cell. At this point, the mother cell dies away, leaving the spore protected by those two membranes and a protein coat.

When conditions improve and the spore germinates, part of its protective protein coat cracks open, allowing the new cell to outgrow, or exit, the protein coat. Unlike a single-membraned bacterium, which would at this point shed its outer membrane, the double-membraned bacterium retains it. Jensen's group found that the new outer membrane has all of the structure and function of a typical outer membrane, even though it originated as part of the mother cell's inner membrane.

"When the bacterium outgrows, one of its two membranes has to be converted from an inner to an outer membrane," Tocheva says. "This is very intriguing, and we don't know how it happens."

To prove that the membrane becomes converted to an outer membrane once it outgrows, the researchers had to show that Acetonema longum's outer membrane was like those seen in other double-membraned bacteria. They achieved this in a few different ways. First they showed that the outer membrane had one of the hallmarks of a true outer membrane—the presence of an immunologically important macromolecule called lipopolysaccharide (LPS). Then they calculated the density of the membranes and found that the outer membrane was slightly denser than the inner membrane, as would be expected of a true outer membrane. Finally, they searched the genomes of about one thousand bacteria and found that A. longum had a full complement of the genes that commonly correlate with having a double membrane and having the ability to sporulate.

With all of this evidence in hand, the researchers were able to say that they were indeed seeing an outer membrane that was once part of an inner membrane. "We were rewarded with this kind of rich data of a sporulating bacterium," Tocheva says. "Acetonema was just the ideal subject."

The group's paper, "Peptidoglycan Remodeling and Conversion of an Inner Membrane into an Outer Membrane During Sporulation," appears online and will be published in the September 2 issue of the journal Cell. In addition to Jensen, Leadbetter, and Tocheva, Caltech postdoctoral scholar Eric Matson and graduate student Dylan Morris participated in the study, along with Farshid Moussavi of Stanford University. The ECT imaging was made possible by a gift from the Gordon and Betty Moore Foundation. The analysis of LPS was performed at the Complex Carbohydrate Research Center and was supported by a Department of Energy grant. The work was also supported by a Natural Sciences and Engineering Research Council of Canada postdoctoral fellowship, by the National Science Foundation, and by the Howard Hughes Medical Foundation.


Kimm Fesenmaier

HIV Havens: Caltech Researchers Find New Clues About How HIV Reservoirs May Form

PASADENA, Calif.— Much like cities organize contingency plans and supplies for emergencies, chronic infectious diseases like HIV form reservoirs that ensure their survival in adverse conditions. But these reservoirs—small populations of viruses or bacteria of a specific type that persist despite attack by the immune system or drug treatment—are not always well understood. Now, however, researchers at the California Institute of Technology (Caltech) believe they have begun to decode how a reservoir of infection can persist in HIV-positive populations. 

The research team—led by David Baltimore, Robert Andrews Millikan Professor of Biology and recipient of the 1975 Nobel Prize in Physiology or Medicine—proposes that a type of HIV infection that uses infected cells to get close to uninfected cells and then discharge a large load of virus on them, may be the reason small populations of HIV-infected cells persist even when antiretroviral drug treatment has been successful in suppressing most other infections within an individual.   

Their findings were reported in the advance online publication of the journal Nature on August 17. 

For chronic infections such as HIV, the end game for scientists is to remove "chronic" from the disease's name—by finding a cure. Many believe that better understanding of viral reservoirs may be the key to eradicating them, and thus the disease. So the research team started at the beginning of the process, looking for clues into how an HIV reservoir might be formed in the first place.

There are two known ways that HIV can infect cells, and thus the human body: cell-free transmission, in which the virus infects immune system cells called T cells it encounters while floating free in plasma; and cell-to-cell transmission, in which the virus moves between T cells by using an infected “donor” cell as its vehicle. Once an uninfected target cell is found, the donor cell can then directionally discharge its viral load upon the target. To replicate both types of transmission, the team infected target cells using both cell-free HIV and previously infected donor cells. They used donor cells that lack a natural marker, HLA-A2, usually used in matching human organ donors to recipients. The target cells did have the marker, and this helped the scientists keep track of which cells were the donors and which were the targets. The target cells were infected either in the absence or in the presence of antiretroviral drugs.

What the researchers found was that while the antiretroviral drugs caused a steep drop in the number of newly infected cells infected via cell-free transmission, the decrease in the number of newly infected cells for the cell-to-cell infected T cells was much more moderate, even when they had large doses of the drugs thrown at them.

"We saw that with cell-to-cell infection, you wind up with a lot more virus infecting a single cell," explains Alex Sigal, a postdoctoral scholar in Baltimore's laboratory and lead author of the study. "When this happens, the chance of at least a single virus getting past the drugs is much larger."

This may explain why, while antiretroviral drugs work very well, they do not eradicate the infection completely. The drugs are probabilistic by nature, meaning that they don't kill 100 percent of the virus. So, as the number of transmitted viruses gets larger, the chance of at least one virus slipping by the drugs and infecting another cell becomes greater. "And you only need one virus to infect a cell and keep the cycle going, forming a reservoir of infection," says Sigal.

Another possibility for why HIV cannot be eradicated is that it goes into latency. A latent reservoir would consist of cells that contain the HIV virus in their DNA, but are not currently making any virus and therefore are not affected by drugs. Sigal says it's possible that both types of reservoirs are present and interact with each other.

"It's important to determine whether or not cell-to-cell replication is causing a reservoir, particularly in terms of finding a cure," he says. "You can't treat it the same way as you would a latent reservoir." Theoretically, virus in a latent reservoir could be eradicated by flushing out the virus from the cells by activating it, and treating the patient with a lot of drugs at the same time so that the released virus can’t enter new cells. This would not work if the virus could get into new cells anyway, despite the drugs.

"For us, the next step is to look at the process on a more physiological level by looking at how HIV infects in organs such as lymph nodes where cell-to-cell transmission actually happens," says Sigal. The team will do so by collaborating with the UCLA Center for AIDS Research, which provided the cells for virus infection from anonymous healthy volunteers in this study. The two groups will work together to increase the level of complexity in additional studies that will aim for a deeper understanding of the reservoir. "We're really looking for a cure, but to get to a cure, you have to fully understand the disease first," he says.

The Nature paper is titled "Cell-to-cell spread of HIV permits ongoing replication despite antiretroviral therapy." Jocelyn T. Kim, an infectious disease doctor and a graduate student in the Caltech-UCLA STAR Program; Alejandro B. Balazs, a postdoctoral scholar in Baltimore's laboratory; and Erez Dekel, Avi Mayo, and Ron Milo of the Weizmann Institute of Science in Israel also contributed to the study.

The research was supported by the National Institutes of Allergy and Infectious Diseases and the Bill & Melinda Gates Foundation. 

Katie Neith

New Faculty Member Brings Quantitative Approaches to Biology

Lea Goentoro remembers the precise moment that biology made an impression on her. It was 2002 and she was a PhD candidate in chemical engineering at Princeton. During a presentation at the Institute for Advanced Study, developmental biologist and Nobel laureate Eric Wieschaus—who shared the 1995 Nobel Prize with the late Edward Lewis, Caltech alumnus and former Morgan Professor of Biology—showed a movie of a live fly embryo under a microscope that was undergoing gastrulation, a phase early in development where a single layer of cells is reorganized into a three-layer structure. "The embryonic cells undergo massive rearrangement and things fold in—and it was all on this big screen," says Goentoro. "It was amazing. For me, that was when I thought, 'Wow. I really want to study this.'"

Now, nine years after that fateful day in New Jersey, Goentoro is Caltech's newest faculty member in the Division of Biology. She joined the division in late July as an assistant professor.

"Caltech is a wonderful place to be—the right place," says Goentoro. "When I saw that the division was looking for someone with a quantitative background and a research program that would combine quantitative approaches with biology, I thought it could be a perfect fit."

Although her educational background is in chemical engineering—like her PhD, she also earned her BS in the field from the University of Wisconsin-Madison in 2001—Goentoro has been applying her math and engineering skills to biology problems for the past 10 years. Previous to arriving at Caltech, she was a postdoctoral fellow at the Systems Biology Department at Harvard Medical School.

Her current research focuses on Weber's Law applied at a cellular level. The law, first discovered by an experimental psychologist in the early 1800s, is the idea that we sense our world in a relative way. Imagine that you spend the day outside in the bright sunlight. When you come indoors, it takes a few moments for your eyes to adjust to a different background. The idea of the law is that you will always be able to adjust to these types of changes.

"From my previous work with Marc Kirschner at Harvard, we found that there is a strong suggestion that this concept also applies to individual cells in our body," says Goentoro. "The way our brain processes sensory information seems to apply to the way individual cells process signals."

She hopes to find out whether or not this idea is true. Do individual cells communicate this way, and if so, how is it implemented at the molecular level? Goentoro uses mathematical modeling as one strategy to address these questions. "Not only does it give us new tools, it gives us new ways of thinking about problems in biology," she says. In addition, Goentoro and others in her lab do experiments in human cell cultures and study embryo development in African frogs to try to understand how individual cells are communicating with each other.

"The signaling pathway that we are studying is highly conserved across all animal cells, so that's why we can jump between one system and another," she explains. "Each system allows us to do different experiments. By combining all of them, we can get more information."

When she's not attending to frogs in her lab, or studying cells under a microscope, Goentoro says she'll be exploring her new hometown. An aspiring hiker, she looks forward to tackling local trails. "I've been promised that there is a really good hiking scene here," she says. In addition, she enjoys scouring used bookstores for hidden treasures. Like research, you never know what you might discover.  

Katie Neith

Caltech Researchers Increase the Potency of HIV-Battling Proteins

PASADENA, Calif.—If one is good, two can sometimes be better. Researchers at the California Institute of Technology (Caltech) have certainly found this to be the case when it comes to a small HIV-fighting protein.

The protein, called cyanovirin-N (CV-N), is produced by a type of blue-green algae and has gained attention for its ability to ward off several diseases caused by viruses, including HIV and influenza. Now Caltech researchers have found that a relatively simple engineering technique can boost the protein's battling prowess.

"By linking two cyanovirins, we were able to make significantly more potent HIV-fighting molecules," says Jennifer Keeffe, a staff scientist at Caltech and first author of a new paper describing the study in the Proceedings of the National Academy of Sciences (PNAS). "One of our linked molecules was 18 times more effective at preventing infection than the naturally occurring, single protein."

The team's linked pairs, or dimers, were able to neutralize all 33 subtypes of HIV that they were tested against. The researchers also found the most successful dimer to be similar or more potent than seven well-studied anti-HIV antibodies that are known to be broadly neutralizing.

CV-N binds well to certain carbohydrates, such as the kind found in high quantities connected to the proteins on the envelope that surrounds the HIV virus. Once attached, CV-N prevents a virus from infecting cells, although the mechanism by which it accomplishes this is not well understood.

What is known is that each CV-N protein has two binding sites where it can bind to a carbohydrate and that both sites are needed to neutralize HIV.

Once the Caltech researchers had linked two CV-Ns together, they wanted to know if the enhanced ability of their engineered dimers to ward off HIV was related to the availability of additional binding sites. So they engineered another version of the dimers—this time with one or more of the binding sites knocked out—and tested their ability to neutralize HIV.

It turns out that the dimers' infection-fighting potency increased with each additional binding site—three sites are better than two, and four are better than three. The advantages seemed to stop at four sites, however; the researchers did not see additional improvements when they linked three or four CV-N molecules together to create molecules with six to eight binding sites.

Although CV-N has a naturally occurring dimeric form, it isn't stable at physiological temperatures, and thus mainly exists in single-copy form. To create dimers that would be stable under such conditions, the researchers covalently bound together two CV-N molecules in a head-to-tail fashion, using flexible polypeptide linkers of varying lengths.

Interestingly, by stabilizing the dimers and locking them into a particular configuration, it seems that the group created proteins with distances between binding sites that are very similar to those between the carbohydrate binding sites in a broadly neutralizing anti-HIV antibody.

"It is possible that we have created a dimer that has its carbohydrate binding sites optimally positioned to block infection," says Stephen Mayo, Bren Professor of Biology and Chemistry, chair of the Division of Biology, and corresponding author of the new paper.

Because it is active against multiple disease-causing viruses, including multiple strains of HIV, CV-N holds unique promise for development as a drug therapy. Other research groups have already started investigating its potential application in prophylactic gels and suppositories.

"Our hope is that those who are working to make prophylactic treatments using cyanovirin will see our results and will use CVN2L0 instead of naturally occurring cyanovirin," Keeffe says. "It has higher potency and may be more protective." 

The paper, entitled "Designed oligomers of cyanovirin-N show enhanced HIV neutralization," was published in the online edition of PNAS. In addition to Keeffe and Mayo, other authors on the paper include research technician Priyanthi N.P. Gnanapragasam, former biology graduate student Sarah K. Gillespie, biology graduate student John Yong, and Pamela J. Bjorkman, the Max Delbruck Professor of Biology at Caltech and a Howard Hughes Medical Institute investigator. 

The work was funded by the National Security Science and Engineering Faculty Fellowship program, the Defense Advanced Research Projects Agency Protein Design Processes program, and the Bill and Melinda Gates Foundation through the Grand Challenges in Global Health Initiative.

Kimm Fesenmaier

Sharper, Deeper, Faster: Interdisciplinary Team Develops Advanced Live-Imaging Approach

PASADENA, Calif.— For modern biologists, the ability to capture high-quality, three-dimensional (3D) images of living tissues or organisms over time is necessary to answer problems in areas ranging from genomics to neurobiology and developmental biology. The better the image, the more detailed the information that can be drawn from it. Looking to improve upon current methods of imaging, researchers from the California Institute of Technology (Caltech) have developed a novel approach that could redefine optical imaging of live biological samples by simultaneously achieving high resolution, high penetration depth (for seeing deep inside 3D samples), and high imaging speed.

The imaging technique is explained in a paper in the advance online publication of the journal Nature Methods, released on July 14. It will also appear in an upcoming print version of the journal.  

"Before our work, the state-of-the-art imaging techniques typically excelled in only one of three key parameters: resolution, depth, or speed. With our technique, it's possible to do well in all three and, critically, without killing, damaging, or adversely affecting the live biological samples," says biologist Scott Fraser, director of the Biological Imaging Center at Caltech's Beckman Institute and senior author of the study. 

The research team achieved this imaging hat trick by first employing an unconventional imaging method called light-sheet microscopy, where a thin, flat sheet of light is used to illuminate a biological sample from the side, creating a single illuminated optical section through the sample. The light given off by the sample is then captured with a camera oriented perpendicularly to the light sheet, harvesting data from the entire illuminated plane at once. This allows millions of image pixels to be captured simultaneously, reducing the light intensity that needs to be used for each pixel. This not only enables fast imaging speed but also decreases the light-induced damage to the living samples, which the teams demonstrated using the embryos of fruit fly and zebrafish.

To achieve sharper image resolution with light-sheet microscopy deep inside the biological samples, the team used a process called two-photon excitation for the illumination. This process has been used previously to allow deeper imaging of biological samples; however, it usually is used to collect the image one pixel at a time by focusing the exciting light to a single small spot.

"The conceptual leap for us was to realize that two-photon excitation could also be carried out in sheet-illumination mode," says Thai Truong, a postdoctoral fellow in Fraser's laboratory and first author of the paper. This novel side-illumination with a two-photon illumination is the topic of a pending patent.

"With this approach, we believe that we can make a contribution to advancing biological imaging in a meaningful way," continues Truong, who did his PhD training in physics. "We did not want to develop a fanciful optical imaging technique that excels only in one niche area, or that places constraints on the sample so severe that the applications will be limited. With a balanced high performance in resolution, depth, and speed, all achieved without photo-damage, two-photon light-sheet microscopy should be applicable to a wide variety of in vivo imaging applications." He credits this emphasis on wide applicability to the interdisciplinary nature of the team—which includes two biologists, two physicists, and one electrical engineer.

"We believe the performance of this imaging technique will open up many applications in life sciences and biomedical research—wherever it is useful to observe, non-invasively, dynamic biological process in 3D and with cellular or subcellular resolution," says Willy Supatto, co–author of the paper and a former postdoctoral fellow in Fraser's laboratory (now at the Centre National de la Recherche Scientifique, in France).

One example of such an application would be to construct 3D movies of the entire embryonic development of an organism, covering the entire embryo in space and time. These movies could capture what individual cells are doing, as well as important genes' spatiotemporal expression patterns—elucidating the activation of those genes within specific tissues at specific times during development.

"The goal is to create 'digital embryos,' providing insights into how embryos are built, which is critical not only for basic understanding of how biology works but also for future medical applications such as robotic surgery, tissue engineering, or stem-cell therapy," says Fraser. The team's first attempt at this can be seen in the accompanying movie, in which the cell divisions and movements that built the entire fruit fly embryo were captured without perturbing its development. 

The Nature Methods paper is titled "Deep and fast live imaging with two-photon scanned light-sheet microscopy." David Koos, senior research scientist at Caltech's Beckman Institute, and John Choi, a former postdoctoral fellow in Fraser's laboratory, also contributed to the study.

The research was supported by the Beckman Institute and the U.S. National Institutes of Health.


Katie Neith

Neutralizing HIV

Each time a virus invades a healthy individual, antibodies created by the body fight to fend off the intruders. For some viruses, like HIV, the antibodies are very specific and are generated too slowly to combat the rapidly changing virus. However, in the past few years, scientists have found that some HIV-positive people develop highly potent antibodies that can neutralize different subtypes of the HIV virus.

Now, a study involving researchers at Caltech points to the possibility of using these neutralizing antibodies in the development of a vaccine. The paper, published in the July 14 issue of Science Express, describes a group of novel antibodies that were isolated from HIV-infected individuals using a new cloning approach.

These antibodies are the most potent anti-HIV antibodies targeting the CD4 binding site— a functional site on the surface of HIV needed for cell entry and infection—that have ever been identified, says Ron Diskin, a post-doctoral scholar at Caltech who worked on the paper. David Ho (BS '74), scientific director of the Aaron Diamond AIDS Research Center in New York, also contributed to the study, which was led by researchers at Rockefeller University.

At Caltech, the researchers conducted structural studies and were able to show, based on similarity to a previously known antibody (VRC01), that the new antibodies indeed target the CD4 receptor binding site. CD4 positive cells are the point of HIV infection and where the virus multiplies.  

This study is important for several different reasons, according to Diskin. "First, it provides extremely useful reagents that can be used for passive immunization to treat infected individuals," he says. "Second, it demonstrates that a comparable and highly effective anti-HIV immune response was elicited in different individuals, which strongly supports the idea that an effective vaccine will be feasible to develop."

Next, researchers at Caltech will address the structural mechanisms that make those antibodies so potent. In fact, they are currently investigating those structural aspects of the neutralization mechanisms.

"We're very excited to have the opportunity to use structural biology to learn what makes these new antibodies so potent against HIV," says Pamela Bjorkman, Caltech's Delbruck Professor of Biology and a co-author of the study. "We hope that visualizing how these antibodies interact with HIV proteins will allow the design of even more potent anti-HIV reagents and provide critical information for vaccine design."

Katie Neith
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Tracking Bacteriophages

Viruses are parasites; they need to live inside another organism to survive. Figuring out just who a virus's host is can be difficult, however. Especially when you're talking about bacteriophages—a particularly pervasive group of bacteria-infecting viruses.

The problem lies in identifying which bacteriophages are infecting which bacteria, without having to culture either the viruses or their hosts in the lab, which can often be a difficult if not impossible task.

To address that issue, a team led by Caltech biophysicist Rob Phillips has created a genetic-analysis technique—called microfluidic digital polymerase chain reaction (PCR)—that can "physically link single bacterial cells harvested from a natural environment with a viral marker genes," the scientists report in the July 1 issue of the journal Science.

The team looked at the bacterial community found in the hindgut of a termite—specifically, a termite collected in 2005 in Costa Rica. The typical termite hindgut contains over 250 different species of bacteria, "making it ideally suited to explore many potential, diverse phage-host interactions," the scientists say.

What they found was that microfluidic digital PCR allowed them to see interactions between the viruses and their bacterial hosts in uncultivated cells harvested directly from the hindgut. They noted, for instance, that variants of a viral packaging gene had made their way into hosts across an entire genus of bacteria; in another gene-tracking experiment, however, they found little evidence of lateral gene transfer or switching of the gene from one host to another, despite plenty of opportunity.

"Our approach does not require culturing hosts or viruses," the researchers write, "and provides a method for examining virus-bacteria interactions in many environments."

For more on the new technology and its potential applications, check out the paper's abstract or this press release from the National Science Foundation, one of the funders of the research.

Lori Oliwenstein