Understanding Olfaction: An Interview with Elizabeth Hong

You walk by a bakery, smell the scent of fresh cookies, and are immediately reminded of baking with your grandmother as a child. The seemingly simple act of learning to associate a smell with a good or bad outcome is actually quite a complicated behavior—one that can begin as a single synapse, or junction, where a signal is passed between two neurons in the brain.

Assistant Professor of Neuroscience Betty Hong is interested in how animals sense cues in their environment, process that information in the brain, and then use that information to guide behaviors. To study the processing of information from synapse to behavior, her work focuses on olfaction—or chemical sensing via smell—in fruit flies.

Hong, who received her bachelor's degree from Caltech in 2002 and her doctorate from Harvard in 2009, came from a postdoctoral position at Harvard Medical School to join the Caltech faculty in June. We spoke with her recently about her work, her life outside the laboratory, and why she is looking forward to being back at Caltech.


How did you initially become interested in your field?

It's rather circuitous. I was initially drawn to neuroscience because I was interested in disease. I had family who passed away from Alzheimer's disease, and it's clear that with the current demographic of our country, diseases associated with aging—like Alzheimer's—are going to have a large impact on society in the next 20 to 30 years. Working at the Children's Hospital Boston in graduate school, I also became increasingly interested in understanding the rise of neurodevelopmental disorders like autism.

I really wanted to understand the mechanistic basis for neurological disease. And then it became clear to me that part of the problem of trying to understand neurological disorders was that we really had no idea how the brain is supposed to work. If you were a mechanic who didn't know how cars work, how could you fix a broken car? That led me to study increasingly more basic mechanisms of how the brain functions.  


Why did you decide to focus your research on olfaction?

Although we humans have evolved to move away from olfaction—humans and primates are very visual—the whole rest of the animal kingdom relies on olfaction heavily for all of its daily survival and functions. Even the lowliest microbe relies on chemical sensing to navigate its way through the environment. We study olfaction in an invertebrate model—the fruit fly Drosophila. We do that for a couple of reasons. One is that it has a very small brain, and so its circuits are very compact, and that small size and numerical simplicity lets us get a global overview of what's happening—a view that you could never get if you're looking at a big circuit, like a mouse brain or a human brain.

The other reason is that there are versatile genetic tools and new technologies that have allowed us to make high-resolution electrical and optical recordings of neural activity in the brains of fruit flies. That very significant technical hurdle had to be crossed in order to make it a worthwhile experimental model. With electrophysiological access to the brain, and genetic tools that allow you to manipulate the circuits, you can watch brain activity as it's happening and ask what happens to neural activity when you tweak the properties of the system in specific ways. And the fly also has a robust and flexible set of behaviors that you can relate to all of this. 


What are some of the behaviors that you are interested in studying?

We're very interested in understanding how flies can associate an odor with a pleasant or unpleasant outcome. So, in the same way that you might associate wonderful baking smells with something from your childhood, flies can learn to arbitrarily associate odors with different outcomes. And to know "when I smell this odor, I should run away," or "based on what happened to me the last time I smelled this odor, this might be an indicator of food"—that's actually a fairly sophisticated behavior that is a basic building block for more complex higher-order cognitive tasks that emerge in vertebrates.

There are many animals that are inflexibly wired. In other words, they smell something, and through evolution, their circuits have evolved to tell them to move toward it or go away from it. Even if they are in an unusual environment, they can't flexibly alter that behavior. The ability to flexibly adapt our behavior to new and unfamiliar environments was a key transition in the evolution of the nervous system.


You are also a Caltech alum. What drew you back as a faculty member?

Yes, it seems like such a long time ago, but I was an undergraduate here—a biology major in Page House—from 1998 to 2002. I was also a SURF student with [Professor of Biology] Bruce Hay and later with David Baltimore [president emeritus and Robert Andrews Millikan Professor of Biology]. It's kind of wild to have as your colleagues people who were your mentors a decade ago, but I think the main reason I chose Caltech was the community of scholars here—on the level of faculty, undergraduate students, graduate students, and postdocs—that I will be able to interact with. In the end, you mainly just want to be with smart, motivated people who want to use science to make a difference in the world. And I think that encapsulates what Caltech does.


Do you have any interests or hobbies that are outside of the lab?

I used to play horn in the wind ensemble and orchestra, including the time when I was here as an undergraduate. But these days, any time that I'm not in the office, I'm with my two young kids. Right now, we're really excited about exploring all the fun and exciting things to do outdoors in Southern California. We've done a lot of hiking and exploring the natural beauty here. The kids have gotten into fishing lately, so our latest thing has been scoping out the best places to fish. I would love to hear from members of the community what their favorite spots are!

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Long-Term Contraception in a Single Shot

Caltech biologists have developed a nonsurgical method to deliver long-term contraception to both male and female animals with a single shot. The technique—so far used only in mice—holds promise as an alternative to spaying and neutering feral animals.

The approach was developed in the lab of Bruce Hay, professor of biology and biological engineering at Caltech, and is described in the October 5 issue of Current Biology. The lead author on the paper is postdoctoral scholar Juan Li.

Hay's team was inspired by work conducted in recent years by David Baltimore and others showing that an adeno-associated virus (AAV)—a small, harmless virus that is unable to replicate on its own, that has been useful in gene-therapy trials—can be used to deliver sequences of DNA to muscle cells, causing them to produce specific antibodies that are known to fight infectious diseases, such as HIV, malaria, and hepatitis C.

Li and her colleagues thought the same approach could be used to produce infertility. They used an AAV to deliver a gene that directs muscle cells to produce an antibody that neutralizes gonadotropin-releasing hormone (GnRH) in mice. GnRH is what the researchers refer to as a "master regulator of reproduction" in vertebrates—it stimulates the release of two hormones from the pituitary that promote the formation of eggs, sperm, and sex steroids. Without it, an animal is rendered infertile.

In the past, other teams have tried neutralizing GnRH through vaccination. However, the loss of fertility that was seen in those cases was often temporary. In the new study, Hay and his colleagues saw that the mice—both male and female—were unable to conceive after about two months, and the majority remained infertile for the remainder of their lives.

"Inhibiting GnRH is an ideal way to inhibit fertility and behaviors caused by sex steroids, such as aggression and territoriality," says Hay. He notes that in the study, his team also shows that female mice can be rendered infertile using a different antibody that targets a binding site for sperm on the egg. "This target is ideal when you want to inhibit fertility but want to leave the individual otherwise completely normal in terms of reproductive behaviors and hormonal cycling."

Hay's team has dubbed the new approach "vectored contraception" and says that there are many other proteins that are thought to be important for reproduction that might also be targeted by this technique.

The researchers are particularly excited about the possibility of replacing spay–neuter programs with single injections. "Spaying and neutering of animals to control fertility, unwanted behavior, and population numbers of feral animals is costly and time consuming, and therefore often doesn't happen," says Hay. "There is a strong desire in many parts of the world for quick, nonsurgical approaches to inhibiting fertility. We think vectored contraception provides such an approach."

As a next step, Hay's team is working with Bill Swanson, director of animal research at the Cincinnati Zoo's Center for Conservation and Research of Endangered Wildlife, to try this approach in female domestic cats. Swanson's team spends much of its time working to promote fertility in endangered cat species, but it is also interested in developing humane ways of managing populations of feral domestic cats through inhibition of fertility, as these animals are often otherwise trapped and euthanized.

Additional Caltech authors on the paper, "Vectored antibody gene delivery mediates long-term contraception," are Alejandra I. Olvera, Annie Moradian, Michael J. Sweredoski, and Sonja Hess. Omar S. Akbari is also a coauthor on the paper and is now at UC Riverside. Some of the work was completed in the Proteome Exploration Laboratory at Caltech, which is supported by the Gordon and Betty Moore Foundation, the Beckman Institute, and the National Institutes of Health. Olvera was supported by a Gates Millennium Scholar Award.

Kimm Fesenmaier
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Capturing the Right Odors to Study the Brain

Hong and colleagues aim to reveal neural mechanisms related to olfaction

Over the summer, Betty Hong, assistant professor of neuroscience, spent a week at the Janelia Research Campus in Ashburn, Virginia, interacting and brainstorming with other researchers from around the country interested in olfaction, our sense of smell. Invited to participate by the National Science Foundation (NSF), these 30 computational and experimental neuroscientists came up with innovative ways to approach some of the mysteries about how the brain processes odors and uses that information to guide behavior.

The five-day session was an example of the agency's new funding mechanism, the Ideas Lab. At these meetings, a multidisciplinary group of researchers is charged with generating potentially transformative proposals on a focused research topic. Now the NSF has awarded $15 million to three projects from the Olfactory Ideas Lab. Hong is coprincipal investigator on one titled "Using natural odor stimuli to crack the olfactory code." The awards expand NSF's investments in President Obama's BRAIN Initiative.

"I am grateful to have had the opportunity to be thrown together for a week with such a smart, diverse group of scientists who approach olfaction from so many different angles," says Hong (BS '02), adding that without the Ideas Lab, it is unlikely that she would have ever established collaborations with her coinvestigators. "I am also extremely grateful to the NSF for including junior investigators like myself who are just kicking off their research program. This unique funding mechanism will enable us to tackle really challenging and innovative research right at the start of our careers."

Olfactory scientists typically use simple synthetic odors involving single molecules for their experiments because natural odors—those that we smell around us every day—are too difficult to reproduce in a reliable way under controlled conditions. However, those simplified stimuli may not trigger the full range of neural computations that constitute olfaction.

Therefore, Hong and her colleagues aim to use comprehensive chemical analysis and computational methods to construct reproducible synthetic odorants in the lab that mimic naturally occurring smells in terms of eliciting typical behavioral responses in honey bees, fruit flies, and fly larvae. (Hong specializes in studies of the fruit fly Drosophila.) These synthetic odor blends can then be used to investigate how the brain processes smells and orders specific adaptive behaviors.

"We believe probing the olfactory circuit with naturalistic stimuli will reveal long-hidden computational features of the circuit," Hong explains. "Much as higher-order visual neurons only respond to complex stimuli like faces or hands, and not to simple bars and dots, we hypothesize that naturalistic odor stimuli will reveal novel features of odor space that the olfactory system encodes, which may only become apparent once appropriate sets of stimuli are used."

Along with Hong, additional principal investigators on the project are Brian Smith of Arizona State University; Aravinthan Samuel of Harvard University; and Tatyana Sharpee of the Salk Institute for Biological Studies. The project will receive $3.6 million over three years.

Kimm Fesenmaier
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Flowing Electrons Help Ocean Microbes Gulp Methane

Good communication is crucial to any relationship, especially when partners are separated by distance. This also holds true for microbes in the deep sea that need to work together to consume large amounts of methane released from vents on the ocean floor. Recent work at Caltech has shown that these microbial partners can still accomplish this task, even when not in direct contact with one another, by using electrons to share energy over long distances.

This is the first time that direct interspecies electron transport—the movement of electrons from a cell, through the external environment, to another cell type—has been documented in microorganisms in nature.

The results were published in the September 16 issue of the journal Nature.

"Our lab is interested in microbial communities in the environment and, specifically, the symbiosis—or mutually beneficial relationship—between microorganisms that allows them to catalyze reactions they wouldn't be able to do on their own," says Professor of Geobiology Victoria Orphan, who led the recent study. For the last two decades, Orphan's lab has focused on the relationship between a species of bacteria and a species of archaea that live in symbiotic aggregates, or consortia, within deep-sea methane seeps. The organisms work together in syntrophy (which means "feeding together") to consume up to 80 percent of methane emitted from the ocean floor—methane that might otherwise end up contributing to climate change as a greenhouse gas in our atmosphere.

Previously, Orphan and her colleagues contributed to the discovery of this microbial symbiosis, a cooperative partnership between methane-oxidizing archaea called anaerobic methanotrophs (or "methane eaters") and a sulfate-reducing bacterium (organisms that can "breathe" sulfate instead of oxygen) that allows these organisms to consume methane using sulfate from seawater. However, it was unclear how these cells share energy and interact within the symbiosis to perform this task.

Because these microorganisms grow slowly (reproducing only four times per year) and live in close contact with each other,  it has been difficult for researchers to isolate them from the environment to grow them in the lab. So, the Caltech team used a research submersible, called Alvin, to collect samples containing the methane-oxidizing microbial consortia from deep-ocean methane seep sediments and then brought them back to the laboratory for analysis.

The researchers used different fluorescent DNA stains to mark the two types of microbes and view their spatial orientation in consortia. In some consortia, Orphan and her colleagues found the bacterial and archaeal cells were well mixed, while in other consortia, cells of the same type were clustered into separate areas.

Orphan and her team wondered if the variation in the spatial organization of the bacteria and archaea within these consortia influenced their cellular activity and their ability to cooperatively consume methane. To find out, they applied a stable isotope "tracer" to evaluate the metabolic activity. The amount of the isotope taken up by individual archaeal and bacterial cells within their microbial "neighborhoods" in each consortia was then measured with a high-resolution instrument called nanoscale secondary ion mass spectrometry (nanoSIMS) at Caltech. This allowed the researchers to determine how active the archaeal and bacterial partners were relative to their distance to one another.

To their surprise, the researchers found that the spatial arrangement of the cells in consortia had no influence on their activity. "Since this is a syntrophic relationship, we would have thought the cells at the interface—where the bacteria are directly contacting the archaea—would be more active, but we don't really see an obvious trend. What is really notable is that there are cells that are many cell lengths away from their nearest partner that are still active," Orphan says.

To find out how the bacteria and archaea were partnering, co-first authors Grayson Chadwick (BS '11), a graduate student in geobiology at Caltech and a former undergraduate researcher in Orphan's lab, and Shawn McGlynn, a former postdoctoral scholar, employed spatial statistics to look for patterns in cellular activity for multiple consortia with different cell arrangements. They found that populations of syntrophic archaea and bacteria in consortia had similar levels of metabolic activity; when one population had high activity, the associated partner microorganisms were also equally active—consistent with a beneficial symbiosis. However, a close look at the spatial organization of the cells revealed that no particular arrangement of the two types of organisms—whether evenly dispersed or in separate groups—was correlated with a cell's activity.

To determine how these metabolic interactions were taking place even over relatively long distances, postdoctoral scholar and coauthor Chris Kempes, a visitor in computing and mathematical sciences, modeled the predicted relationship between cellular activity and distance between syntrophic partners that are dependent on the molecular diffusion of a substrate. He found that conventional metabolites—molecules previously predicted to be involved in this syntrophic consumption of methane—such as hydrogen—were inconsistent with the spatial activity patterns observed in the data. However, revised models indicated that electrons could likely make the trip from cell to cell across greater distances.

"Chris came up with a generalized model for the methane-oxidizing syntrophy based on direct electron transfer, and these model results were a better match to our empirical data," Orphan says. "This pointed to the possibility that these archaea were directly transferring electrons derived from methane to the outside of the cell, and those electrons were being passed to the bacteria directly."

Guided by this information, Chadwick and McGlynn looked for independent evidence to support the possibility of direct interspecies electron transfer. Cultured bacteria, such as those from the genus Geobacter, are model organisms for the direct electron transfer process. These bacteria use large proteins, called multi-heme cytochromes, on their outer surface that act as conductive "wires" for the transport of electrons.

Using genome analysis—along with transmission electron microscopy and a stain that reacts with these multi-heme cytochromes—the researchers showed that these conductive proteins were also present on the outer surface of the archaea they were studying. And that finding, Orphan says, can explain why the spatial arrangement of the syntrophic partners does not seem to affect their relationship or activity.

"It's really one of the first examples of direct interspecies electron transfer occurring between uncultured microorganisms in the environment. Our hunch is that this is going to be more common than is currently recognized," she says.

Orphan notes that the information they have learned about this relationship will help to expand how researchers think about interspecies microbial interactions in nature. In addition, the microscale stable isotope approach used in the current study can be used to evaluate interspecies electron transport and other forms of microbial symbiosis occurring in the environment.

These results were published in a paper titled, "Single cell activity reveals direct electron transfer in methanotrophic consortia." The work was funded by the Department of Energy Division of Biological and Environmental Research and the Gordon and Betty Moore Foundation Marine Microbiology Initiative.

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Bar-Coding Technique Opens Up Studies Within Single Cells

All of the cells in a particular tissue sample are not necessarily the same—they can vary widely in terms of genetic content, composition, and function. Yet many studies and analytical techniques aimed at understanding how biological systems work at the cellular level treat all of the cells in a tissue sample as identical, averaging measurements over the entire cellular population. It is easy to see why this happens. With the cell's complex matrix of organelles, signaling chemicals, and genetic material—not to mention its miniscule scale—zooming in to differentiate what is happening within each individual cell is no trivial task.

"But being able to do single-cell analysis is crucial to understanding a lot of biological systems," says Long Cai, assistant professor of chemistry at Caltech. "This is true in brains, in biofilms, in embryos . . . you name it."

Now Cai's lab has developed a method for simultaneously imaging and identifying dozens of molecules within individual cells. This technique could offer new insight into how cells are organized and interact with each other and could eventually improve our understanding of many diseases.

The imaging technique that Cai and his colleagues have developed allows researchers not only to resolve a large number of molecules—such as messenger RNA species (mRNAs)—within a single cell, but also to systematically label each type of molecule with its own unique fluorescent "bar code" so it can be readily identified and measured without damaging the cell.

"Using this technique, there is essentially no limit on how many different types of molecules you can detect within a single cell," explains Cai.

The new method uses an innovative sequential bar-coding scheme that takes fluorescence in situ hybridization (FISH), a well-known procedure for detecting specific sequences of DNA or RNA in a sample, to the next level. Cai and his colleagues have dubbed their technique FISH Sequential Coding anALYSis (FISH SCALYS). 

FISH makes use of molecular probes—short fragments of DNA bound to fluorescent dyes, or fluorophores. These probes bind, or hybridize, to DNA or RNA with complementary sequences. When a hybridized sample is imaged with microscopy, the fluorophore lights up, pinpointing the target molecule's location.

There are a handful of fluorophores that can be used in these probes, and researchers typically use them to identify only a few different genes. For example, they will use a red dye to label all of the probes that target a specific type of mRNA. And when they image the sample, they will see a bunch of red dots in the cell. Then they will take another set of probes that target a different type of mRNA, label them with a blue fluorophore, and see glowing blue spots. And so on.

But what if a researcher wants to image more types of molecules than there are fluorophores? In the past, they have tried to mix the dyes together, making both red and blue probes for a particular gene, so that when both probes bind to the gene, the resulting dot would look purple. It was an imperfect solution and could still only label about 30 different types of molecules.

Cai's team realized that the same handful of fluorophores could be used in sequential rounds of hybridization to create thousands of unique fluorescent bar codes that could clearly identify many types of molecules (see graphic at right).

"With our technique, each tagged molecule remains just one single color in each round but we build up a bar code through multiple rounds, so the colors remain distinguishable. Using additional colors and extra rounds of hybridization, you can scale up easily to identify tens of thousands of different molecules," says Cai.

The number of bar codes available is potentially immense: FN, where F is the number of fluorophores and N is the number of rounds of hybridization. So with four dyes and eight rounds of hybridization, scientists would have more than enough bar codes (48=65,536) to cover all of the approximately 20,000 RNA molecules in a cell.

Cai says FISH SCALYS could be used to determine molecular identities of various types of cells, including embryonic stem cells. "One subset of genes will be turned on for one type of cell and off for another," he explains. It could also provide insight into the way that diseases alter cells, allowing researchers to compare the expression differences for a large number of genes in normal tissue versus diseased tissue.

Cai has recently been funded by the McKnight Endowment Fund for Neuroscience to adapt the technique to identify different types of neurons in samples from the hippocampus, a part of the brain associated with memory and learning.

Cai is also leading a program through Caltech's Beckman Institute that is helping other researchers on campus apply the imaging method to diverse biological questions.

Cai and his team describe the technique in a Nature Methods paper titled "Single-cell in situ RNA profiling by sequential hybridization." Caltech graduate student Eric Lubeck and postdoctoral scholar Ahmet Coskun are lead authors on the paper. Additional coauthors include Timur Zhiyentayev, a former Caltech graduate student, and Mubhij Ahmad, a former research technician in the Cai lab. The work has been funded by the National Institutes of Health's Single Cell Analysis Program.

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An Antibody That Can Attack HIV in New Ways

Proteins called broadly neutralizing antibodies (bNAbs) are a promising key to the prevention of infection by HIV, the virus that causes AIDS. bNAbs have been found in blood samples from some HIV patients whose immune systems can naturally control the infection. These antibodies may protect a patient's healthy cells by recognizing a protein called the envelope spike, present on the surface of all HIV strains and inhibiting, or neutralizing, the effects of the virus. Now Caltech researchers have discovered that one particular bNAb may be able to recognize this signature protein, even as it takes on different conformations during infection—making it easier to detect and neutralize the viruses in an infected patient.

The work, from the laboratory of Pamela Bjorkman, Centennial Professor of Biology, was published in the September 10 issue of the journal Cell.

The process of HIV infection begins when the virus comes into contact with human immune cells called T cells that carry a particular protein, CD4, on their surface. Three-part (or "trimer") proteins called envelope spikes on the surface of the virus recognize and bind to the CD4 proteins. The spikes can be in either a closed or an open conformation, going from closed to open when the spike binds to CD4. The open conformation then triggers fusion of the virus with the target cell, allowing the HIV virus to deposit its genetic material inside the host cell, forcing it to become a factory for making new viruses that can go on to infect other cells.

The bNAbs recognize the envelope spike on the surface of HIV, and most known bNAbs only recognize the spike in the closed conformation. Although the only target of neutralizing antibodies is the envelope spike, each bNAb actually functions by recognizing just one specific target, or epitope, on this protein. Some targets allow more effective neutralization of the virus, and, therefore, some bNAbs are more effective against HIV than others. In 2014, Bjorkman and her collaborators at Rockefeller University reported initial characterization of a potent bNAb called 8ANC195 in the blood of HIV patients whose immune systems could naturally control their infections. They also discovered that this antibody could neutralize the HIV virus by targeting a different epitope than any other previously identified bNAb.

In the work described in the recent Cell paper, they investigated how 8ANC195 functions—and how its unique properties could be beneficial for HIV therapies.

"In Pamela's lab we use X-ray crystallography and electron microscopy to study protein–protein interactions on a molecular level," says Louise Scharf, a postdoctoral scholar in Bjorkman's laboratory and the first author on the paper. "We previously were able to define the binding site of this antibody on a subunit of the HIV envelope spike, so in this study we solved the three-dimensional structure of this antibody in complex with the entire spike, and showed in detail exactly how the antibody recognizes the virus."

What they found was that although most bNAbs recognize the envelope spike in its closed conformation, 8ANC195 could recognize the viral protein in both the closed conformation and a partially open conformation. "We think it's actually an advantage if the antibody can recognize these different forms," Scharf says.

The most common form of HIV infection is when a virus in the bloodstream attaches to a T cell and infects the cell. In this instance, the spikes on the free-floating virus would be predominantly in the closed conformation until they made contact with the host cell. Most bNAbs could neutralize this virus. However, HIV also can spread directly from one cell to another. In this case, because the antibody already is attached to the host cell, the spike is in an open conformation. But 8ANC195 could still recognize and attach to it.

A potential medical application of this antibody is in so-called combination therapies, in which a patient is given a cocktail of several antibodies that work in different ways to fight off the virus as it rapidly changes and evolves. "Our collaborators at Rockefeller have studied this extensively in animal models, showing that if you administer a combination of these antibodies, you greatly reduce how much of the virus can escape and infect the host," Scharf says. "So 8ANC195 is one more antibody that we can use therapeutically; it targets a different epitope than other potent antibodies, and it has the advantage of being able to recognize these multiple conformations."

The idea of bNAb therapeutics might not be far from a clinical reality. Scharf says that the same collaborators at Rockefeller University are already testing bNAbs in a human treatment in a clinical trial. Although the initial trial will not include 8ANC195, the antibody may be included in a combination therapy trial in the near future, Scharf says.

Furthermore, the availability of complete information about how 8ANC195 binds to the viral spike will allow Scharf, Bjorkman, and their colleagues to begin engineering the antibody to be more potent and able to recognize more strains of HIV.

"In addition to supporting the use of 8ANC195 for therapeutic applications, our structural studies of 8ANC195 have revealed an unanticipated new conformation of the HIV envelope spike that is relevant to understanding the mechanism by which HIV enters host cells and bNAbs inhibit this process," Bjorkman says.

These results were published in a journal article titled "Broadly Neutralizing Antibody 8ANC195 Recognizes Closed and Open States of HIV-1 Env." In addition to Scharf and Bjorkman, other Caltech coauthors include graduate student Haoqing Wang, research technician Han Gao, research scientist Songye Chen, and Beckman Institute resource director Alasdair W. McDowall. Funding for the work was provided by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health; the Bill and Melinda Gates Foundation; and the American Cancer Society. Crystallography and electron microscopy were done at the Molecular Observatory at Caltech, supported by the Gordon and Betty Moore Foundation.

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Making Nanowires from Protein and DNA

The ability to custom design biological materials such as protein and DNA opens up technological possibilities that were unimaginable just a few decades ago. For example, synthetic structures made of DNA could one day be used to deliver cancer drugs directly to tumor cells, and customized proteins could be designed to specifically attack a certain kind of virus. Although researchers have already made such structures out of DNA or protein alone, a Caltech team recently created—for the first time—a synthetic structure made of both protein and DNA. Combining the two molecule types into one biomaterial opens the door to numerous applications.

A paper describing the so-called hybridized, or multiple component, materials appears in the September 2 issue of the journal Nature.

There are many advantages to multiple component materials, says Yun (Kurt) Mou (PhD '15), first author of the Nature study. "If your material is made up of several different kinds of components, it can have more functionality. For example, protein is very versatile; it can be used for many things, such as protein–protein interactions or as an enzyme to speed up a reaction. And DNA is easily programmed into nanostructures of a variety of sizes and shapes."

But how do you begin to create something like a protein–DNA nanowire—a material that no one has seen before?

Mou and his colleagues in the laboratory of Stephen Mayo, Bren Professor of Biology and Chemistry and the William K. Bowes Jr. Leadership Chair of Caltech's Division of Biology and Biological Engineering, began with a computer program to design the type of protein and DNA that would work best as part of their hybrid material. "Materials can be formed using just a trial-and-error method of combining things to see what results, but it's better and more efficient if you can first predict what the structure is like and then design a protein to form that kind of material," he says.

The researchers entered the properties of the protein–DNA nanowire they wanted into a computer program developed in the lab; the program then generated a sequence of amino acids (protein building blocks) and nitrogenous bases (DNA building blocks) that would produce the desired material.

However, successfully making a hybrid material is not as simple as just plugging some properties into a computer program, Mou says. Although the computer model provides a sequence, the researcher must thoroughly check the model to be sure that the sequence produced makes sense; if not, the researcher must provide the computer with information that can be used to correct the model. "So in the end, you choose the sequence that you and the computer both agree on. Then, you can physically mix the prescribed amino acids and DNA bases to form the nanowire."

The resulting sequence was an artificial version of a protein–DNA coupling that occurs in nature. In the initial stage of gene expression, called transcription, a sequence of DNA is first converted into RNA. To pull in the enzyme that actually transcribes the DNA into RNA, proteins called transcription factors must first bind certain regions of the DNA sequence called protein-binding domains.

Using the computer program, the researchers engineered a sequence of DNA that contained many of these protein-binding domains at regular intervals. They then selected the transcription factor that naturally binds to this particular protein-binding site—the transcription factor called Engrailed from the fruit fly Drosophila. However, in nature, Engrailed only attaches itself to the protein-binding site on the DNA. To create a long nanowire made of a continuous strand of protein attached to a continuous strand of DNA, the researchers had to modify the transcription factor to include a site that would allow Engrailed also to bind to the next protein in line.

"Essentially, it's like giving this protein two hands instead of just one," Mou explains. "The hand that holds the DNA is easy because it is provided by nature, but the other hand needs to be added there to hold onto another protein."

Another unique attribute of this new protein–DNA nanowire is that it employs coassembly—meaning that the material will not form until both the protein components and the DNA components have been added to the solution. Although materials previously could be made out of DNA with protein added later, the use of coassembly to make the hybrid material was a first. This attribute is important for the material's future use in medicine or industry, Mou says, as the two sets of components can be provided separately and then combined to make the nanowire whenever and wherever it is needed.

This finding builds on earlier work in the Mayo lab, which, in 1997, created one of the first artificial proteins, thus launching the field of computational protein design. The ability to create synthetic proteins allows researchers to develop proteins with new capabilities and functions, such as therapeutic proteins that target cancer. The creation of a coassembled protein–DNA nanowire is another milestone in this field.

"Our earlier work focused primarily on designing soluble, protein-only systems. The work reported here represents a significant expansion of our activities into the realm of nanoscale mixed biomaterials," Mayo says.

Although the development of this new biomaterial is in the very early stages, the method, Mou says, has many promising applications that could change research and clinical practices in the future.

"Our next step will be to explore the many potential applications of our new biomaterial," Mou says. "It could be incorporated into methods to deliver drugs into cells—to create targeted therapies that only bind to a certain biomarker on a certain cell type, such as cancer cells. We could also expand the idea of protein–DNA nanowires to protein–RNA nanowires that could be used for gene therapy applications. And because this material is brand-new, there are probably many more applications that we haven't even considered yet."  

The work was published in a paper titled, "Computational design of co-assembling protein-DNA nanowires." In addition to Mou and Mayo, other Caltech coauthors include former graduate students Jiun-Yann Yu (PhD '14) and Timothy M. Wannier (PhD '15), as well as Chin-Lin Guo from Academia Sinica in Taiwan. The work was funded by the Defense Advanced Research Projects Agency Protein Design Processes Program, a National Security Science and Engineering Faculty Fellowship, and the Caltech Programmable Molecular Technology Initiative funded by the Gordon and Betty Moore Foundation.

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Caltech Chemists Solve Major Piece of Cellular Mystery

Team determines the architecture of a second subcomplex of the nuclear pore complex

Not just anything is allowed to enter the nucleus, the heart of eukaryotic cells where, among other things, genetic information is stored. A double membrane, called the nuclear envelope, serves as a wall, protecting the contents of the nucleus. Any molecules trying to enter or exit the nucleus must do so via a cellular gatekeeper known as the nuclear pore complex (NPC), or pore, that exists within the envelope.

How can the NPC be such an effective gatekeeper—preventing much from entering the nucleus while helping to shuttle certain molecules across the nuclear envelope? Scientists have been trying to figure that out for decades, at least in part because the NPC is targeted by a number of diseases, including some aggressive forms of leukemia and nervous system disorders such as a hereditary form of Lou Gehrig's disease. Now a team led by André Hoelz, assistant professor of biochemistry at Caltech, has solved a crucial piece of the puzzle.

In February of this year, Hoelz and his colleagues published a paper describing the atomic structure of the NPC's coat nucleoporin complex, a subcomplex that forms what they now call the outer rings (see illustration). Building on that work, the team has now solved the architecture of the pore's inner ring, a subcomplex that is central to the NPC's ability to serve as a barrier and transport facilitator. In order to the determine that architecture, which determines how the ring's proteins interact with each other, the biochemists built up the complex in a test tube and then systematically dissected it to understand the individual interactions between components. Then they validated that this is actually how it works in vivo, in a species of fungus.

For more than a decade, other researchers have suggested that the inner ring is highly flexible and expands to allow large macromolecules to pass through. "People have proposed some complicated models to explain how this might happen," says Hoelz. But now he and his colleagues have shown that these models are incorrect and that these dilations simply do not occur.

"Using an interdisciplinary approach, we solved the architecture of this subcomplex and showed that it cannot change shape significantly," says Hoelz. "It is a relatively rigid scaffold that is incorporated into the pore and basically just sits as a decoration, like pom-poms on a bicycle. It cannot dilate."

The new paper appears online ahead of print on August 27 in Science Express. The four co-lead authors on the paper are Caltech postdoctoral scholars Tobias Stuwe, Christopher J. Bley, and Karsten Thierbach, and graduate student Stefan Petrovic.

Crystal Structure of Fungal Channel Nucleoporin Complex
This video features a rotating three-dimensional crystal structure of the fungal channel nucleoporin complex bound to the adaptor nucleoporin Nic96. This interaction is the complex's sole site of attachment to the rest of the inner ring of the NPC. The channel nucleoporin complex borders the central transport channel and fills it with filamentous structures (phenylalanine-glycine repeats) that form a diffusion barrier and provide docking sites for proteins that ferry molecules across the nuclear envelope. Credit: Andre Hoelz/Caltech and Science

Together, the inner and outer rings make up the symmetric core of the NPC, a structure that includes 21 different proteins. The symmetric core is so named because of its radial symmetry (the two remaining subcomplexes of the NPC are specific to either the side that faces the cell's cytoplasm or the side that faces the nucleus and are therefore not symmetric). Having previously solved the structure of the coat nucleoporin complex and located it in the outer rings, the researchers knew that the remaining components that are not membrane anchored must make up the inner ring.

They started solving the architecture by focusing on the channel nucleoporin complex, or channel, which lines the central transport channel and is made up of three proteins, accounting for about half of the inner ring. This complex produces filamentous structures that serve as docking sites for specific proteins that ferry molecules across the nuclear envelope.

The biochemists employed bacteria to make the proteins associated with the inner ring in a test tube and mixed various combinations until they built the entire subcomplex. Once they had reconstituted the inner ring subcomplex, they were able to modify it to investigate how it is held together and which of its components are critical, and to determine how the channel is attached to the rest of the pore.

Hoelz and his team found that the channel is attached at only one site. This means that it cannot stretch significantly because such shape changes require multiple attachment points. Hoelz notes that a new electron microscopy study of the NPC published in 2013 by Martin Beck's group at the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany, indicated that the central channel is bigger than previously thought and wide enough to accommodate even the largest cargoes known to pass through the pore.

When the researchers introduced mutations that effectively eliminated the channel's single attachment, the complex could no longer be incorporated into the inner ring. After proving this in the test tube, they also showed this to be true in living cells.

"This whole complex is a very complicated machine to assemble. The cool thing here is that nature has found an elegant way to wait until the very end of the assembly of the nuclear pore to incorporate the channel," says Hoelz. "By incorporating the channel, you establish two things at once: you immediately form a barrier and you generate the ability for regulated transport to occur through the pore." Prior to the channel's incorporation, there is simply a hole through which macromolecules can freely pass.

Next, Hoelz and his colleagues used X-ray crystallography to determine the structure of the channel nucleoporin subcomplex bound to the adaptor nucleoporin Nic96, which is its only nuclear pore attachment site. X-ray crystallography involves shining X-rays on a crystallized sample and analyzing the pattern of rays reflected off the atoms in the crystal. Because the NPC is a large and complex molecular machine that also has many moving parts, they used an engineered antibody to essentially "superglue" many copies of the complex into place to form a nicely ordered crystalline sample. Then they analyzed hundreds of samples using Caltech's Molecular Observatory—a facility developed with support from the Gordon and Betty Moore Foundation that includes an automated X-ray beam line at the Stanford Synchrotron Radiation Laboratory that can be controlled remotely from Caltech—and the GM/CA beam line at the Advanced Photon Source at the Argonne National Laboratory. Eventually, they were able to determine the size, shape, and position of all the atoms of the channel nucleoporin subcomplex and its location within the full NPC.

"The crystal structure nailed it," Hoelz says. "There is no way that the channel is changing shape. All of that other work that, for more than 10 years, suggested it was dilating was wrong."

The researchers also solved a number of crystal structures from other parts of the NPC and determined how they interact with components of the inner ring. In doing so they demonstrated that one such interaction is critical for positioning the channel in the center of the inner ring. They found that exact positioning is needed for the proper export from the nucleus of mRNA and components of ribosomes, the cell's protein-making complexes, rendering it critical in the flow of genetic information from DNA to mRNA to protein.

Hoelz adds that now that the architectures of the inner and outer rings of the NPC are known, getting an atomic structure of the entire symmetric core is "a sprint to the summit."

"When I started at Caltech, I thought it might take another 10, 20 years to do this," he says. "In the end, we have really only been working on this for four and a half years, and the thing is basically tackled. I want to emphasize that this kind of work is not doable everywhere. The people who worked on this are truly special, talented, and smart; and they worked day and night on this for years."

Ultimately, Hoelz says he would like to understand how the NPC works in great detail so that he might be able to generate therapies for diseases associated with the dysfunction of the complex. He also dreams of building up an entire pore in the test tube so that he can fully study it and understand what happens as it is modified in various ways. "Just as they did previously when I said that I wanted to solve the atomic structure of the nuclear pore, people will say that I'm crazy for trying to do this," he says. "But if we don't do it, it is likely that nobody else will."

The paper, "Architecture of the fungal nuclear pore inner ring complex," had a number of additional Caltech authors: Sandra Schilbach (now of the Max Planck Institute of Biophysical Chemistry), Daniel J. Mayo, Thibaud Perriches, Emily J. Rundlet, Young E. Jeon, Leslie N. Collins, Ferdinand M. Huber, and Daniel H. Lin. Additional coauthors include Marcin Paduch, Akiko Koide, Vincent Lu, Shohei Koide, and Anthony A. Kossiakoff of the University of Chicago; and Jessica Fischer and Ed Hurt of Heidelberg University.



Kimm Fesenmaier
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NSF BRAIN Funding Awarded to Caltech Neuroscientist

On August 12, in support of President Obama's Brain Research through Advancing Innovative Neurotechnology—or BRAIN—Initiative, the National Science Foundation (NSF) announced 16 new grants for fundamental brain research. A cognitive neuroengineering project co-led by Richard Andersen, the James G. Boswell Professor of Neuroscience, was selected as a recipient for one of these grants.

Designed to bring together interdisciplinary teams of scientists and engineers from diverse fields, the grants represent two themes: neuroengineering and brain-inspired concepts and designs, and individuality and variation. Each grants provides up to $1 million in funding over two to four years.

Andersen, whose work falls under the first theme, plans to use his grant to improve the functionality of neural prosthetic devices—devices that, when implanted in the brain, can allow patients with amputations or paralysis to control the movement of a robotic limb. The work is a collaboration with Charles Y. Liu, of Keck Medicine of USC, and Kapil Katyal of Johns Hopkins University.

In a clinical trial earlier this year, Andersen showed that a neural prosthetic device implanted in the brain's center for intentions—the posterior parietal cortex—could allow a tetraplegic patient to control a robotic arm with only his thoughts. The new work will build on this idea, Andersen says. "We are developing a shared control system in which we can record the intent of a tetraplegic patient and immediately communicate that intent to a smart robotic limb that can handle the details of the movement. This enables more effortless control by the patients," he says.

The grants are funded by the NSF Integrative Strategies of Understanding Neural and Cognitive Systems program and the NSF Computer & Information Science & Engineering Directorate. The NSF Directorates for Engineering and for Education and Human Resources also support the grants.

Andersen, who also received a grant from the state-funded Cal-BRAIN program for work in improving neural prosthetics, joins six other Caltech projects associated with the BRAIN Initiative that were funded by the National Institutes of Health last fall.

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Mosquitoes Use Smell to See Their Hosts

On summer evenings, we try our best to avoid mosquito bites by dousing our skin with bug repellents and lighting citronella candles. These efforts may keep the mosquitoes at bay for a while, but no solution is perfect because the pests have evolved to use a triple threat of visual, olfactory, and thermal cues to home in on their human targets, a new Caltech study suggests.

The study, published by researchers in the laboratory of Michael Dickinson, the Esther M. and Abe M. Zarem Professor of Bioengineering, appears in the July 17 online version of the journal Current Biology.

When an adult female mosquito needs a blood meal to feed her young, she searches for a host—often a human. Many insects, mosquitoes included, are attracted by the odor of the carbon dioxide (CO2) gas that humans and other animals naturally exhale. However, mosquitoes can also pick up other cues that signal a human is nearby. They use their vision to spot a host and thermal sensory information to detect body heat.

But how do the mosquitoes combine this information to map out the path to their next meal?

To find out how and when the mosquitoes use each type of sensory information, the researchers released hungry, mated female mosquitoes into a wind tunnel in which different sensory cues could be independently controlled. In one set of experiments, a high-concentration CO2 plume was injected into the tunnel, mimicking the signal created by the breath of a human. In control experiments, the researchers introduced a plume consisting of background air with a low concentration of CO2. For each experiment, researchers released 20 mosquitoes into the wind tunnel and used video cameras and 3-D tracking software to follow their paths.

When a concentrated CO2 plume was present, the mosquitos followed it within the tunnel as expected, whereas they showed no interest in a control plume consisting of background air.

"In a previous experiment with fruit flies, we found that exposure to an attractive odor led the animals to be more attracted to visual features," says Floris van Breugel, a postdoctoral scholar in Dickinson's lab and first author of the study. "This was a new finding for flies, and we suspected that mosquitoes would exhibit a similar behavior. That is, we predicted that when the mosquitoes were exposed to CO2, which is an indicator of a nearby host, they would also spend a lot of time hovering near high-contrast objects, such as a black object on a neutral background."

To test this hypothesis, van Breugel and his colleagues did the same CO2 plume experiment, but this time they provided a dark object on the floor of the wind tunnel. They found that in the presence of the carbon dioxide plumes, the mosquitoes were attracted to the dark high-contrast object. In the wind tunnel with no CO2 plume, the insects ignored the dark object entirely.

While it was no surprise to see the mosquitoes tracking a CO2 plume, "the new part that we found is that the CO2 plume increases the likelihood that they'll fly toward an object. This is particularly interesting because there's no CO2 down near that object—it's about 10 centimeters away," van Breugel says. "That means that they smell the CO2, then they leave the plume, and several seconds later they continue flying toward this little object. So you could think of it as a type of memory or lasting effect."

Next, the researchers wanted to see how a mosquito factors thermal information into its flight path. It is difficult to test, van Breugel says. "Obviously, we know that if you have an object in the presence of a CO2 plume—warm or cold—they will fly toward it because they see it," he says. "So we had to find a way to separate the visual attraction from the thermal attraction."

To do this, the researchers constructed two glass objects that were coated with a clear chemical substance that made it possible to heat them to any desired temperature. They heated one object to 37 degrees Celsius (approximately human body temperature) and allowed one to remain at room temperature, and then placed them on the floor of the wind tunnel with and without CO2 plumes, and observed mosquito behavior. They found that mosquitoes showed a preference for the warm object. But contrary to the mosquitoes' visual attraction to objects, the preference for warmth was not dependent on the presence of CO2.

"These experiments show that the attraction to a visual feature and the attraction to a warm object are separate. They are independent, and they don't have to happen in order, but they do often happen in this particular order because of the spatial arrangement of the stimuli: a mosquito can see a visual feature from much further away, so that happens first. Only when the mosquito gets closer does it detect an object's thermal signature," van Breugel says.

Information gathered from all of these experiments enabled the researchers to create a model of how the mosquito finds its host over different distances. They hypothesize that from 10 to 50 meters away, a mosquito smells a host's CO2 plume. As it flies closer—to within 5 to 15 meters—it begins to see the host. Then, guided by visual cues that draw it even closer, the mosquito can sense the host's body heat. This occurs at a distance of less than a meter.

"Understanding how brains combine information from different senses to make appropriate decisions is one of the central challenges in neuroscience," says Dickinson, the principal investigator of the study. "Our experiments suggest that female mosquitoes do this in a rather elegant way when searching for food. They only pay attention to visual features after they detect an odor that indicates the presence of a host nearby. This helps ensure that they don't waste their time investigating false targets like rocks and vegetation. Our next challenge is to uncover the circuits in the brain that allow an odor to so profoundly change the way they respond to a visual image."

The work provides researchers with exciting new information about insect behavior and may even help companies design better mosquito traps in the future. But it also paints a bleak picture for those hoping to avoid mosquito bites.

"Even if it were possible to hold one's breath indefinitely," the authors note toward the end of the paper, "another human breathing nearby, or several meters upwind, would create a CO2 plume that could lead mosquitoes close enough to you that they may lock on to your visual signature. The strongest defense is therefore to become invisible, or at least visually camouflaged. Even in this case, however, mosquitoes could still locate you by tracking the heat signature of your body . . . The independent and iterative nature of the sensory-motor reflexes renders mosquitoes' host seeking strategy annoyingly robust."

These results were published in a paper titled "Mosquitoes use vision to associate odor plumes with thermal targets." In addition to Dickinson and van Breugel, the other authors are Jeff Riffell and Adrienne Fairhall from the University of Washington. The work was funded by a grant from the National Institutes of Health.

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