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38 Winter 2015 / HHMI Bulletin Chronicle / Lab Book

the inside can replicate and spread. But chronic infections can be lengthy, and after a few weeks of sustained action, cellular exhaustion often sets in. The T cells become less effective at slaying their targets and begin producing proteins that prevent them from recognizing infected cells. One of these proteins is called The immune system’s T lymphocytes, like this one, programmed cell death protein 1, or PD-1. need downtime to do their job properly. Susan Kaech, an HHMI early career scientist at , discovered a feedback Kaech concluded that T cells need this respite T Cell Burnout loop by which PD-1 activation leads to an to function properly. “They have to turn down increase in a protein called FOXO1. FOXO1, their response or else they’ll get over-activated, Some immune cells in turn, produces factors that promote T cell and we think that causes the cells to deteriorate function better after exhaustion, including more PD-1. Kaech and die,” she says. “We’re starting to appreciate suspected that eliminating FOXO1 might that exhaustion is an important process that is taking a break. curtail T cells’ exhaustion and make helping to maintain this precious pool of T cells.” every living being needs to rest. them better killers. The fndings, published November 20, 2014, Even our immune cells enter a dysfunctional What she found was the exact opposite. in Immunity, could lead to drugs that modulate state called T cell exhaustion if they’re When her team created mice lacking the FOXO1 FOXO1 and help reinvigorate the immune overworked. In fact, this fatigue and the gene, the rodents’ T cells did produce less PD-1 response of patients being treated for chronic ensuing downtime are important parts compared to animals with the gene. But the viral infections or even . “There may of the immune process. cells weren’t better at controlling the viral be a point where you can stop the cells from Killer T cells get their name from their infection. Instead, without the rest afforded fully entering the exhausted state – where you function. They target sickened cells involved them in their exhausted state, the cells died, suppress PD-1, but not so much that the cells in chronic infections, killing them before and viral replication increased. die,” Kaech says. – Nicole Kresge

IN BRIEF

SEEING RED neuronal signaling. The resulting neighboring cells to proteins have similar stretches As a fish swims, nerve cells fire in its indelibly tags firing shore up their defenses. of five amino acids that became brain, sending signals racing along neurons with a red glow in the These alerts are sent tagged with phosphate groups. a neural network ending in muscles presence of violet . via a family of proteins As the team reported March 13, that make its fins flap and its tail “Ideally, we would flip the [violet] called interferons, 2015, in , the addition of swish. By using a molecule called

light switch on while an animal is which are produced a phosphate molecule to that Parsons

CaMPARI to permanently mark doing a behavior that we care about, when surveillance stretch of amino acids causes Leif neurons as they fire, scientists can then flip the switch off as soon proteins in the the adaptors to activate IRF3. now watch as signals light up such as the animal stops the behavior,” infected cell detect Now that they know neural networks in live animals. Schreiter explains. “So we’re a pathogen. Although several how the interferon pathways

CaMPARI came out of a capturing a snapshot of neural different surveillance proteins scout converge, Chen and his team are Illustration: collaborative project spearheaded activity that occurs only while the for signs of pathogens, new research examining them in more detail. by Eric Schreiter, a senior scientist animal is doing that behavior.” shows how the proteins all activate Eventually, they hope to develop in Group Leader Loren Looger’s lab The scientists published their a single molecule called IRF3 to turn small that treat immune Diseases; at the Janelia Research Campus. The results February 13, 2015, in Science. on interferon production. disorders by interfering with the team started with a protein called Although they are still tinkering There are three known pathways. Eos, which emits a green glow until with CaMPARI to make it more pathways that trigger type 1 Infectious it’s exposed to violet light. The light sensitive and reliable, they’ve interferon production. In each A CAT TALE and changes the molecule’s structure, already made it available to scientists case, the individual pathway’s A central tenet of biology is that causing it to glow red. By combining Allergy

on Addgene and the Bloomington unique surveillance protein uses each in a protein is

Eos with the calcium-sensitive Drosophila Stock Center. Janelia its own adaptor protein to relay specified by a three-letter code of protein calmodulin, the researchers Group Leader Misha Ahrens is also the message that an invader is found in RNA (mRNA). were able to couple Eos’s color distributing CaMPARI-expressing present and interferon is needed. That may be true most of the time, Institute change to the burst of calcium zebrafish. Siqi Liu, a graduate student in but HHMI Investigator Jonathan that accompanies HHMI Investigator Zhijian “James” Weissman at the University of

IMMUNE RALLY CRY Chen’s lab at the University of California, San Francisco (UCSF), National Like a Good Samaritan, a cell that’s Texas Southwestern Medical Center, along with Onn Brandman at

been attacked by a warns noticed that all three adaptor Stanford University and UCSF Photo: HHMI Bulletin / Spring 2015 39

Watch a mouse begin to drink thirstily when prompted by a blue light. Go to hhmi.org/bulletin/spring-2015. Researchers have long suspected that intake by about 80 percent. The researchers the signals driving animals to drink originate published their fndings online January 26, in the brain’s subfornical organ, or SFO. Located 2015, in . outside the -brain barrier, where it has According to Zuker, the opposing neurons the opportunity to directly sense the electrolyte likely work together to ensure animals take balance in body fluids, the SFO shows increased in enough to maintain fluid homeostasis, activity in dehydrated animals. Yuki Oka, including , electrolyte balance, a postdoctoral fellow in Zuker’s Columbia and cell volume. It remains to be seen whether University lab, took a closer look at the SFO in the same circuit controls thirst in ; if so, mice and identifed two types of nerve cells: the fndings could one day help people with an excitatory CAMKII-expressing neurons and impaired sense of thirst. – Nicole Kresge Got Thirst? inhibitory VGAT-expressing neurons. Using Here’s Why optogenetics, Oka added a light-sensitive The mouse brain contains CAMKII neurons (red) that protein to the cells in the animals’ SFOs, trigger thirst and VGAT neurons (green) that quench thirst. Scientists have allowing him to selectively activate them with blue light. When he flipped the switch and pinpointed the neurons activated the CAMKII neurons, the rodents in the brain that drank with gusto. “You have a water-satiated animal that control thirst. is happily wandering around, with zero humans can typically survive only interest in drinking,” says Zuker. “Activate three to fve days without water. But we’ve all this group of SFO neurons, and the mouse said “I’m dying of thirst” well before we’ve just beelines to the water spout. As long as the been waterless that long – albeit with little light is on, that mouse keeps on drinking.” understanding of the mechanism behind the Oka showed that the animals became such urge. But now the brain signals that govern thirst avid drinkers that they consumed as much as are no longer a mystery. A team led by HHMI 8 percent of their body weight in water – the Investigator Charles Zuker has fgured out that equivalent of 1.5 gallons for humans. When the motivation to drink water is controlled Oka used the same technique to stimulate by two sets of neurons: one that provokes the VGAT neurons, thirsty animals immediately stimulus to sip and one that quenches it. stopped drinking and reduced their water

colleague Adam Frost, has published their findings Janelia Group Leader David Stern sites that don’t look like good Hox discovered an exception to the rule. January 2, 2015, in Science. recently revealed the answer to binding sites,” explains Stern. Generally, protein synthesis They don’t yet know the this long-standing paradox. requires three components: function of CAT tails, but Stern’s team, in collaboration A TARGET FOR DYSKINESIA an mRNA template, a to they have some ideas with Richard Mann’s lab at Some diseases occur read the template and assemble they’re pursuing. For Columbia University Medical episodically in people who appear the protein, and transfer RNA example, the extra Center, figured out that otherwise healthy. One of these (tRNA) molecules to supply the amino acids may trigger Hox proteins activate genes disorders is paroxysmal nonkine- necessary amino acids. Weissman a stress response in the via weak interactions at sigenic dyskinesia (PNKD). and his colleagues discovered that cell, or they could tag an previously unrecognized The neurological disease’s symp- sometimes, when protein synthesis incomplete peptide for DNA binding sites. The toms – involuntary movements stalls, elongation can still continue destruction. scientists discovered this by in the limbs, torso, and face – are without the mRNA template. This showing that a Hox protein triggered by coffee, alcohol, and type of synthesis relies on only two HOX PARADOX called Ubx controls expression of stress. In 2004, HHMI Investigator components: a part of the ribosome NO MORE a gene called shavenbaby by binding Louis J. Ptáçek at the University of called the 60s subunit and a protein During development in animals, the weakly to two enhancer regions on California, San Francisco, discov- named Rqc2 that recruits tRNA Hox gene family directs the formation DNA near the gene. These low- ered the gene that causes PNKD.

Parsons molecules. With these components, of segment-specific anatomy along affinity binding sites conferred Now, just over a decade later, he’s

cells will continue to randomly add the head-to-tail axis. The genes are shavenbaby’s specificity for Ubx over figured out what the gene does. Leif amino acids to the stalled peptide, responsible for putting the head, other Hox proteins. To deduce the function of the with one caveat. Rqc2 recognizes only thorax, abdomen, and other body The findings, published January PNKD gene, Ptáçek’s team raised alanine and threonine (abbreviated parts in the right places. Surprisingly, 15, 2015, in Cell, explain why scientists antibodies against the PNKD protein.

Illustration: “A” and “T”), so it creates what all Hox proteins bind with high have been unable to predict where This allowed them to isolate PNKD

lab; Weissman refers to as “CAT tails.” affinity to very similar stretches of Hox proteins bind to DNA simply and its binding partners from mouse “This is a mode of protein DNA, which begs the question: how by looking at its sequence. “They’re brain tissue. From these isolates,

Zuker synthesis that we’ve missed for can Hox proteins find and activate not regulating genes by binding to they discovered that PNKD interacts 50 years. It’s totally unprecedented,” their target genes if all binding the sites that everybody thought with two other proteins, called

Photo: says Weissman. He and his colleagues sequences look essentially the same? they were – they’re binding to these RIM1 and RIM2, that are involved 40 Spring 2015 / HHMI Bulletin Chronicle / Lab Book

Brunger’s team used a technique called single- cryomicroscopy to freeze NSF molecules bound to SNARE complexes at several stages and then capture images of them. The resulting snapshots provide near-atomic or better detail of the SNARE disassembly process. Like a series of movie stills, the images show NSF latching onto a SNARE complex, then NSF bound to ATP – the molecule that powers the salvage operation. Salvaging SNAREs Yet another image captured NSF after it had fnished working, bound to an energy- Scientists catch depleted form of ATP, called ADP. a protein-recycling The SNARE complex resembles a rope with a left-handed twist; the team’s images machine in the act. revealed that NSF uses adapter proteins called cells are huge proponents of recycling. SNAPs to grasp the “rope” in multiple places. They reuse everything from tiny The SNAPs wrap around the SNARE complex to massive molecular complexes. Despite this with a right-handed twist, suggesting that the penchant for salvage, scientists have never disassembly occurs via a simple unwinding been able to see one form of cellular recycling motion that frees the zipped SNARE proteins. in action – until now. A group led by HHMI A cell’s NSF complex uses SNAP proteins (orange) to The results, published February 5, 2015, Investigator Axel Brunger recently captured grasp and unwind membrane-embedded SNARE proteins in Nature, raise other questions the team a protein in the act of pulling apart a spent (red and blue helices). is eager to pursue. “There is a lot to be done cluster of membrane fusion molecules. in order to understand the motions and “Membrane fusion is a process akin to cells use membrane-bound SNARE proteins. conformational changes needed to disassemble the merging of soap bubbles into larger ones,” During fusion, SNAREs located on opposing the SNARE complex,” says Brunger. says Brunger, a structural biologist at Stanford membranes zip together into a stable complex, “Our electron microscope structures now University. “But that’s where the analogy linking the membranes. When the two enable us to design follow-up biophysical ends, since biological membranes do not membranes have become one, a protein called experiments to answer these questions at a merge all that easily.” To facilitate the process, NSF recycles the SNARE components. very deep level.” – Nicole Kresge

IN BRIEF 2015; in neurotransmitter release. As the as preventing the formation of new December 23, 2014, in eLife, these two Early Career Scientist Jeffrey Kieft scientists reported March 10, 2015, blood vessels, protecting cells in proteins confer cell-surface binding at the University of Colorado Proceedings of the National Academy Denver wonder if a universal in the the retina and brain from damage, to PEDF, transduce PEDF signals into copyright of Sciences, PNKD normally suppresses and stopping cancer cells from distinct types of cells that respond to start signal existed that could be 61-67, neurotransmitter release, but the growing. Despite considerable effort PEDF, and function in a cell type- recognized by both eukaryotic 518, mutant form found in the disease by scientists in academia and industry, specific manner. Because of the critical and prokaryotic . does so less effectively. The result the identity of the receptors had, role of these cell-surface receptors A structured region of an RNA is excessive synaptic transmission, until recently, remained a mystery. in PEDF signaling, they have high molecule dubbed an internal ribosome Nature

which leads to the involuntary This long-standing puzzle was so potential as therapeutic targets for entry site (IRES), which is used by Ltd: movements experienced by people intractable that it took HHMI Early cancer and other diseases. some viruses to initiate translation in with the disease. Career Scientist Hui and his , fit the bill. Taking a close According to Ptá ek, RIM1 and team at the University of California, UNIVERSAL look at the molecule, Kieft’s team ç Publishers RIM2 may be important targets for Los Angeles, seven years to solve. PROTEIN SYNTHESIS discovered that it could also initiate developing better medications for “We were initially intrigued The process of gene expression protein synthesis in prokaryotes. This PNKD and other dyskinesias, such as by this problem because of PEDF’s is, at its core, universal. But particular IRES, it turns out, binds both Macmillan those seen in Parkinson’s disease. broad medical value in treating major different use different bacterial and eukaryotic ribosomes

diseases,” says Sun. “But the hunt methods to carry out the “DNA in a similar structure-dependent from THE LONG LOST RECEPTOR for the receptor turned out to be an makes RNA and RNA makes manner, but the bacterial interaction In our cells, there is a widely expressed extremely challenging adventure.” protein” recipe. For example, the appears transient and weaker.

secreted factor called pigment His team methodically searched for signals used to recruit ribosomes The findings, published March 5, permission epithelium-derived factor (PEDF), PEDF’s binding partner by looking and start protein synthesis in 2015, in Nature, suggest there might be by which affects a bewildering array of at various native tissues and cells, as bacteria are not the same signals other naturally occurring structured bodily processes. The well as in databases of molecules used by eukaryotes. Yet much of that can initiate bacterial Parsons

cellular receptors with unknown functions. the structure and function of the protein synthesis. One of Kieft’s next Reprinted that bind PEDF Finally, they found two ribosome, the molecular machine steps will be to determine if other such Leif are at the root proteins that fit the bill – that translates RNA into proteins, structures do indeed exist, and, if so, image: of many useful PLXDC1 and PLXDC2. is conserved in both types of what they mean in terms of evolution Science activities, such As Sun’s team reported organisms. This made HHMI and gene regulation. Illustration: