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 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.
A new partnership will support translational sciences and health technology at Caltech thanks to a three-year commitment from Heritage Medical Research Institute (HMRI), a nonprofit founded and led by Caltech trustee Richard N. Merkin.
With this gift, the Institute and HMRI have created the Heritage Research Institute for the Advancement of Medicine and Science at Caltech. Eight Caltech faculty members from three academic divisions have been selected for the inaugural cohort of Heritage researchers, with a ninth yet to be named. These scientists and engineers—who will hold the title of Heritage Principal Investigators—will receive salary and research support as well as opportunities to learn from and collaborate with each other and with practicing physicians in the local community.
"Dick Merkin's insights into the changing landscape of modern medicine, his devotion to supporting young talent, and his exceptional generosity have come together to create an innovative program to advance translational research," says President Thomas F. Rosenbaum, holder of the Sonja and William Davidow Presidential Chair and professor of physics. "The generous support of HMRI, through Dick's vision, will provide the freedom and resources for faculty from across the divisions to tackle difficult science and engineering problems for the betterment of the human condition."
As a physician and a healthcare executive, Merkin has witnessed the rapid evolution of medicine and patient care in recent decades—and says he sees monumental changes on the horizon.
"I think some of the greatest breakthroughs this century will occur in biology, and I think Caltech is particularly positioned to be a leader in this area," Merkin says. "Our biggest problems are our biggest opportunities, and Caltech is gifted in looking at the world not as it is, but as it could be."
Caltech is uniquely suited to accelerating progress due to its highly collaborative environment, Merkin adds. The convergence of multidisciplinary science and technology, he says, is driving innovation at an exponential rate, particularly in the areas of implantable sensors and precision medicine.
Many of Caltech's new Heritage Principal Investigators have already deepened our understanding of how the human body works—from the microbes in our gut to the chemicals in our brain—and are advancing the study of diseases such as diabetes, autism, and cancer. As a trustee and benefactor, Merkin has been energized by the potential impact of their investigations.
"The most imaginative scientists on the globe are concentrated at Caltech," Merkin says. "They are dedicated to understanding the world around us. Just being able to interact with so many passionate, hardworking, and brilliant people is inspiring. I'm very grateful to be part of the Institute."
Adds Stephen Mayo, the William K. Bowes Jr. Leadership Chair of the Division of Biology and Biological Engineering and Bren Professor of Biology and Chemistry: "As a valued friend of the Institute and a physician, Richard Merkin knows that the treatments of tomorrow begin in the lab today. This gift will embolden the Heritage Principal Investigators—some of whom are in the early stages of their careers—to pursue their most promising ideas and, in turn, quicken the pace of discovery in the biosciences."
A graduate of the University of Miami, Merkin began his career as a physician before creating what is now known as Heritage Provider Network (HPN) in 1979. Merkin serves as HPN's president and chief executive officer and has overseen its growth into one of California's largest healthcare provider networks. In 2012, Fast Company magazine named HPN one of the most innovative healthcare companies for embracing techniques such as data mining and predictive modeling to better the well-being of patients and improve the nation's healthcare system.
Merkin's philanthropy focuses on medical research, the arts, and children, with a special emphasis on the people of Southern California. He has served on the Caltech Board of Trustees since 2007 and also sits on the boards of the Los Angeles County Museum of Art and United Friends of the Children, as well as educational institutions, including the Keck School of Medicine of USC and Alliance College-Ready Public Schools. The latter runs 27 charter schools in the greater Los Angeles area, including one site named after him—the Richard Merkin Middle School.
In 2003, Merkin founded HMRI, a nonprofit that also has supported the Dana Farber Cancer Institute and the Prostate Cancer Foundation. In deciding where to direct HMRI's research funds, making a pledge to Caltech made sense for Merkin.
"Watching the Institute's stewardship of resources as a trustee makes me very comfortable investing as a benefactor," Merkin says. "Supporting Caltech and its faculty and students is a much broader investment in a better future—not just for the local community, not just for the United States, but, really, for the world."
Generating and storing renewable energy, such as solar or wind power, is a key barrier to a clean-energy economy. When the Joint Center for Artificial Photosynthesis (JCAP) was established at Caltech and its partnering institutions in 2010, the U.S. Department of Energy (DOE) Energy Innovation Hub had one main goal: a cost-effective method of producing fuels using only sunlight, water, and carbon dioxide, mimicking the natural process of photosynthesis in plants and storing energy in the form of chemical fuels for use on demand. Over the past five years, researchers at JCAP have made major advances toward this goal, and they now report the development of the first complete, efficient, safe, integrated solar-driven system for splitting water to create hydrogen fuels.
"This result was a stretch project milestone for the entire five years of JCAP as a whole, and not only have we achieved this goal, we also achieved it on time and on budget," says Caltech's Nate Lewis, George L. Argyros Professor and professor of chemistry, and the JCAP scientific director.
The new solar fuel generation system, or artificial leaf, is described in the August 27 online issue of the journal Energy and Environmental Science. The work was done by researchers in the laboratories of Lewis and Harry Atwater, director of JCAP and Howard Hughes Professor of Applied Physics and Materials Science.
"This accomplishment drew on the knowledge, insights and capabilities of JCAP, which illustrates what can be achieved in a Hub-scale effort by an integrated team," Atwater says. "The device reported here grew out of a multi-year, large-scale effort to define the design and materials components needed for an integrated solar fuels generator."
Solar Fuels Prototype in Operation A fully integrated photoelectrochemical device performing unassisted solar water splitting for the production of hydrogen fuel. Credit: Erik Verlage and Chengxiang Xiang/Caltech
The new system consists of three main components: two electrodes—one photoanode and one photocathode—and a membrane. The photoanode uses sunlight to oxidize water molecules, generating protons and electrons as well as oxygen gas. The photocathode recombines the protons and electrons to form hydrogen gas. A key part of the JCAP design is the plastic membrane, which keeps the oxygen and hydrogen gases separate. If the two gases are allowed to mix and are accidentally ignited, an explosion can occur; the membrane lets the hydrogen fuel be separately collected under pressure and safely pushed into a pipeline.
Semiconductors such as silicon or gallium arsenide absorb light efficiently and are therefore used in solar panels. However, these materials also oxidize (or rust) on the surface when exposed to water, so cannot be used to directly generate fuel. A major advance that allowed the integrated system to be developed was previous work in Lewis's laboratory, which showed that adding a nanometers-thick layer of titanium dioxide (TiO2)—a material found in white paint and many toothpastes and sunscreens—onto the electrodes could prevent them from corroding while still allowing light and electrons to pass through. The new complete solar fuel generation system developed by Lewis and colleagues uses such a 62.5-nanometer-thick TiO2 layer to effectively prevent corrosion and improve the stability of a gallium arsenide–based photoelectrode.
Another key advance is the use of active, inexpensive catalysts for fuel production. The photoanode requires a catalyst to drive the essential water-splitting reaction. Rare and expensive metals such as platinum can serve as effective catalysts, but in its work the team discovered that it could create a much cheaper, active catalyst by adding a 2-nanometer-thick layer of nickel to the surface of the TiO2. This catalyst is among the most active known catalysts for splitting water molecules into oxygen, protons, and electrons and is a key to the high efficiency displayed by the device.
The photoanode was grown onto a photocathode, which also contains a highly active, inexpensive, nickel-molybdenum catalyst, to create a fully integrated single material that serves as a complete solar-driven water-splitting system.
A critical component that contributes to the efficiency and safety of the new system is the special plastic membrane that separates the gases and prevents the possibility of an explosion, while still allowing the ions to flow seamlessly to complete the electrical circuit in the cell. All of the components are stable under the same conditions and work together to produce a high-performance, fully integrated system. The demonstration system is approximately one square centimeter in area, converts 10 percent of the energy in sunlight into stored energy in the chemical fuel, and can operate for more than 40 hours continuously.
"This new system shatters all of the combined safety, performance, and stability records for artificial leaf technology by factors of 5 to 10 or more ," Lewis says.
"Our work shows that it is indeed possible to produce fuels from sunlight safely and efficiently in an integrated system with inexpensive components," Lewis adds, "Of course, we still have work to do to extend the lifetime of the system and to develop methods for cost-effectively manufacturing full systems, both of which are in progress."
Because the work assembled various components that were developed by multiple teams within JCAP, coauthor Chengxiang Xiang, who is co-leader of the JCAP prototyping and scale-up project, says that the successful end result was a collaborative effort. "JCAP's research and development in device design, simulation, and materials discovery and integration all funneled into the demonstration of this new device," Xiang says.
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.