Vaccine signatures in humanized mice point to better understanding of infectious diseases

white laboratory mouse

Infectious diseases kill millions of people each year, but the search for treatments is hampered by the fact that laboratory mice are not susceptible to some human viruses, including killers like human immunodeficiency virus (HIV). For decades, researchers have turned to mice whose immune systems have been “humanized” to respond in a manner similar to humans.

Now a team at Princeton University has developed a comprehensive way to evaluate how immune responses of humanized mice measure up to actual humans. The research team looked at the mouse and human immune responses to one of the strongest vaccines known, a yellow fever vaccine called YFV-17D. The comparison of these “vaccine signatures” showed that a newly developed humanized mouse developed at Princeton shares significant immune-system responses with humans. The study was published in the journal Nature Communications.

“Understanding immune responses to human pathogens and potential vaccines remains challenging due to differences in the way our human immune system responds to stimuli, as compared to for example that of conventional mice, rats or other animals,” said Alexander Ploss, associate professor of molecular biology at Princeton. “Until now a rigorous method for testing the functionality of the human immune system in such a model has been missing. Our study highlights an experimental paradigm to address this gap.”

Humanized mice have been used in infectious disease research since the late 1980s. Yet without rigorous comparisons, researchers know little about how well the mice predict human responses such as the production of infection-fighting cells and antibodies.

To address this issue, researchers exposed the mice to the YFV-17D vaccine, which is made from a weakened, or attenuated, living yellow fever virus. Vaccines protect against future infection by provoking the production of antibodies and immune-system cells.

In previous work, the researchers explored the effect of YFV-17D on conventional humanized mice. But the researchers found that the mice responded only weakly. This led them to develop a mouse with responses that are more similar to those of humans.

To do so, the researchers introduced additional human genes for immune system components — such as molecules that detect foreign invaders and chemical messengers called cytokines — so that the complexity of the engrafted human immune system reflected that of humans. They found that the new mice have responses to YFV-17D that are very similar to the responses seen in humans. For example, the pattern of gene expression that occurs in response to YFV-17D in the mice shared significant similarities to that of humans. This signature gene expression pattern, reflected in the “transcriptome,” or total readout of all of the genes of the organism, translated into better control of the yellow fever virus infection and to immune responses that were more specific to yellow fever.

The researchers also looked at two other types of immune responses: the cellular responses, involving production of cytotoxic T cells and natural killer cells that attack and kill infected cells, and the production of antibodies specific to the virus. By evaluating these three types of responses – transcriptomic, cellular, and antibody – in both mice and humans, the researchers produced a reliable platform for evaluating how well the mice can serve as proxies for humans.

Florian Douam, a postdoctoral research associate and the first author on the study, hopes that the new testing platform will help researchers explore exactly how vaccines induce immunity against pathogens, which in many cases is not well understood.

“Many vaccines have been generated empirically without profound knowledge of how they induce immunity,” Douam said. “The next generation of mouse models, such as the one we introduced in our study, offer unprecedented opportunities for investigating the fundamental mechanisms that define the protective immunity induced by live-attenuated vaccines.”

Mice bearing human cells or human tissues have the potential to aid research on treatments for many diseases that infect humans but not other animals, such as – in addition to HIV – Epstein Barr Virus, human T-cell leukemia virus, and Karposi sarcoma-associated herpes virus.

“Our study highlights the importance of human biological signatures for guiding the development of mouse models of disease,” said Ploss. “It also highlights a path toward developing better models for human immune responses.”

The study involved contributions from Florian Douam, Gabriela Hrebikova, Jenna Gaska, Benjamin Winer and Brigitte Heller in Princeton University’s Department of Molecular Biology; Robert Leach, Lance Parsons and Wei Wang in Princeton University’s Lewis Sigler Institute for Integrative Genomics; Bruno Fant at the University of Pennsylvania; Carly G. K. Ziegler and Alex K. Shalek of Massachusetts Institute of Technology and Harvard Medical School; and Alexander Ploss in Princeton University’s Department of Molecular Biology.

The research was supported the National Institutes of Health (NIH, R01AI079031 and R01AI107301, to A.P) and an Investigator in Pathogenesis Award by the Burroughs Wellcome Fund (to A.P.). Additionally, A.K.S. was supported by the Searle Scholars Program, the Beckman Young Investigator Program, the NIH (1DP2OD020839, 5U24AI118672, 1U54CA217377, 1R33CA202820, 2U19AI089992, 1R01HL134539, 2RM1HG006193, 2R01HL095791, P01AI039671), and the Bill & Melinda Gates Foundation (OPP1139972). C.G.K.Z. was supported by a grant from the National Institute of General Medical Sciences (NIGMS, T32GM007753). J.M.G. and B.Y.W. were supported by a pre-doctoral training grant from the NIGMS (T32GM007388). B.Y.W. was also a recipient of a pre-doctoral fellowship from the New Jersey Commission on Cancer Research.

By Catherine Zandonella, Office of the Dean for Research

How rabies virus moves through nerve cells, and how it might be stopped

Cells in red against black background

To successfully infect its host, the rabies virus must move from the nerve ending to the nerve cell body where it can replicate. In a study published July 20 in the journal PLoS Pathogens, researchers from Princeton University reveal that the rabies virus moves differently compared to other neuron-invading viruses and that its journey can be blocked by a drug commonly used to treat amoebic dysentery.

Most viruses only infect the nervous system accidentally when the immune system is compromised. But some “neurotropic” viruses have evolved to target neurons as part of their normal infectious cycle. The rabies virus, for example, is transmitted when an infected animal bites into a host’s muscle. It then spreads into the end terminals of motor neurons innervating the muscle and travels along the neurons’ long axon fibers to the neuronal cell bodies. From there, the virus can spread throughout the central nervous system and into the salivary glands, where it can be readily transmitted to other hosts. Though rabies infections in humans are rare in the United States, the virus kills nearly 60,000 people annually.

Alpha herpesviruses, such as herpes simplex viruses, also enter peripheral nerve terminals and move along axons to the neuronal cell body, where they can lie dormant for the life of the host.

“Transport to the neuronal cell body is not a passive process, but an active one relying on the neuron’s own motor proteins and microtubule tracks,” said Lynn Enquist, Princeton’s Henry L. Hillman Professor in Molecular Biology, a professor of molecular biology and the Princeton Neuroscience Institute, and the study’s senior author. “Virus particles must engage this machinery for efficient transport in axons, otherwise infection cannot start.”

Viruses moving in a narrow nerve cell
Twenty-four hours after infecting nerve terminals, the rabies virus (red) reaches the cell bodies (green) of control neurons (left) but not neurons treated with emetine (right). Image credit: MacGibeny et al., 2018.

Enquist and colleagues previously found that alpha herpesviruses engage the neuronal transport machinery by stimulating protein synthesis at infected nerve terminals. Viral transport to the cell body can therefore be blocked by drugs that inhibit protein synthesis, as well as by cellular antiviral proteins called interferons.

In the current study, Enquist and colleagues investigated how the rabies virus engages the neuronal transport machinery. The researchers infected neurons with a virulent strain of the virus tagged with a red fluorescent protein, allowing the researchers to observe viral transport in real time by live-cell fluorescence microscopy.

The study was led by Margaret MacGibeny, who earned her Ph.D. in 2018, and associate research scholar Orkide Koyuncu, at Princeton, with contributions from research associate Christoph Wirblich and Matthias Schnell, professor and chair of microbiology and immunology at Thomas Jefferson University.

In contrast to alpha herpesvirus infections, the team found that interferons had no effect on rabies virus transport, perhaps because, until it reaches the neuronal cell body, the rabies virus hides out inside cellular structures called endosomes.

“We also couldn’t detect increased protein synthesis in axons upon rabies virus infection,” MacGibeny said. “But, to our surprise, we saw that a protein synthesis inhibitor called emetine efficiently blocked rabies virus transport to the cell body.”

Emetine had no effect on the transport of endosomes devoid of the rabies virus. But endosomes carrying the virus were either completely immobilized, or were only able to move short distances at slower-than-normal speeds.

Other protein synthesis inhibitors did not block rabies virus transport, however, suggesting that emetine works by inhibiting a different process in infected neurons.

“Emetine has been used to treat amoebic dysentery,” Koyuncu said. “In the laboratory it is widely used to inhibit protein synthesis but there are recent reports indicating that emetine has anti-viral effects that are independent of protein synthesis inhibition. Our study shows that this drug can inhibit rabies virus invasion of the nervous system through a novel mechanism that hasn’t been reported before.”

“The manuscript by MacGibeny et al. both advances and complicates our understanding of how neurotropic viruses make their way from the axon terminus to the cell body,” said Professor Glenn Rall, an expert in neurotropic virus infections at Fox Chase Cancer Center, who was not involved in the study. “Revealing variations in the axonal transport of neurotropic viruses, coupled with intriguing insights into new roles for well-known drugs, has both mechanistic and clinical implications for these life-threatening infections.

“Our next step is to figure out how emetine disrupts rabies virus transport in axons,” Enquist says. “Does it inhibit cell signaling pathways after rabies virus entry, or does it directly block the recruitment of motor proteins to virus-carrying endosomes?”

This study was funded by the US National Institutes of Health (grants P40 OD010996, RO1 NS33506, and F30 NS090640).

The study, Retrograde axonal transport of rabies virus is unaffected by interferon treatment but blocked by emetine locally in axons, by Margaret A. MacGibeny, Orkide O. Koyuncu, Christoph Wirblich, Matthias J. Schnell, and Lynn W. Enquist was published in PLoS Pathogens. 2018. DOI: 10.1371/journal.ppat.1007188

Text courtesy of the Department of Molecular Biology

An immune signaling pathway for control of Yellow Fever Virus infection

By the Department of Molecular Biology

Princeton University researchers have uncovered a critical role for a new immune signaling pathway in controlling infection by the flavivirus Yellow Fever Virus (YFV).  The paper describing this discovery was published today in the journal mBio.

Infection with YFV causes a devastating illness with a mortality rate of up to 50%.  Like other members of its viral family—which includes West Nile Virus, Dengue Virus and Zika Virus—YFV is transmitted to humans by mosquitos that are expanding into new areas across the globe, exposing more people to these dangerous viruses. Fortunately, there is an effective vaccine for YFV: a live-attenuated strain of the virus, called YFV-17D, which differs by only a few amino acids from the virulent viral strain YFV-Asibi, but nonetheless provokes a potent and durable protective immune response in humans.

“An improved understanding of the complex mechanisms regulating YFV-17D attenuation will provide insights into key viral-host interactions that regulate host immune responses and infection outcomes, [and] open novel avenues for the development of innovative vaccine strategies,” said Alexander Ploss, assistant professor at Princeton’s Department of Molecular Biology, who led the study with first author Florian Douam, a postdoctoral research associate. However, research efforts have been hampered due to the fact that mice, which are used in the study of viral infections, are resistant to YFV infection. Nonetheless, recent mouse experiments have pointed to an important role for cytokines called interferons (IFN) in controlling the virus.

Mice, like humans, possess three types of interferons, molecules produced by the immune system during infection: type I interferons, which signal through the widely distributed IFN-α/β receptor; type II interferons that act on IFN-γ receptors present in most tissues; and type III interferons, which activate signaling by IFN-λ receptors found on epithelial cells. Mice lacking type I receptors die after infection by YFV-Asibi, but survive YFV-17D infection despite extensive viral replication at early stage of infection.Type II IFN signaling has also been shown to be important for clearing up late stage YFV-Asibi and YFV-17D infection when type I IFN signaling is defective. By contrast, the contribution of type III IFN signaling to control of YFV infection was unknown.

To address this question, Douam and colleagues studied YFV-17D infection in mice lacking the type III receptor. Initial experiments showed that these mice were able to control viral replication and rapidly cleared YFV-17D, indicating that type III signaling alone wasn’t necessary for resistance to YFV-17D. However, mice lacking both type I and type III receptors succumbed after YFV-17D infection, suggesting type III signaling does contribute to the antiviral immune response.

To find out more, the authors examined YFV-17D levels in various tissues. Early in the infection, the virus was present in every tissue of each mouse model examined. However, although viral loads were low in wild-type mice and type III receptor-deficient mice, they were much higher in type I and type I/III receptor-deficient mice. Surprisingly, the viral loads in brains of type I/III receptor-deficient mice increased over time in comparison to type I receptor-deficient mice, showing that loss of type III IFN signaling enhances the susceptibility of type I-receptor deficient animals to brain infection. This is significant because the presence of viruses in the brain can cause brain damage such as spongiosis or encephalitis. The low level of YFV-17FD brain invasion in wild-type mice caused mild spongiosis, whereas type I/III receptor-deficient mice had severe spongiosis—potentially explaining YFV-17D lethality in those animals.  However, this raised the question of why YFV-17 was present at such high levels in the animals’ brains.

Brain tissues
Left panel: Loss of Type I IFN signaling leads to active replication of the attenuated YFV strain (YFV-17D), which is accompanied by viral invasion of the brain and damage to brain tissues (spongiosis). Right panel: The additional loss of Type III IFN signaling in Type I IFN-deficient mice impairs the integrity of the blood-brain-barrier and alters immune cell function, which aggravates spongiosis and is ultimately lethal. Image Credit: Florian Douam and Alexander Ploss

Another study recently showed that type III IFN signaling affects the epithelial cells that make up the blood brain barrier (BBB), and modulates BBB integrity during infection by another flavivirus, West Nile Virus. Consistent with this, Ploss’s group observed that the BBB of type I/III receptor-deficient mice was especially leaky to a blue dye. But this wasn’t the only way that loss of type III IFN signaling impaired the body’s response to YFV; the researchers also found evidence that type III receptor deficiency provokes strong imbalances in several different kinds of immune cells during YFV-17D infection. In particular, type I/III receptor-deficient mice were defective in the activation of T cells, critical immune cells that control YFV-17D infection.

“We uncovered a critical role of type III IFN-mediated signaling in preserving the integrity of the blood brain barrier and preventing viral brain invasion,” Ploss said. More work is needed to explore how type III IFN signaling affects YFV infection in primates, but this study already provides important new insights about a poorly understood immune signaling pathway.

The study was supported by grants from National Institutes of Health (R01 AI107301, R21AI117213 to Alexander Ploss and R01 AI104669 to Sergei Kotenko). Additional funding included a Research Scholar Award from the American Cancer Society (RSG-15-048-01-MPC), the Princeton Environmental Institute‘s Grand Health Challenge program from Princeton University, and an Investigator in Pathogenesis Award by the Burroughs Wellcome Fund (all to Alexander Ploss).

The study, “Type III Interferon-mediated signaling is critical for controlling live attenuated Yellow Fever Virus infection in vivo.,” by Florian Douam, Yentil E. Soto-Albrecht, Gabriela Hrebikova, Evita Sadimin, Christian Davidson, Sergei V. Kotenko and Alexander Ploss was published in the journal mBio.  (2017). doi:10.1128/mBio.00819-17

‘Acidic patch’ regulates access to genetic information

Histone image

By Pooja Makhijani for the Department of Chemistry

Chromatin remodelers — protein machines that pack and unpack chromatin, the tightly wound DNA-protein complex in cell nuclei — are essential and powerful regulators for critical cellular processes, such as replication, recombination and gene transcription and repression. In a new study published Aug. 2 in the journal Nature, a team led by researchers from Princeton University unravels more details on how a class of ATP-dependent chromatin remodelers, called ISWI, regulate access to genetic information.

The researchers reported that ISWI remodelers use a structural feature of the nucleosome, known as the “acidic patch,” to remodel chromatin. The nucleosome is the fundamental structural subunit of chromatin, and is often compared to thread wrapped around a spool.

Geoffrey Dann
Geoffrey Dann. Photo by C. Todd Reichart

“The acidic patch is a negatively charged surface, presented on each face of the nucleosome disc, that is formed by amino acids contributed by two different histone proteins, H2A and H2B,” said Geoffrey Dann, a graduate student in the Department of Molecular Biology at Princeton and the study’s lead author. “Histone proteins are overall very positively charged, which makes the negatively charged acidic patch region of the nucleosome very unique. Recognition of the acidic patch has never before been implicated in chromatin remodeling.”

The research was conducted in the laboratory of Tom Muir, the Van Zandt Williams Jr. Class of 1965 Professor of Chemistry and chair of the Department of Chemistry. Research in the Muir group centers on elucidating the physiochemical basis of protein functions in biomedically relevant systems.

Because ISWI remodelers are known to interact extensively with nucleosomes, the researchers hypothesized that signals, in the form of chemical modifications on histone proteins embedded within nucleosomes, communicate to the remodelers on which nucleosome to act. Using high throughput screening technology, an assay process often used in drug discovery, allowed the researchers to quickly conduct tens of thousands of biochemical measurements to test their assumptions. “The number of chromatin modifications known to exist in vivo is astronomical,” Dann said.

Not only did the experiments reveal that ISWI remodelers use the “acidic patch” to remodel chromatin, but also determined that remodeling enzymes outside the family of ISWI remodelers also use this structural feature, “suggesting that this feature may be a general requirement for chromatin remodeling to occur,” Dann said.

Certain chemical modifications that act on histone proteins that are adjacent to the acidic patch also have the ability to enhance or inhibit ISWI remodeling activity, he explained. “A handful of other proteins are known to engage the acidic patch in their interaction with chromatin as well, and we also found that the biochemistry of several of these proteins was affected by such modifications. Interestingly, each protein tested had its own signature response to this collection of modifications.”

The high throughput screening technology method also generated a vast library of data to drive the design of future studies geared toward further understanding ISWI regulation. “This study generated an immense amount of data pointing to many other novel regulatory inputs, in the form of chromatin modifications, into ISWI remodeling activity,” Dann said. “A long-term goal in our lab is to use this data resource as a launch pad for additional studies investigating how chromatin modifications affect ISWI remodeling, and how this plays into the various roles ISWI remodelers assume in the cell.”

histone diagram
Diagram depicting all histone modifications, mutants, and variants present in the 115-member nucleosome library used in this study. Residues modified or mutated were mapped on to the nucleosome in black. H2A (light yellow), H2B (light red), H3 (light blue), and H4 (light green) modification and mutation locations are indicated by boxes and lines. For clarity, connections are only shown to a single copy of each histone protein.

Their findings may also identify a new instrument in cells’ molecular repertoire of chromatin-remodeling tools and spur investigations into potential cancer therapeutic targets. “Mutations in the acidic patch are known to occur in certain types of human cancers, which underscores the emerging importance of the acidic patch in chromatin biology,” Dann said.

The study, “ISWI chromatin remodellers sense nucleosome modifications to determine substrate preference,” was published Aug. 2 by Nature. doi:10.1038/nature23671

The authors at Princeton University were Geoffrey P. Dann, Glen Liszczak, John D. Bagert, Manuel M. Müller, Uyen T. T. Nguyen, Felix Wojcik, Zachary Z. Brown, Jeffrey Bos, Rasmus Pihl, Samuel B. Pollock, Katharine L. Diehl and Tom W. Muir. Also contributing to the study were Tatyana Panchenko & C. David Allis at The Rockefeller University.

The research was funded in part by the German Research Foundation and the National Institutes of Health (GM112365, R01 GM107047).

Read the full article here:


Princeton researchers report new system to study chronic hepatitis B

A co-culture of human hepatocytes
A co-culture of human hepatocytes and non-parenchymal stromal cells self-assembles into liver-like structures that can be infected for extended periods with the hepatitis B virus. Image courtesy of Benjamin Winer and Alexander Ploss, Princeton University Department of Molecular Biology.

By the Department of Molecular Biology

Scientists from Princeton University‘s Department of Molecular Biology have successfully tested a cell-culture system that will allow researchers to perform laboratory-based studies of long-term hepatitis B virus (HBV) infections. The technique, which is described in a paper published July 25 in the journal Nature Communications, will aid the study of viral persistence and accelerate the development of antiviral drugs to cure chronic hepatitis B, a condition that affects over 250 million people worldwide and can cause severe liver disease, including liver cancer.

HBV specifically infects the liver by binding to a protein called sodium-taurocholate co-transporting polypeptide (NTCP) that is only present on the surface of liver cells. Once inside the cell, HBV hijacks its host’s cellular machinery to convert the virus’s DNA into a stable “mini-chromosome.” This allows the virus to establish persistent, long-term infections that can ultimately cause liver fibrosis, cirrhosis and hepatocellular carcinoma. The World Health Organization estimates that 600,000 people die every year as a result of HBV infection.

Researchers have so far failed to develop drugs that can cure chronic HBV infections, partly because they have not been able to study the long-term infection of liver cells grown in the laboratory. Liver cells—also known as hepatocytes—lose their function within days of being isolated from donor livers, preventing researchers from studying anything other than the acute stage of HBV infection. Hepatocytes can be maintained for longer when they are co-cultured with other, supportive cells.

“In previous studies using hepatocytes and cells known as fibroblasts grown on micro-patterned surfaces, HBV infections worked with only a few donors, and infection lasted for no longer than 14-19 days and required the suppression of antiviral cell signaling pathways, which poses problems for studying host-cell responses to HBV and for antiviral drug testing,” said Alexander Ploss, an assistant professor of molecular biology at Princeton University.

Dr. Ploss and colleagues at Princeton and the Hurel Corporation, led by graduate student Benjamin Winer, tested a different system, in which primary human hepatocytes are co-cultured with non-parenchymal stromal cells, which are cells that support the function of the parenchymal hepatocytes in the liver. When plated in collagen-coated labware, the co-cultures self-assemble into liver-like structures. These self-assembling liver-like cultures could be persistently infected with HBV for over 30 days, without the aid of antiviral signaling inhibitors. Moreover, the system worked with hepatocytes grown from a variety of donors and with viruses isolated from chronically-infected patients, which are harder to work with than lab-grown strains of HBV.

“The establishment of a co-culturing system of human primary hepatocytes and non-parenchymal stromal cells for extended HBV infection is a valuable addition to the armamentarium of cell culture model systems for the study of HBV biology and therapeutic development, which has been hampered by a relative lack of efficient infectious cell culture systems,” said T. Jake Liang, a senior investigator at the National Institute of Diabetes and Digestive and Kidney Diseases, who was not involved in the research.

Ploss and colleagues were able to scale down their co-culture infections to volumes as small as a few hundred microliters. This will be important for future high-throughput screens for anti-HBV drug candidates. As a proof-of-principle for these screens, the researchers found that they could block HBV infections in their co-culture system using drugs that either prevent the virus’ entry into hepatocytes or inhibit a viral enzyme that is essential for the virus’ replication. “The platform presented here may aid the identification and testing of novel therapeutic regimens,” Ploss said.

This study is supported in part by grants from the National Institutes of Health (R21AI117213 to Alexander Ploss and R37GM086868 to Tom W. Muir), a Burroughs Wellcome Fund Award for Investigators in Pathogenesis (to Alexander Ploss) and funds from Princeton University (to Alexander Ploss). Benjamin Y. Winer is a recipient of F31 NIH/NRSA Ruth L. Kirschstein Predoctoral awarded from the National Institute of Allergy and Infectious Diseases. Felix Wojcik is supported by a German Research Foundation (DFG) postdoctoral fellowship.

The study, “Long-term hepatitis B infection in a scalable hepatic co-culture system,” by Benjamin Y. Winer, Tiffany S. Huang, Eitan Pludwinski, Brigitte Heller, Felix Wojcik, Gabriel E. Lipkowitz, Amit Parekh, Cheul Cho, Anil Shrirao, Tom W. Muir, Eric Novik, Alexander Ploss, was published in Nature Communications on July 25, 2017. DOI: 10.1038/s41467-017-00200-8.

Read more in this commentary in Nature Microbiology.

How TPX2 helps microtubules branch out

By Staff, Department of Molecular Biology

Branching microtubules, which are structures involved in cell division, form in response to a protein known as TPX2, according to a study conducted at Princeton University in the laboratory of Sabine Petry, assistant professor of molecular biology. The image was featured on the cover of the Journal of Cell Biology. Image credit: Alfaro-Aco et al.

A new study has revealed insights into how new microtubules branch from the sides of existing ones. Researchers at Princeton University investigated proteins that control the formation of the thin, hollow tubes, which play an essential role in cellular structure and cell division. In a study published in the Journal of Cell Biology in March, the team found that one of these microtubule regulators—a protein called TPX2—controls the formation of new microtubule branches.

“TPX2 is often overexpressed in various cancers, and, in many cases, serves as a prognostic indicator,” said Raymundo Alfaro-Aco, a graduate student in the Department of Molecular Biology. Aco conducted the study with graduate student Akanksha Thawani in the Department of Chemical and Biological Engineering in the laboratory of Sabine Petry, assistant professor of molecular biology. “Therefore, elucidating the role of TPX2 in cell division in general can have important implications in our understanding of human diseases,” Alfaro-Aco said.

Microtubules are formed by the polymerization of two proteins, α- and ß-tubulin, but a third form of tubulin—γ-tubulin—helps to initiate (or “nucleate”) microtubule polymerization inside cells. γ-Tubulin combines with several other proteins to form γ-tubulin ring complexes (γ-TuRCs) that localize, for example, to the cell’s centrosomes, which nucleate and organize most of the microtubules that assemble into the mitotic spindle, the cellular structure that segregates chromosomes into newly forming daughter cells during cell division.

While a postdoc at the University of California-San Francisco, Petry demonstrated that spindles also contain microtubules that are nucleated from the sides of other microtubules (Petry et al., Cell. 152: 768-777, 2013). This “branching nucleation” process depends, in part, on a microtubule-binding protein called TPX2. Petry and Alfaro-Aco decided to investigate exactly how this protein stimulates branching microtubule nucleation.

To explore this question, the researchers used cell-free extracts prepared from frog eggs, which are capable of forming functional spindles in vitro, Alfaro-Aco said. “This powerful system allows us to easily add or remove factors, such as proteins or small molecules, to probe different aspects of spindle assembly,” he said. “Combining this extract system with a powerful imaging method — known as total internal reflection fluorescence microscopy — allows us to observe and measure microtubule events, such as nucleation, at the level of single microtubules.”

By adding different fragments of TPX2 to egg extracts and observing their effects on microtubules, Alfaro-Aco found that a fragment containing three of the protein’s seven alpha-helical domains was the smallest piece capable of stimulating branching microtubule nucleation.

This minimal fragment contained three short stretches of amino acids that are similar to sequences found in proteins that bind and activate γ-TuRC. The researchers found that deleting or mutating these sequences eliminated the TPX2 fragment’s capacity to stimulate microtubule branching, without affecting the protein’s ability to bind to microtubules.

The team also found that this region of TPX2 binds to γ-TuRC. Mutating the three sequences found in other γ-TuRC-binding proteins didn’t inhibit this interaction but, because these mutants no longer stimulate branching microtubule nucleation, Alfaro-Aco and colleagues think that the sequences are required to activate γ-TuRC. TPX2 may therefore bind to existing spindle microtubules and then bind and activate γ-TuRC to initiate the formation of a new microtubule branch. This process is crucial for spindle assembly and the accurate segregation of chromosomes.

This work was supported by the National Institutes of Health/National Institute of General Medical Sciences (grant # 4R00GM100013), the Pew Scholars Program in the Biomedical Sciences, the Sidney Kimmel Foundation, and the David and Lucile Packard Foundation. In addition, Alfaro-Aco received support from the Howard Hughes Medical Institute and the National Science Foundation.

Alfaro-Aco, R., A. Thawani, and S. Petry. Structural analysis of the role of TPX2 in branching microtubule nucleation. Journal of Cell Biology, 216: 983-997, 2017. DOI: 10.1083/jcb.201607060 | Published March 6, 2017.

Come together: Nucleolus forms via combination of active and passive processes

Movie caption: Researchers at Princeton studied the temperature dependence of the formation of the nucleolus, a cellular organelle. The movie shows the nuclei of intact fly cells as they are subjected to temperature changes in the surrounding fluid. As the temperature is shifted from low to high, the spontaneously assembled proteins dissolve, as can be seen in the disappearance of the bright spots.

By Catherine Zandonella, Office of the Dean for Research

Researchers at Princeton found that the nucleolus, a cellular organelle involved in RNA synthesis, assembles in part through the passive process of phase separation – the same type of process that causes oil to separate from water. The study, published in the journal Proceedings of the National Academy of Sciences, is the first to show that this happens in living, intact cells.

Understanding how cellular structures form could help explain how organelles change in response to diseases. For example, a hallmark of cancer cells is the swelling of the nucleolus.

To explore the role of passive processes – as opposed to active processes that involve energy consumption – in nucleolus formation, Hanieh Falahati, a graduate student in Princeton’s Lewis-Sigler Institute for Integrative Genomics, looked at the behavior of six nucleolus proteins under different temperature conditions. Phase separation is enhanced at lower temperatures, which is why salad dressing containing oil and vinegar separates when stored in the refrigerator. If phase separation were driving the assembly of proteins, the researchers should see the effect at low temperatures.

Falahati showed that four of the six proteins condensed and assembled into the nucleolus at low temperatures and reverted when the temperature rose, indicating that the passive process of phase separation was at work. However, the assembly of the other two proteins was irreversible, indicating that active processes were in play.

“It was kind of a surprising result, and it shows that cells can take advantage of spontaneous processes for some functions, but for other things, active processes may give the cell more control,” said Falahati, whose adviser is Eric Wieschaus, Princeton’s Squibb Professor in Molecular Biology and a professor of molecular biology and the Lewis-Sigler Institute for Integrative Genomics, and a Howard Hughes Medical Institute researcher.

The research was funded in part by grant 5R37HD15587 from the National Institute of Child Health and Human Development (NICHD), and by the Howard Hughes Medical Institute.

The study, “Independent active and thermodynamic processes govern the nucleolus assembly in vivo,” by Hanieh Falahatia and Eric Wieschaus, was published online ahead of print in the journal Proceedings of the National Academy of Sciences on January 23, 2017, doi: 10.1073/pnas.1615395114.