Select Publications Archive

Accurate annotation of plant genomes remains complex due to the presence of many pseudogenes arising from whole-genome duplication-generated redundancy or the capture and movement of gene fragments by transposable elements. Machine learning on genome-wide epigenetic marks, informed by transcriptomic and proteomic training data, could be used to improve annotations through classification of all putative protein-coding genes as either constitutively silent or able to be expressed. Expressed genes were subclassified as able to express both mRNAs and proteins or only RNAs, and CG gene body methylation was associated only with the former subclass. More than 60,000 protein-coding genes have been annotated in the reference genome of maize inbred B73. About two-thirds of these genes are transcribed and are designated the filtered gene set (FGS). Classification of genes by our trained random forest algorithm was accurate and relied only on histone modifications or DNA methylation patterns within the gene body; promoter methylation was unimportant. Other inbred lines are known to transcribe significantly different sets of genes, indicating that the FGS is specific to B73. We accurately classified the sets of transcribed genes in additional inbred lines, arising from inbred-specific DNA methylation patterns. This approach highlights the potential of using chromatin information to improve annotations of functional genes.

Immune genes are under intense, pathogen-induced pressure, which causes these genes to diversify over evolutionary time and become species-specific. Through a forward genetic screen we recently described a C. elegans-specific gene called pals-22 to be a repressor of "Intracellular Pathogen Response" or IPR genes. Here we describe pals-25, which, like pals-22, is a species-specific gene of unknown biochemical function. We identified pals-25 in a screen for suppression of pals-22 mutant phenotypes and found that mutations in pals-25 suppress all known phenotypes caused by mutations in pals-22. These phenotypes include increased IPR gene expression, thermotolerance, and immunity against natural pathogens, including Nematocida parisii microsporidia and the Orsay virus. Mutations in pals-25 also reverse the reduced lifespan and slowed growth of pals-22 mutants. Transcriptome analysis indicates that pals-22 and pals-25 control expression of genes induced not only by natural pathogens of the intestine, but also by natural pathogens of the epidermis. Indeed, in an independent forward genetic screen we identified pals-22 as a repressor and pals-25 as an activator of epidermal defense gene expression. In summary, the species-specific pals-22 and pals-25 genes act as a switch to regulate a program of gene expression, growth, and defense against diverse natural pathogens in C. elegans.

Synthetic gene drive systems possess enormous potential to replace, alter, or suppress wild populations of significant disease vectors and crop pests; however, their utility in diverse populations remains to be demonstrated. Here, we report the creation of a synthetic Medea gene drive system in a major worldwide crop pest, Drosophila suzukii We demonstrate that this drive system, based on an engineered maternal "toxin" coupled with a linked embryonic "antidote," is capable of biasing Mendelian inheritance rates with up to 100% efficiency. However, we find that drive resistance, resulting from naturally occurring genetic variation and associated fitness costs, can be selected for and hinder the spread of such a drive. Despite this, our results suggest that this gene drive could maintain itself at high frequencies in a wild population and spread to fixation if either its fitness costs or toxin resistance were reduced, providing a clear path forward for developing future such systems in this pest.

A large body of evidence demonstrates that strategies that promote student interactions and cognitively engage students with content (1) lead to gains in learning and attitudinal outcomes for students in science, technology, engineering, and mathematics (STEM) courses (1, 2). Many educational and governmental bodies have called for and supported adoption of these student-centered strategies throughout the undergraduate STEM curriculum. But to the extent that we have pictures of the STEM undergraduate instructional landscape, it has mostly been provided through self-report surveys of faculty members, within a particular STEM discipline [e.g., (3–6)]. Such surveys are prone to reliability threats and can underestimate the complexity of classroom environments, and few are implemented nationally to provide valid and reliable data (7). Reflecting the limited state of these data, a report from the U.S. National Academies of Sciences, Engineering, and Medicine called for improved data collection to understand the use of evidence-based instructional practices (8). We report here a major step toward a characterization of STEM teaching practices in North American universities based on classroom observations from over 2000 classes taught by more than 500 STEM faculty members across 25 institutions.

Lysine acetylation is a key posttranslational modification that regulates diverse proteins involved in a range of biological processes. The role of histone acetylation in plant defense is well established, and it is known that pathogen effector proteins encoding acetyltransferases can directly acetylate host proteins to alter immunity. However, it is unclear whether endogenous plant enzymes can modulate protein acetylation during an immune response. Here, we investigate how the effector molecule HC-toxin (HCT), a histone deacetylase inhibitor produced by the fungal pathogen Cochliobolus carbonum race 1, promotes virulence in maize through altering protein acetylation. Using mass spectrometry, we globally quantified the abundance of 3,636 proteins and the levels of acetylation at 2,791 sites in maize plants treated with HCT as well as HCT-deficient or HCT-producing strains of C. carbonum. Analyses of these data demonstrate that acetylation is a widespread posttranslational modification impacting proteins encoded by many intensively studied maize genes. Furthermore, the application of exogenous HCT enabled us to show that the activity of plant-encoded enzymes (histone deacetylases) can be modulated to alter acetylation of nonhistone proteins during an immune response. Collectively, these results provide a resource for further mechanistic studies examining the regulation of protein function by reversible acetylation and offer insight into the complex immune response triggered by virulent C. carbonum.

Ribosomes that experience terminal stalls during translation are resolved by ribosome-associated quality control (QC) pathways that oversee mRNA and nascent chain destruction and recycle ribosomal subunits. The proximal factors that sense stalled ribosomes and initiate mammalian ribosome-associated QC events remain undefined. We demonstrate that the ZNF598 ubiquitin ligase and the 40S ribosomal protein RACK1 help to resolve poly(A)-induced stalled ribosomes. They accomplish this by regulating distinct and overlapping regulatory 40S ribosomal ubiquitylation events. ZNF598 primarily mediates regulatory ubiquitylation of RPS10 and RPS20, whereas RACK1 regulates RPS2, RPS3, and RPS20 ubiquitylation. Gain or loss of ZNF598 function or mutations that block RPS10 or RPS20 ubiquitylation result in defective resolution of stalled ribosomes and subsequent readthrough of poly(A)-containing stall sequences. Together, our results indicate that ZNF598, RACK1, and 40S regulatory ubiquitylation plays a pivotal role in mammalian ribosome-associated QC pathways.

Transverse (T)-tubules make-up a specialized network of tubulated muscle cell membranes involved in excitation-contraction coupling for power of contraction. Little is known about how T-tubules maintain highly organized structures and contacts throughout the contractile system despite the ongoing muscle remodeling that occurs with muscle atrophy, damage and aging. We uncovered an essential role for autophagy in T-tubule remodeling with genetic screens of a developmentally regulated remodeling program in Drosophila abdominal muscles. Here, we show that autophagy is both upregulated with and required for progression through T-tubule disassembly stages. Along with known mediators of autophagosome-lysosome fusion, our screens uncovered an unexpected shared role for Rab2 with a broadly conserved function in autophagic clearance. Rab2 localizes to autophagosomes and binds to HOPS complex members, suggesting a direct role in autophagosome tethering/fusion. Together, the high membrane flux with muscle remodeling permits unprecedented analysis both of T-tubule dynamics and fundamental trafficking mechanisms.

Microbial pathogens often establish infection within particular niches of their host for replication. Determining how infection occurs preferentially in specific host tissues is a key aspect of understanding host-microbe interactions. Here, we describe the discovery of a natural microsporidian parasite of the nematode Caenorhabditis elegans that displays a unique tissue tropism compared to previously described parasites of this host. We characterize the life cycle of this new species, Nematocida displodere, including pathogen entry, intracellular replication, and exit. N. displodere can invade multiple host tissues, including the epidermis, muscle, neurons, and intestine of C. elegans. Despite robust invasion of the intestine very little replication occurs there, with the majority of replication occurring in the muscle and epidermis. This feature distinguishes N. displodere from two closely related microsporidian pathogens, N. parisii and N. sp. 1, which exclusively invade and replicate in the intestine. Comparison of the N. displodere genome with N. parisii and N. sp. 1 reveals that N. displodere is the earliest diverging species of the Nematocida genus. Over 10% of the proteins encoded by the N. displodere genome belong to a single species-specific family of RING-domain containing proteins of unknown function that may be mediating interactions with the host. Altogether, this system provides a powerful whole-animal model to investigate factors responsible for pathogen growth in different tissue niches.

On December 18, 2014, a yellow female fly quietly emerged from her pupal case. What made her unique was that she had only one parent carrying a mutant allele of this classic recessive locus. Then, one generation later, after mating with a wild-type male, all her offspring displayed the same recessive yellow phenotype. Further analysis of other such yellow females revealed that the construct causing the mutation was converting the opposing chromosome with 95% efficiency. These simple results, seen also in mosquitoes and yeast, open the door to a new era of genetics wherein the laws of traditional Mendelian inheritance can be bypassed for a broad variety of purposes. Here, we consider the implications of this fundamentally new form of "active genetics," its applications for gene drives, reversal and amplification strategies, its potential for contributing to cell and gene therapy strategies, and ethical/biosafety considerations associated with such active genetic elements. Also watch the Video Abstract.

Accurate interpretation of forward genetic screens of chromosomes exposed in mature spermatozoa to a mutagenic chemical requires understanding-incomplete to date-of how exposed chromosomes and their replicas proceed through early development stages from the fertilized ovum to establishment of the germ line of the treated male's offspring. We describe a model for early embryonic development and establishment of the germ line of Drosophila melanogaster and a model-validating experiment. Our model proposes that, barring repair, DNA strands modified by treatment with alkylating agents are stable and mutagenic. Each replication of an alkylated strand can result in misreplication and a mutant-bearing daughter nucleus. Daughter nuclei thenceforth replicate faithfully and their descendants comprise the embryonic syncytium. Of the 256 nuclei present after the eighth division, several migrate into the polar plasm at the posterior end of the embryo to found the germ line. Based upon distribution of descendants of the alkylated strands, the misreplication rate, and the number of nuclei selected as germline progenitors, the frequency of gonadal mosaicism is predictable. Experimentally, we tracked chromosomes 2 and 3 from EMS-treated sperm through a number of generations, to characterize autosomal recessive lethal mutations and infer gonadal genetic content of the sons of treated males. Over 50% of 106 sons bore germ lines that were singly, doubly, or triply mosaic for chromosome 2 or chromosome 3. These findings were consistent with our model, assuming a rate of misreplication between .65 and .80 at each replication of an alkylated strand. Crossing treated males to mismatch-repair-deficient females had no apparent effect on mutation rate.

An appreciable fraction of the Drosophila melanogaster genome is dedicated to male fertility. One approach to characterizing this subset of the genome is through the study of male-sterile mutations. We studied the relation between vital and male-fertility genes in three large autosomal regions that were saturated for lethal and male-sterile mutations. The majority of male-sterile mutations affect genes that are exclusively expressed in males. These genes are required only for male fertility, and several mutant alleles of each such gene were encountered. A few male-sterile mutations were alleles of vital genes that are expressed in both males and females. About one-fifth of the genes in Drosophila melanogaster show male-specific expression in adults. Although some earlier studies found a paucity of genes on the X chromosome showing male-biased expression, we did not find any significant differences between the X chromosome and the autosomes either in the relative frequencies of mutations to male sterility or in the frequencies of genes with male-specific expression in adults. Our results suggest that as much as 25% of the Drosophila genome may be dedicated to male fertility.

Microsporidia comprise a large phylum of obligate intracellular eukaryotes that are fungal-related parasites responsible for widespread disease, and here we address questions about microsporidia biology and evolution. We sequenced three microsporidian genomes from two species, Nematocida parisii and Nematocida sp1, which are natural pathogens of Caenorhabditis nematodes and provide model systems for studying microsporidian pathogenesis. We performed deep sequencing of transcripts from a time course of N. parisii infection. Examination of pathogen gene expression revealed compact transcripts and a dramatic takeover of host cells by Nematocida. We also performed phylogenomic analyses of Nematocida and other microsporidian genomes to refine microsporidian phylogeny and identify evolutionary events of gene loss, acquisition, and modification. In particular, we found that all microsporidia lost the tumor-suppressor gene retinoblastoma, which we speculate could accelerate the parasite cell cycle and increase the mutation rate. We also found that microsporidia acquired transporters that could import nucleosides to fuel rapid growth. In addition, microsporidian hexokinases gained secretion signal sequences, and in a functional assay these were sufficient to export proteins out of the cell; thus hexokinase may be targeted into the host cell to reprogram it toward biosynthesis. Similar molecular changes appear during formation of cancer cells and may be evolutionary strategies adopted independently by microsporidia to proliferate rapidly within host cells. Finally, analysis of genome polymorphisms revealed evidence for a sexual cycle that may provide genetic diversity to alleviate problems caused by clonal growth. Together these events may explain the emergence and success of these diverse intracellular parasites.

Auxin regulates every aspect of plant growth and development. Previous genetic studies demonstrated that YUCCA (YUC) flavin-containing monooxygenases (FMOs) catalyze a rate-limiting step in auxin biosynthesis and that YUCs are essential for many developmental processes. We proposed that YUCs convert indole-3-pyruvate (IPA) to indole-3-acetate (IAA). However, the exact biochemical mechanism of YUCs has remained elusive. Here we present the biochemical characterization of recombinant Arabidopsis YUC6. Expressed in and purified from E. coli, YUC6 contains FAD as a cofactor, which has peaks at 448 nm and 376 nm in the UV-Visible spectrum. We show that YUC6 uses NADPH and oxygen to convert IPA to IAA. The first step of the YUC6-catalyzed reaction is the reduction of the FAD cofactor to FADH by NADPH. Subsequently, FADH reacts with oxygen to form a flavin-C4a-(hydro)peroxy intermediate3, which we show has a maximum absorbance at 381 nm in its UV-Visible spectrum. The final chemical step is the reaction of the C4a-intermediate with IPA to produce IAA. Although the sequences of the YUC enzymes are related to those of the mammalian FMOs, which oxygenate nucleophilic substrates, YUC6 oxygenates an electrophilic substrate (IPA). Nevertheless, both classes of enzymes form quasi-stable C4a-(hydro)peroxyl FAD intermediates. The YUC6 intermediate has a half-life of ~20 s whereas that of some FMOs is more than 30 min. This work reveals the catalytic mechanism of the first known plant flavin monooxygenase and provides a foundation for further investigating how YUC activities are regulated in plants.

The discovery of large supramolecular complexes such as the purinosome suggests that subcellular organization is central to enzyme regulation. A screen of the yeast GFP strain collection to identify proteins that assemble into visible structures identified four novel filament systems comprised of glutamate synthase, guanosine diphosphate-mannose pyrophosphorylase, cytidine triphosphate (CTP) synthase, or subunits of the eIF2/2B translation factor complex. Recruitment of CTP synthase to filaments and foci can be modulated by mutations and regulatory ligands that alter enzyme activity, arguing that the assembly of these structures is related to control of CTP synthase activity. CTP synthase filaments are evolutionarily conserved and are restricted to axons in neurons. This spatial regulation suggests that these filaments have additional functions separate from the regulation of enzyme activity. The identification of four novel filaments greatly expands the number of known intracellular filament networks and has broad implications for our understanding of how cells organize biochemical activities in the cytoplasm.

Reversible phosphoinositide phosphorylation provides a dynamic membrane code that balances opposing cell functions. However, in vivo regulatory relationships between specific kinases, phosphatases, and phosphoinositide subpools are not clear. We identified myotubularin (mtm), a Drosophila melanogaster MTM1/ MTMR2 phosphoinositide phosphatase, as necessary and sufficient for immune cell protrusion formation and recruitment to wounds. Mtm-mediated turnover of endosomal phosphatidylinositol 3-phosphate (PI(3)P) pools generated by both class II and III phosphatidylinositol 3-kinases (Pi3K68D and Vps34, respectively) is needed to down-regulate membrane influx, promote efflux, and maintain endolysosomal homeostasis. Endocytosis, but not endolysosomal size, contributes to cortical remodeling by mtm function. We propose that Mtm-dependent regulation of an endosomal PI(3)P pool has separable consequences for endolysosomal homeostasis and cortical remodeling. Pi3K68D depletion (but not Vps34) rescues protrusion and distribution defects in mtm-deficient immune cells and restores functions in other tissues essential for viability. The broad interactions between mtm and class II Pi3K68D suggest a novel strategy for rebalancing PI(3)P-mediated cell functions in MTM-related human disease.

During fasting, mammals maintain normal glucose homeostasis by stimulating hepatic gluconeogenesis. Elevations in circulating glucagon and epinephrine, two hormones that activate hepatic gluconeogenesis, trigger the cAMP-mediated phosphorylation of cAMP response element-binding protein (Creb) and dephosphorylation of the Creb-regulated transcription coactivator-2 (Crtc2)-two key transcriptional regulators of this process. Although the underlying mechanism is unclear, hepatic gluconeogenesis is also regulated by the circadian clock, which coordinates glucose metabolism with changes in the external environment. Circadian control of gene expression is achieved by two transcriptional activators, Clock and Bmal1, which stimulate cryptochrome (Cry1 and Cry2) and Period (Per1, Per2 and Per3) repressors that feed back on Clock-Bmal1 activity. Here we show that Creb activity during fasting is modulated by Cry1 and Cry2, which are rhythmically expressed in the liver. Cry1 expression was elevated during the night-day transition, when it reduced fasting gluconeogenic gene expression by blocking glucagon-mediated increases in intracellular cAMP concentrations and in the protein kinase A-mediated phosphorylation of Creb. In biochemical reconstitution studies, we found that Cry1 inhibited accumulation of cAMP in response to G protein-coupled receptor (GPCR) activation but not to forskolin, a direct activator of adenyl cyclase. Cry proteins seemed to modulate GPCR activity directly through interaction with G(s)α. As hepatic overexpression of Cry1 lowered blood glucose concentrations and improved insulin sensitivity in insulin-resistant db/db mice, our results suggest that compounds that enhance cryptochrome activity may provide therapeutic benefit to individuals with type 2 diabetes.

PMID: 20852621

Alarm communication is a key adaptation that helps social groups resist predation and rally defenses. In Asia, the world’s largest hornet, Vespa mandarinia, and the smaller hornet, Vespa velutina, prey upon foragers and nests of the Asian honey bee, Apis cerana. We attacked foragers and colony nest entrances with these predators and provide the first evidence, in social insects, of an alarm signal that encodes graded danger and attack context. We show that, like Apis mellifera, A. cerana possesses a vibrational “stop signal,” which can be triggered by predator attacks upon foragers and inhibits waggle dancing. Large hornet attacks were more dangerous and resulted in higher bee mortality. Per attack at the colony level, large hornets elicited more stop signals than small hornets. Unexpectedly, stop signals elicited by large hornets (SS large hornet) had a significantly higher vibrational fundamental frequency than those elicited by small hornets (SS small hornet) and were more effective at inhibiting waggle dancing. Stop signals resulting from attacks upon the nest entrance (SS nest) were produced by foragers and guards and were significantly longer in pulse duration than stop signals elicited by attacks upon foragers (SS forager). Unlike SS forager, SS nest were targeted at dancing and non-dancing foragers and had the common effect, tuned to hornet threat level, of inhibiting bee departures from the safe interior of the nest. Meanwhile, nest defenders were triggered by the bee alarm pheromone and live hornet presence to heat-ball the hornet. In A. cerana, sophisticated recruitment communication that encodes food location, the waggle dance, is therefore matched with an inhibitory/alarm signal that encodes information about the context of danger and its threat level.

Human activities have resulted in rising temperatures and the introduction or extirpation of top predators worldwide. Both processes generate cascading impacts throughout food webs and can jeopardize important ecosystem services. We examined the impact of fish stocking on communities and ecosystems in California mountain lakes across an elevation (temperature and dissolved organic carbon) gradient to determine how trophic cascades and ecosystem function vary with climate. Here, we show that the impact of fish on the pelagic consumer-to-producer biomass ratio strengthened at low elevation, while invertebrate community composition and benthic ecosystem rates (periphyton production and litter decomposition) were most influenced by predators at high elevation. A warming climate may therefore alter the stability of lake ecosystems by shifting the strength of top-down control by introduced predators over food web structure and function.

Alarm communication is a key adaptation that helps social groups resist predation and rally defenses. In Asia, the world’s largest hornet, Vespa mandarinia, and the smaller hornet, Vespa velutina, prey upon foragers and nests of the Asian honey bee, Apis cerana. We attacked foragers and colony nest entrances with these predators and provide the first evidence, in social insects, of an alarm signal that encodes graded danger and attack context. We show that, like Apis mellifera, A. cerana possesses a vibrational “stop signal,” which can be triggered by predator attacks upon foragers and inhibits waggle dancing. Large hornet attacks were more dangerous and resulted in higher bee mortality. Per attack at the colony level, large hornets elicited more stop signals than small hornets. Unexpectedly, stop signals elicited by large hornets (SS large hornet) had a significantly higher vibrational fundamental frequency than those elicited by small hornets (SS small hornet) and were more effective at inhibiting waggle dancing. Stop signals resulting from attacks upon the nest entrance (SS nest) were produced by foragers and guards and were significantly longer in pulse duration than stop signals elicited by attacks upon foragers (SS forager). Unlike SS forager, SS nest were targeted at dancing and non-dancing foragers and had the common effect, tuned to hornet threat level, of inhibiting bee departures from the safe interior of the nest. Meanwhile, nest defenders were triggered by the bee alarm pheromone and live hornet presence to heat-ball the hornet. In A. cerana, sophisticated recruitment communication that encodes food location, the waggle dance, is therefore matched with an inhibitory/alarm signal that encodes information about the context of danger and its threat level.

Understanding the conditions that allow speciation to occur is difficult because most research has focused on either long-lived organisms or asexual microorganisms. We propagated bacteriophage λ, a virus with rapid generations and frequent recombination, on two Escherichia coli host genotypes that expressed either the LamB or OmpF receptor. When supplied with either single host (allopatry), phage λ improved its binding to the available receptor while losing its ability to use the alternative. When evolving on both hosts together (sympatry), the viruses split into two lineages with divergent receptor preferences. Although the level of divergence varied among replicates, some lineages evolved reproductive isolation via genetic incompatibilities. This outcome indicates that, under suitable conditions, allopatric and sympatric speciation can occur with similar ease.

Trade-offs are thought to arise from inevitable, biophysical limitations that prevent organisms from optimizing multiple traits simultaneously. A leading explanation for biodiversity maintenance is a theory that if the shape, or geometry, of a trade-off is right, then multiple species can coexist. Testing this theory, however, is difficult as trait data is usually too noisy to discern shape, or trade-offs necessary for the theory are not observed in vivo. To address this, we infer geometry directly from the biophysical mechanisms that cause trade-offs, deriving the geometry of two by studying nutrient uptake and metabolic properties common to all living cells. To test for their presence in vivo we isolated Escherichia coli mutants that vary in a nutrient transporter, LamB, and found evidence for both trade-offs. Consistent with data, population genetics models incorporating the trade-offs successfully predict the co-maintenance of three distinct genetic lineages, demonstrating that trade-off geometry can be deduced from fundamental principles of living cells and used to predict stable genetic polymorphisms.

Warming experiments are increasingly relied on to estimate plant responses to global climate change. For experiments to provide meaningful predictions of future responses, they should reflect the empirical record of responses to temperature variability and recent warming, including advances in the timing of flowering and leafing. We compared phenology (the timing of recurring life history events) in observational studies and warming experiments spanning four continents and 1,634 plant species using a common measure of temperature sensitivity (change in days per degree Celsius). We show that warming experiments underpredict advances in the timing of flowering and leafing by 8.5-fold and 4.0-fold, respectively, compared with long-term observations. For species that were common to both study types, the experimental results did not match the observational data in sign or magnitude. The observational data also showed that species that flower earliest in the spring have the highest temperature sensitivities, but this trend was not reflected in the experimental data. These significant mismatches seem to be unrelated to the study length or to the degree of manipulated warming in experiments. The discrepancy between experiments and observations, however, could arise from complex interactions among multiple drivers in the observational data, or it could arise from remediable artifacts in the experiments that result in lower irradiance and drier soils, thus dampening the phenological responses to manipulated warming. Our results introduce uncertainty into ecosystem models that are informed solely by experiments and suggest that responses to climate change that are predicted using such models should be re-evaluated.

Identifying traits that affect rates of speciation and extinction and, hence, explain differences in species diversity among clades is a major goal of evolutionary biology. Detecting such traits is especially difficult when they undergo frequent transitions between states. Self-incompatibility, the ability of hermaphrodites to enforce outcrossing, is frequently lost in flowering plants, enabling self-fertilization. We show, however, that in the nightshade plant family (Solanaceae), species with functional self-incompatibility diversify at a significantly higher rate than those without it. The apparent short-term advantages of potentially self-fertilizing individuals are therefore offset by strong species selection, which favors obligate outcrossing.

PMID: 20966249 [PubMed - indexed for MEDLINE]

Variation in invasion success may result from the divergent evolutionary histories of introduced species compared to those of native taxa. The vulnerability of native biotas to ecological disruption may be especially great on oceanic islands invaded by continental species with unique ecological traits. In part because Hawaii lacks native eusocial insects, social invaders may threaten endemic taxa that are ecologically similar but solitary. Using a combination of field manipulations, molecular analyses, physiological data, and behavioral assays, we identify the mechanisms underlying the displacement of two genera of native solitary Hymenoptera in Hawaii by a social continental invader, the western yellowjacket (Vespula pensylvanica). Experimental removal of V. pensylvanica colonies resulted in increased densities of native Hymenoptera. Endemic Hylaeus bees directly suffer through predation by yellowjackets, and perhaps as a consequence, avoid floral resources occupied by V. pensylvanica. Native Nesodynerus wasps also avoid V. pensylvanica but are negatively affected by yellowjackets not through predation, but through exploitative competition for caterpillar prey. Displacement of native solitary Hymenoptera may be heightened by the ability of V. pensylvanica to prey upon and scavenge honey bees and to rob their honey stores, resources unavailable to endemic bees and wasps because of their specialized niches. Our study provides a unique example of an ecologically generalized social invader that restructures native assemblages of solitary Hymenoptera by interacting with endemic taxa on multiple trophic levels.

PMID: 21141190 [PubMed - indexed for MEDLINE]

Deleterious mutations appearing in a population increase in frequency until stopped by natural selection. The ensuing equilibrium creates a stable frequency of deleterious mutations or the mutational load. Here I develop the comparable concept of a damage load, which is caused by harmful non-heritable changes to the phenotype. A damage load also ensues when the increase of damage is opposed by selection. The presence of a damage load favors the evolution of asymmetrical transmission of damage by a mother to her daughters. The asymmetry is beneficial because it increases fitness variance, but it also leads to aging or senescence. A mathematical model based on microbes reveals that a cell lineage dividing symmetrically is immortal if lifetime damage rates do not exceed a threshold. The evolution of asymmetry allows the lineage to persist above the threshold, but the lineage becomes mortal. In microbes with low genomic mutation rates, it is likely that the damage load is much greater than the mutational load. In metazoans with higher genomic mutation rates, the damage and the mutational load could be of the same magnitude. A fit of the model to experimental data shows that Escherichia coli cells experience a damage rate that is below the threshold and are immortal under the conditions examined. The model estimates the asymmetry level of E. coli to be low but sufficient for persisting at higher damage rates. The model also predicts that increasing asymmetry results in diminishing fitness returns, which may explain why the bacterium has not evolved higher asymmetry.

PMID: 20865171

Intestinal barrier leakage constitutes a potential therapeutic target for many inflammatory diseases and represents a disease progression marker during chronic viral infections. However, the causes of altered gut barrier remain mostly unknown. Using murine infection with lymphocytic choriomeningitis virus, we demonstrate that, in contrast to an acute viral strain, a persistent viral isolate leads to long-term viral replication in hematopoietic and mesenchymal cells, but not epithelial cells (IECs), in the intestine. Viral persistence drove sustained intestinal epithelial barrier leakage, which was characterized by increased paracellular flux of small molecules and was associated with enhanced colitis susceptibility. Type I IFN signaling caused tight junction dysregulation in IECs, promoted gut microbiome shifts and enhanced intestinal CD8 T cell responses. Notably, both type I IFN receptor blockade and CD8 T cell depletion prevented infection-induced barrier leakage. Our study demonstrates that infection with a virus that persistently replicates in the intestinal mucosa increases epithelial barrier permeability and reveals type I IFNs and CD8 T cells as causative factors of intestinal leakage during chronic infections.

Genetic conflicts arise when the evolutionary interests of two genetic elements are not aligned. Conflicts between genomes (e.g. pathogen versus host) or within the same genome (e.g. internal parasitic DNA sequences versus the rest of the host genome) can both foster ‘molecular arms races’, in which genes on both sides of the conflict rapidly evolve due to bouts of adaptation and counter-adaptation. Importantly, a source of genetic novelty is needed to fuel these arms races. In this review, we highlight gene conversion as a major force in generating the novel alleles on which selection can act. Using examples from both intergenomic and intragenomic conflicts, we feature the mechanisms by which gene conversion facilitates the rapid evolution of genes in conflict.

During cell division, the inheritance of a functional endoplasmic reticulum (ER) is ensured by the endoplasmic reticulum stress surveillance (ERSU) pathway. Activation of ERSU causes the septin ring to mislocalize, which blocks ER inheritance and cytokinesis. Here, we uncover that the septin ring in fact translocates to previously utilized cell division sites called cytokinetic remnants (CRMs). This unconventional translocation requires Nba1, a negative polarity regulator that normally prevents repolarization and re-budding at CRMs. Furthermore, septin ring translocation relies on the recruitment and activation of a key ERSU component Slt2 by Bem1, without activating Cdc42. Failure to transfer all septin subunits to CRMs delays the cell’s ability to re-enter the cell cycle when ER homeostasis is restored and hinders cell growth after ER stress recovery. Thus, these deliberate but unprecedented rearrangements of cell polarity factors during ER stress safeguard cell survival and the timely cell-cycle re-entry upon ER stress recovery.

Immunotherapy with checkpoint inhibitors for liver cancer, while active in many clinical trials worldwide, may have uncertain outcomes due to the unique immunotolerant microenvironment of the liver. In previous experiments, we unexpectedly identified a robust liver tumor-preventive effect of a synthetic double-stranded RNA, polyinosinic-polycytidylic acid (polyIC), in mice. Herein we further demonstrate that polyIC given at the precancer stage effectively prevented liver tumorigenesis by activating natural killer cells, macrophages, and some T-cell subsets; no inhibitory effect was observed on tumor progression if injected after tumor initiation. Nevertheless, polyIC administration potently induced programmed death ligand 1 (PD-L1) expression in liver sinusoid endothelial cells, which prompted us to test a combined treatment of polyIC and PD-L1 antibody (Ab). Although injecting PD-L1 Ab alone did not show any therapeutic effect, injection of polyIC sensitized the hepatic response to PD-L1 blockade. Combination of polyIC and PD-L1 Ab resulted in sustained accumulation of active cluster of differentiation 8 cytotoxic T cells and robust liver tumor suppression and conferred a survival advantage in mice. These preclinical data in animal models suggest that, despite the low efficacy of PD-L1/PD-1 blockade alone, careful design of mechanism-based combinatorial immunotherapeutic protocols may shift the paradigm in liver cancer treatment by coordinating maximal activation of multiple innate and adaptive immune functions. Conclusion: We provide proof of principle for the development of an efficient prevention strategy of liver tumorigenesis and a powerful combination immunotherapy for primary liver cancer.

Heterozygous deletions within chromosome 20q, or del(20q), are frequent cytogenetic abnormalities detected in hematologic malignancies. To date, identification of genes in the del(20q) common deleted region that contribute to disease development have remained elusive. Through assessment of patient gene expression we have identified STK4 (encoding Hippo kinase MST1) as a 20q gene that is downregulated below haploinsufficient amounts in myelodysplastic syndrome (MDS) and myeloproliferative neoplasm (MPN). Hematopoietic-specific gene inactivation in mice revealed Hippo kinase loss to induce splenomegaly, thrombocytopenia, megakaryocytic dysplasia, and a propensity for chronic granulocytosis; phenotypes that closely resemble those observed in patients harboring del(20q). In a JAK2-V617F model, heterozygous Hippo kinase inactivation led to accelerated development of lethal myelofibrosis, recapitulating adverse MPN disease progression and revealing a novel genetic interaction between these two molecular events. Quantitative serum protein profiling showed that myelofibrotic transformation in mice was associated with cooperative effects of JAK2-V617F and Hippo kinase inactivation on innate immune-associated proinflammatory cytokine production, including IL-1β and IL-6. Mechanistically, MST1 interacted with IRAK1, and shRNA-mediated knockdown was sufficient to increase IRAK1-dependent innate immune activation of NF-κB in human myeloid cells. Consistent with this, treatment with a small molecule IRAK1/4 inhibitor rescued the aberrantly elevated IL-1β production in the JAK2-V617F MPN model. This study identifies Hippo kinase MST1 ( STK4) as having a central role in the biology of del(20q)-associated hematologic malignancies and reveals a novel molecular basis of adverse MPN progression that may be therapeutically exploitable via IRAK1 inhibition.

Human Schlafen 11 (SLFN11) is an interferon stimulated gene (ISG) that we previously have demonstrated to ablate translation of HIV proteins based on the virus’ distinct codon preference. Additionally, lack of SLFN11 expression has been linked to the resistance of cancer cells to DNA damaging agents (DDAs). We recently resolved the underlying mechanism, finding that it involves SLFN11-mediated cleavage of select tRNAs predominantly employed in the translation of the ATR and ATM Ser/Thr kinases, thereby establishing SLFN11 as a novel tRNA endo-nuclease. Even though SLFN11 is thus involved in two of the most prominent diseases of our time, cancer and HIV infection, its regulation remained thus far unresolved. Using mass spectroscopy and bioinformatics-based approaches combined with site-directed mutagenesis, we show here that SLFN11 is phosphorylated at three different sites, which requires dephosphorylation in order for SLFN11 to become fully functionally active. Furthermore, we identified protein phosphatase 1 catalytic subunit gamma (PPP1CC) as the upstream enzyme whose activity is required for SLFN11 to cleave tRNAs and thereby act as a selective translational inhibitor. In summary, our work has identified both the mechanism of SLFN11 activation and PPP1CC as the enzyme responsible for its activation. Our findings open up future studies of the PPP1CC subunit(s) involved in SLFN11 activation and the putative kinase(s) that inactivate SLFN11.

GTPases are a large superfamily of essential enzymes that regulate a variety of cellular processes. They share a common core structure supporting nucleotide binding and hydrolysis, and are potentially descended from the same ancestor. Yet their biological functions diverge dramatically, ranging from cell division and movement to signal transduction and translation. It has been shown that conformational changes through binding to different substrates underlie the regulation of their activities. Here we investigate the conformational dynamics of three typical GTPases by in silico simulation. We find that these three GTPases possess overall similar substrate-associated dynamic features, beyond their distinct functions. Further identification of key common and family-specific elements in these three families helps us understand how enzymes are adapted to acquire distinct functions from a common core structure. Our results provide unprecedented insights into the functional mechanism of GTPases in general, which potentially facilitates novel protein design in the future.

Bacteria exhibit cell-to-cell variability in their resilience to stress, for example, following antibiotic exposure. Higher resilience is typically ascribed to "dormant" non-growing cellular states. Here, by measuring membrane potential dynamics of Bacillus subtilis cells, we show that actively growing bacteria can cope with ribosome-targeting antibiotics through an alternative mechanism based on ion flux modulation. Specifically, we observed two types of cellular behavior: growth-defective cells exhibited a mathematically predicted transient increase in membrane potential (hyperpolarization), followed by cell death, whereas growing cells lacked hyperpolarization events and showed elevated survival. Using structural perturbations of the ribosome and proteomic analysis, we uncovered that stress resilience arises from magnesium influx, which prevents hyperpolarization. Thus, ion flux modulation provides a distinct mechanism to cope with ribosomal stress. These results suggest new approaches to increase the effectiveness of ribosome-targeting antibiotics and reveal an intriguing connection between ribosomes and the membrane potential, two fundamental properties of cells.

Human beings are almost certainly the most diseased species on earth. By one accounting, there are at least 1400 human pathogens, including bacteria, fungi, prions, protozoa, viruses, and worms, and of these, 100-150 appear capable of causing human epidemics. 1, 2 Even this is likely to be an underestimate, as new and sensitive sequencing techniques continue to uncover new viruses at a steady rate. ³ We human beings are remarkable in many ways, but why are we remarkable for playing host to so many infectious agents? Why is it that we must maintain high levels of vaccine coverage to prevent infectious agents from sickening or even killing large swaths of the population? The answers lie in the story of human disease epidemics, and it begins with human cultural and technological ascendance and what we now understand to be its inevitable consequences for pestilence and death. It is about our ingenuity, which has caused the retreat of many infectious diseases, but highlights a central tension in human existence—immediate self-interest vs long-term collective welfare. ⁴ The concept is not just academic; there are real-world implications that we can resolve with an understanding of human disease ecology. The notion is that we are not only culturally connected or genetically connected through a common ancestry. Rather, there is another fundamental concept that is, perhaps, not widely accepted or even understood. We are biologically connected, in the present, through our exchange of infectious agents and our common susceptibility to disease.

Immunotherapy with checkpoint inhibitors for liver cancer, while active in many clinical trials worldwide, may have uncertain outcomes due to the unique immunotolerant microenvironment of the liver. In previous experiments, we unexpectedly identified a robust liver tumor‐preventive effect of a synthetic double‐stranded RNA, polyinosinic‐polycytidylic acid (polyIC), in mice. Herein we further demonstrate that polyIC given at the precancer stage effectively prevented liver tumorigenesis by activating natural killer cells, macrophages, and some T‐cell subsets; no inhibitory effect was observed on tumor progression if injected after tumor initiation. Nevertheless, polyIC administration potently induced programmed death ligand 1 (PD‐L1) expression in liver sinusoid endothelial cells, which prompted us to test a combined treatment of polyIC and PD‐L1 antibody (Ab). Although injecting PD‐L1 Ab alone did not show any therapeutic effect, injection of polyIC sensitized the hepatic response to PD‐L1 blockade. Combination of polyIC and PD‐L1 Ab resulted in sustained accumulation of active cluster of differentiation 8 cytotoxic T cells and robust liver tumor suppression and conferred a survival advantage in mice. These preclinical data in animal models suggest that, despite the low efficacy of PD‐L1/PD‐1 blockade alone, careful design of mechanism‐based combinatorial immunotherapeutic protocols may shift the paradigm in liver cancer treatment by coordinating maximal activation of multiple innate and adaptive immune functions. Conclusion: We provide proof of principle for the development of an efficient prevention strategy of liver tumorigenesis and a powerful combination immunotherapy for primary liver cancer.

The unfolded protein response (UPR) is induced by proteotoxic stress of the endoplasmic reticulum (ER). Here we report that ATF6, a major mammalian UPR sensor, is also activated by specific sphingolipids, dihydrosphingosine (DHS) and dihydroceramide (DHC). Single mutations in a previously undefined transmembrane domain motif that we identify in ATF6 incapacitate DHS/DHC activation while still allowing proteotoxic stress activation via the luminal domain. ATF6 thus possesses two activation mechanisms: DHS/DHC activation and proteotoxic stress activation. Reporters constructed to monitor each mechanism show that phenobarbital-induced ER membrane expansion depends on transmembrane domain-induced ATF6. DHS/DHC addition preferentially induces transcription of ATF6 target lipid biosynthetic and metabolic genes over target ER chaperone genes. Importantly, ATF6 containing a luminal achromatopsia eye disease mutation, unresponsive to proteotoxic stress, can be activated by fenretinide, a drug that upregulates DHC, suggesting a potential therapy for this and other ATF6-related diseases including heart disease and stroke.

The means by which the physicochemical properties of different cellular components together determine bacterial cell shape remain poorly understood. Here, we investigate a programmed cell-shape change during Bacillus subtilis sporulation, when a rod-shaped vegetative cell is transformed to an ovoid spore. Asymmetric cell division generates a bigger mother cell and a smaller, hemispherical forespore. The septum traps the forespore chromosome, which is translocated to the forespore by SpoIIIE. Simultaneously, forespore size increases as it is reshaped into an ovoid. Using genetics, timelapse microscopy, cryo-electron tomography, and mathematical modeling, we demonstrate that forespore growth relies on membrane synthesis and SpoIIIE-mediated chromosome translocation, but not on peptidoglycan or protein synthesis. Our data suggest that the hydrated nucleoid swells and inflates the forespore, displacing ribosomes to the cell periphery, stretching septal peptidoglycan, and reshaping the forespore. Our results illustrate how simple biophysical interactions between core cellular components contribute to cellular morphology.

Global transcriptional silencing is a highly conserved mechanism central to the oocyte-to-embryo transition. We report the unexpected discovery that global transcriptional silencing in oocytes depends on an mRNA decay activator. Oocyte-specific loss of ZFP36L2 an RNA-binding protein that promotes AU-rich element-dependent mRNA decay prevents global transcriptional silencing and causes oocyte maturation and fertilization defects, as well as complete female infertility in the mouse. Single-cell RNA sequencing revealed that ZFP36L2 downregulates mRNAs encoding transcription and chromatin modification regulators, including a large group of mRNAs for histone demethylases targeting H3K4 and H3K9, which we show are bound and degraded by ZFP36L2. Oocytes lacking Zfp36l2 fail to accumulate histone methylation at H3K4 and H3K9, marks associated with the transcriptionally silent, developmentally competent oocyte state. Our results uncover a ZFP36L2-dependent mRNA decay mechanism that acts as a developmental switch during oocyte growth, triggering wide-spread shifts in chromatin modification and global transcription.

Inflammation influences cancer development, progression, and the efficacy of cancer treatments, yet the mechanisms by which immune signaling drives alterations in the cancer cell transcriptome remain unclear. Using ChIP-seq, RNA-seq, and GRO-seq, here we demonstrate a global overlap in the binding of tumor-promoting p53 mutants and the master proinflammatory regulator NFκB that drives alterations in enhancer and gene activation in response to chronic TNF-α signaling. We show that p53 mutants interact directly with NFκB and that both factors impact the other's binding at diverse sets of active enhancers. In turn, the simultaneous and cooperative binding of these factors is required to regulate RNAPII recruitment, the synthesis of enhancer RNAs, and the activation of tumor-promoting genes. Collectively, these findings establish a mechanism by which chronic TNF-α signaling orchestrates a functional interplay between mutant p53 and NFκB that underlies altered patterns of cancer-promoting gene expression.Inflammation is known to affect cancer development, yet the mechanisms by which immune signaling drives transformation remain unclear. Here, the authors provide evidence that chronic TNF-α signaling promotes the enhancer binding and transcriptional interplay between mutant p53 and NFκB.

Dynamic changes in the expression of transcription factors (TFs) can influence the specification of distinct CD8+ T cell fates, but the observation of equivalent expression of TFs among differentially fated precursor cells suggests additional underlying mechanisms. Here we profiled the genome-wide histone modifications, open chromatin and gene expression of naive, terminal-effector, memory-precursor and memory CD8+ T cell populations induced during the in vivo response to bacterial infection. Integration of these data suggested that the expression and binding of TFs contributed to the establishment of subset-specific enhancers during differentiation. We developed a new bioinformatics method using the PageRank algorithm to reveal key TFs that influence the generation of effector and memory populations. The TFs YY1 and Nr3c1, both constitutively expressed during CD8+ T cell differentiation, regulated the formation of terminal-effector cell fates and memory-precursor cell fates, respectively. Our data define the epigenetic landscape of differentiation intermediates and facilitate the identification of TFs with previously unappreciated roles in CD8+ T cell differentiation.

To identify endogenous miRNA-target sites, we isolated AGO-bound RNAs from Caenorhabditis elegans by individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP), which fortuitously also produced miRNA-target chimeric reads. Through the analysis of thousands of reproducible chimeras, pairing to the miRNA seed emerged as the predominant motif associated with functional interactions. Unexpectedly, we discovered that additional pairing to 3' sequences is prevalent in the majority of target sites and leads to specific targeting by members of miRNA families. By editing an endogenous target site, we demonstrate that 3' pairing determines targeting by specific miRNA family members and that seed pairing is not always sufficient for functional target interactions. Finally, we present a simplified method, chimera PCR (ChimP), for the detection of specific miRNA-target interactions. Overall, our analysis revealed that sequences in the 5' as well as the 3' regions of a miRNA provide the information necessary for stable and specific miRNA-target interactions in vivo.

Many transcription factors co-express with their homologs to regulate identical target genes, however the advantages of such redundancies remain elusive. Using single-cell imaging and microfluidics, we study the yeast general stress response transcription factor Msn2 and its seemingly redundant homolog Msn4. We find that gene regulation by these two factors is analogous to logic gate systems. Target genes with fast activation kinetics can be fully induced by either factor, behaving as an 'OR' gate. In contrast, target genes with slow activation kinetics behave as an 'AND' gate, requiring distinct contributions from both factors, upon transient stimulation. Furthermore, such genes become an 'OR' gate when the input duration is prolonged, suggesting that the logic gate scheme is not static but rather dependent on the input dynamics. Therefore, Msn2 and Msn4 enable a time-based mode of combinatorial gene regulation that might be applicable to homologous transcription factors in other organisms.

Microbial pathogens often establish infection within particular niches of their host for replication. Determining how infection occurs preferentially in specific host tissues is a key aspect of understanding host-microbe interactions. Here, we describe the discovery of a natural microsporidian parasite of the nematode Caenorhabditis elegans that displays a unique tissue tropism compared to previously described parasites of this host. We characterize the life cycle of this new species, Nematocida displodere, including pathogen entry, intracellular replication, and exit. N. displodere can invade multiple host tissues, including the epidermis, muscle, neurons, and intestine of C. elegans. Despite robust invasion of the intestine very little replication occurs there, with the majority of replication occurring in the muscle and epidermis. This feature distinguishes N. displodere from two closely related microsporidian pathogens, N. parisii and N. sp. 1, which exclusively invade and replicate in the intestine. Comparison of the N. displodere genome with N. parisii and N. sp. 1 reveals that N. displodere is the earliest diverging species of the Nematocida genus. Over 10% of the proteins encoded by the N. displodere genome belong to a single species-specific family of RING-domain containing proteins of unknown function that may be mediating interactions with the host. Altogether, this system provides a powerful whole-animal model to investigate factors responsible for pathogen growth in different tissue niches.

Lymphomas are relatively common forms of cancer that develop in cells of the immune system. In this study, Cornelis Murre and colleagues identify a previously unsuspected population of immune cells. These cells are missing some of the standard features that define normal immune cells. In experiments performed in mice, it was found that loss of these features leads to the development of a malignancy very similar to human Burkitt lymphoma. This type of lymphoma, named after the scientist who first identified it in populations in Africa in the 1950s, is of particular interest because it is now known to also affect children and patients with weakened immune systems, including organ transplant patients whose immune systems must be actively suppressed to prevent rejection. The progress made in this study is important because it gives new insight into the pathways by which normal immune cells develop and mature and how, when disrupted, may lead to lymphoma.

The development of a complex body plan requires a diversity of regulatory networks. Here we consider the concept of TATA-box-binding protein (TBP) family proteins as "system factors" that each supports a distinct set of transcriptional programs. For instance, TBP activates TATA-box-dependent core promoters, whereas TBP-related factor 2 (TRF2) activates TATA-less core promoters that are dependent on a TCT or downstream core promoter element (DPE) motif. These findings led us to investigate the evolution of TRF2. TBP occurs in Archaea and eukaryotes, but TRF2 evolved prior to the emergence of the bilateria and subsequent to the evolutionary split between bilaterians and nonbilaterian animals. Unlike TBP, TRF2 does not bind to the TATA box and could thus function as a new system factor that is largely independent of TBP. We postulate that this TRF2-based system served as the foundation for new transcriptional programs, such as those involved in triploblasty and body plan development, that facilitated the evolution of bilateria.

During B lymphocyte development, immunoglobulin heavy-chain variable (VH), diversity (DH), and joining (JH) segments assemble to generate a diverse antigen receptor repertoire. Here, we have marked the distal VH and DH-JH-Eμ regions with Tet-operator binding sites and traced their 3D trajectories in pro-B cells transduced with a retrovirus encoding Tet-repressor-EGFP. We found that these elements displayed fractional Langevin motion (fLm) due to the viscoelastic hindrance from the surrounding network of proteins and chromatin fibers. Using fractional Langevin dynamics modeling, we found that, with high probability, DHJH elements reach a VH element within minutes. Spatial confinement emerged as the dominant parameter that determined the frequency of such encounters. We propose that the viscoelastic nature of the nuclear environment causes coding elements and regulatory elements to bounce back and forth in a spring-like fashion until specific genomic interactions are established and that spatial confinement of topological domains largely controls first-passage times for genomic interactions.

The response of cells to changes in their environment often requires coregulation of gene networks, but little is known about how this can occur at the post-transcriptional level. An important example of post-transcriptional coregulation is the selective translational regulation in response to growth conditions of mammalian mRNAs that encode protein biosynthesis factors and contain hallmark 5'-terminal oligopyrimidine tracts (5'TOP). However, the responsible trans-factors and the mechanism by which they coregulate 5'TOP mRNAs have remained elusive. Here we identify stress granule-associated TIA-1 and TIAR proteins as key factors in human 5'TOP mRNA regulation, which upon amino acid starvation assemble onto the 5' end of 5'TOP mRNAs and arrest translation at the initiation step, as evidenced by TIA-1/TIAR-dependent 5'TOP mRNA translation repression, polysome release, and accumulation in stress granules. This requires starvation-mediated activation of the GCN2 (general control nonderepressible 2) kinase and inactivation of the mTOR (mammalian target of rapamycin) signaling pathway. Our findings provide a mechanistic explanation to the long-standing question of how the network of 5'TOP mRNAs are coregulated according to amino acid availability, thereby allowing redirection of limited resources to mount a nutrient deprivation response. This presents a fundamental example of how a group of mRNAs can be translationally coregulated in response to changes in the cellular environment.

Tubulins are essential for the reproduction of many eukaryotic viruses, but historically, bacteriophage were assumed not to require a cytoskeleton. Here, we identify a tubulin-like protein, PhuZ, from bacteriophage 201φ2-1 and show that it forms filaments in vivo and in vitro. The PhuZ structure has a conserved tubulin fold, with an unusual, extended C terminus that we demonstrate to be critical for polymerization in vitro and in vivo. Longitudinal packing in the crystal lattice mimics packing observed by EM of in-vitro-formed filaments, indicating how interactions between the C terminus and the following monomer drive polymerization. PhuZ forms a filamentous array that is required for positioning phage DNA within the bacterial cell. Correct positioning to the cell center and optimal phage reproduction only occur when the PhuZ filament is dynamic. Thus, we show that PhuZ assembles a spindle-like array that functions analogously to the microtubule-based spindles of eukaryotes.

MicroRNAs (miRNAs) comprise a large family of small RNA molecules that post-transcriptionally regulate gene expression in many biological pathways. Most miRNAs are derived from long primary transcripts that undergo processing by Drosha to produce ~65-nucleotide precursors that are then cleaved by Dicer, resulting in the mature 22-nucleotide forms. Serving as guides in Argonaute protein complexes, mature miRNAs use imperfect base pairing to recognize sequences in messenger RNA transcripts, leading to translational repression and destabilization of the target messenger RNAs. Here we show that the miRNA complex also targets and regulates non-coding RNAs that serve as substrates for the miRNA-processing pathway. We found that the Argonaute protein in Caenorhabditis elegans, ALG-1, binds to a specific site at the 3′ end of let-7 miRNA primary transcripts and promotes downstream processing events. This interaction is mediated by mature let-7 miRNA through a conserved complementary site in its own primary transcript, thus creating a positive-feedback loop. We further show that ALG-1 associates with let-7 primary transcripts in nuclear fractions. Argonaute also binds let-7 primary transcripts in human cells, demonstrating that the miRNA pathway targets non-coding RNAs in addition to protein-coding messenger RNAs across species. Moreover, our studies in C. elegans reveal a novel role for Argonaute in promoting biogenesis of a targeted transcript, expanding the functions of the miRNA pathway in gene regulation. This discovery of autoregulation of let-7 biogenesis establishes a new mechanism for controlling miRNA expression.

Multiple inhibitory molecules create a profoundly immunosuppressive environment during chronic viral infections in humans and mice. Therefore, eliciting effective immunity in this context represents a challenge. Here, we report that during a murine chronic viral infection, interleukin-6 (IL-6) was produced by irradiation-resistant cells in a biphasic manner, with late IL-6 being absolutely essential for viral control. The underlying mechanism involved IL-6 signaling on virus-specific CD4 T cells that caused up-regulation of the transcription factor Bcl6 and enhanced T follicular helper cell responses at late, but not early, stages of chronic viral infection. This resulted in escalation of germinal center reactions and improved antibody responses. Our results uncover an antiviral strategy that helps to safely resolve a persistent infection in vivo.

The posterior parietal cortex (PPC) performs many functions, including decision making and movement control. It remains unknown which input and output pathways of PPC support different functions. We addressed this issue in mice, focusing on PPC neurons projecting to the dorsal striatum (PPC-STR) and the posterior secondary motor cortex (PPC-pM2). Projection-specific, retrograde labeling showed that PPC-STR and PPC-pM2 represent largely distinct subpopulations, with PPC-STR receiving stronger inputs from association areas and PPC-pM2 receiving stronger sensorimotor inputs. Two-photon calcium imaging during decision making revealed that the PPC-STR population encodes history-dependent choice bias more strongly than PPC-pM2 or general PPC populations. Furthermore, optogenetic inactivation of PPC-STR neurons or their terminals in STR decreased history-dependent bias, while inactivation of PPC-pM2 neurons altered movement kinematics. Therefore, PPC biases action selection through its STR projection while controlling movements through PPC-pM2 neurons. PPC may support multiple functions through parallel subpopulations, each with distinct input-output connectivity.

Phase separation into liquid-like compartments is an emerging property of proteins containing prion-like domains (PrLDs), yet the in vivo roles of phase separation remain poorly understood. TIA proteins contain a C-terminal PrLD, and mutations in the PrLD are associated with several diseases. Here, we show that the C. elegans TIAR-2/TIA protein functions cell autonomously to inhibit axon regeneration. TIAR-2 undergoes liquid-liquid phase separation in vitro and forms granules with liquid-like properties in vivo. Axon injury induces a transient increase in TIAR-2 granule number. The PrLD is necessary and sufficient for granule formation and inhibiting regeneration. Tyrosine residues within the PrLD are important for granule formation and inhibition of regeneration. TIAR-2 is also serine phosphorylated in vivo. Non-phosphorylatable TIAR-2 variants do not form granules and are unable to inhibit axon regeneration. Our data demonstrate an in vivo function for phase-separated TIAR-2 and identify features critical for its function in axon regeneration.

In the Drosophila antenna, different subtypes of olfactory receptor neurons (ORNs) housed in the same sensory hair (sensillum) can inhibit each other non-synaptically. However, the mechanisms underlying this underexplored form of lateral inhibition remain unclear. Here we use recordings from pairs of sensilla impaled by the same tungsten electrode to demonstrate that direct electrical (“ephaptic”) interactions mediate lateral inhibition between ORNs. Intriguingly, within individual sensilla, we find that ephaptic lateral inhibition is asymmetric such that one ORN exerts greater influence onto its neighbor. Serial block-face scanning electron microscopy of genetically identified ORNs and circuit modeling indicate that asymmetric lateral inhibition reflects a surprisingly simple mechanism: the physically larger ORN in a pair corresponds to the dominant neuron in ephaptic interactions. Thus, morphometric differences between compartmentalized ORNs account for highly specialized inhibitory interactions that govern information processing at the earliest stages of olfactory coding.

Experience-dependent expression of immediate-early gene transcription factors (IEG-TFs) can transiently change the transcriptome of active neurons and initiate persistent changes in cellular function. However, the impact of IEG-TFs on circuit connectivity and function is poorly understood. We investigate the specificity with which the IEG-TF NPAS4 governs experience-dependent changes in inhibitory synaptic input onto CA1 pyramidal neurons (PNs). We show that novel sensory experience selectively enhances somatic inhibition mediated by cholecystokinin-expressing basket cells (CCKBCs) in an NPAS4-dependent manner. NPAS4 specifically increases the number of synapses made onto PNs by individual CCKBCs without altering synaptic properties. Additionally, we find that sensory experience-driven NPAS4 expression enhances depolarization-induced suppression of inhibition (DSI), a short-term form of cannabinoid-mediated plasticity expressed at CCKBC synapses. Our results indicate that CCKBC inputs are a major target of the NPAS4-dependent transcriptional program in PNs and that NPAS4 is an important regulator of plasticity mediated by endogenous cannabinoids.

Editing domains of aminoacyl tRNA synthetases correct tRNA charging errors to maintain translational fidelity. A mutation in the editing domain of alanyl tRNA synthetase (AlaRS) in Aarssti mutant mice results in an increase in the production of serine-mischarged tRNAAla and the degeneration of cerebellar Purkinje cells. Here, using positional cloning, we identified Ankrd16, a gene that acts epistatically with the Aarssti mutation to attenuate neurodegeneration. ANKRD16, a vertebrate-specific protein that contains ankyrin repeats, binds directly to the catalytic domain of AlaRS. Serine that is misactivated by AlaRS is captured by the lysine side chains of ANKRD16, which prevents the charging of serine adenylates to tRNAAla and precludes serine misincorporation in nascent peptides. The deletion of Ankrd16 in the brains of Aarssti/sti mice causes widespread protein aggregation and neuron loss. These results identify an amino-acid-accepting co-regulator of tRNA synthetase editing as a new layer of the machinery that is essential to the prevention of severe pathologies that arise from defects in editing.

Complex spatial working memory tasks have been shown to require both hippocampal sharp-wave ripple (SWR) activity and dentate gyrus (DG) neuronal activity. We therefore asked whether DG inputs to CA3 contribute to spatial working memory by promoting SWR generation. Recordings from DG and CA3 while rats performed a dentate-dependent working memory task on an eight-arm radial maze revealed that the activity of dentate neurons and the incidence rate of SWRs both increased during reward consumption. We then found reduced reward-related CA3 SWR generation without direct input from dentate granule neurons. Furthermore, CA3 cells with place fields in not-yet-visited arms preferentially fired during SWRs at reward locations, and these prospective CA3 firing patterns were more pronounced for correct trials and were dentate-dependent. These results indicate that coordination of CA3 neuronal activity patterns by DG is necessary for the generation of neuronal firing patterns that support goal-directed behavior and memory.

Early life stress (ELS) in the form of child abuse/neglect is associated with an increased risk of developing social dysfunction in adulthood. Little is known, however, about the neural substrates or the neuromodulatory signaling that govern ELS-induced social dysfunction. Here, we show that ELS-induced downregulation of dopamine receptor 3 (Drd3) signaling and its corresponding effects on neural activity in the lateral septum (LS) are both necessary and sufficient to cause social abnormalities in adulthood. Using in vivo Ca ²⁺ imaging, we found that Drd3-expressing-LS (Drd3 ^LS) neurons in animals exposed to ELS show blunted activity in response to social stimuli. In addition, optogenetic activation of Drd3 ^LS neurons rescues ELS-induced social impairments. Furthermore, pharmacological treatment with a Drd3 agonist, which increases Drd3 ^LS neuronal activity, normalizes the social dysfunctions of ELS mice. Thus, we identify Drd3 in the LS as a critical mediator and potential therapeutic target for the social abnormalities caused by ELS.

Changes in social preference of amphibian larvae result from sustained exposure to kinship odorants. To understand the molecular and cellular mechanisms of this neuroplasticity, we investigated the effects of olfactory system activation on neurotransmitter (NT) expression in accessory olfactory bulb (AOB) interneurons during development. We show that protracted exposure to kin or non-kin odorants changes the number of dopamine (DA)- or gamma aminobutyric acid (GABA)-expressing neurons, with corresponding changes in attraction/aversion behavior. Changing the relative number of dopaminergic and GABAergic AOB interneurons or locally introducing DA or GABA receptor antagonists alters kinship preference. We then isolate AOB microRNAs (miRs) differentially regulated across these conditions. Inhibition of miR-375 and miR-200b reveals that they target Pax6 and Bcl11b to regulate the dopaminergic and GABAergic phenotypes. The results illuminate the role of NT switching governing experience-dependent social preference.

In neurons, lysosomes, which degrade membrane and cytoplasmic components, are thought to primarily reside in somatic and axonal compartments, but there is little understanding of their distribution and function in dendrites. Here, we used conventional and two-photon imaging and electron microscopy to show that lysosomes traffic bidirectionally in dendrites and are present in dendritic spines. We find that lysosome inhibition alters their mobility and also decreases dendritic spine number. Furthermore, perturbing microtubule and actin cytoskeletal dynamics has an inverse relationship on the distribution and motility of lysosomes in dendrites. We also find trafficking of lysosomes is correlated with synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors. Strikingly, lysosomes traffic to dendritic spines in an activity-dependent manner and can be recruited to individual spines in response to local activation. These data indicate the position of lysosomes is regulated by synaptic activity and thus plays an instructive role in the turnover of synaptic membrane proteins.

Subcellular localization of ribosomes defines the location and capacity for protein synthesis. Methods for in vivo visualizing ribosomes in multicellular organisms are desirable in mechanistic investigations of the cell biology of ribosome dynamics. Here, we developed an approach using split GFP for tissue-specific visualization of ribosomes in Caenorhabditis elegans. Labeled ribosomes are detected as fluorescent puncta in the axons and synaptic terminals of specific neuron types, correlating with ribosome distribution at the ultrastructural level. We found that axonal ribosomes change localization during neuronal development and after axonal injury. By examining mutants affecting axonal trafficking and performing a forward genetic screen, we showed that the microtubule cytoskeleton and the JIP3 protein UNC-16 exert distinct effects on localization of axonal and somatic ribosomes. Our data demonstrate the utility of tissue-specific visualization of ribosomes in vivo, and provide insight into the mechanisms of active regulation of ribosome localization in neurons.

Motor learning is accompanied by widespread changes within the motor cortex, but it is unknown whether these changes are ultimately funneled through a stable corticospinal output channel or whether the corticospinal output itself is plastic. We investigated the consistency of the relationship between corticospinal neuron activity and movement through in vivo two-photon calcium imaging in mice learning a lever-press task. Corticospinal neurons exhibited heterogeneous correlations with movement, with the majority of movement-modulated neurons decreasing activity during movement. Individual cells changed their activity across days, which led to changed associations between corticospinal activity and movement. Unlike previous observations in layer 2/3, activity accompanying learned movements did not become more consistent with learning; instead, the activity of dissimilar movements became more decorrelated. These results indicate that the relationship between corticospinal activity and movement is dynamic and that the types of activity and plasticity are different from and possibly complementary to those in layer 2/3.

Major depressive disorder (MDD) patients display a common but often variable set of symptoms making successful, sustained treatment difficult to achieve. Separate depressive symptoms may be encoded by differential changes in distinct circuits in the brain, yet how discrete circuits underlie behavioral subsets of depression and how they adapt in response to stress has not been addressed. We identify two discrete circuits of parvalbumin-positive (PV) neurons in the ventral pallidum (VP) projecting to either the lateral habenula or ventral tegmental area contributing to depression. We find that these populations undergo different electrophysiological adaptations in response to social defeat stress, which are normalized by antidepressant treatment. Furthermore, manipulation of each population mediates either social withdrawal or behavioral despair, but not both. We propose that distinct components of the VP PV circuit can subserve related, yet separate depressive-like phenotypes in mice, which could ultimately provide a platform for symptom-specific treatments of depression.

During the lifespans of most animals, reproductive maturity and mating activity are highly coordinated. In Drosophila melanogaster, for instance, male fertility increases with age, and older males are known to have a copulation advantage over young ones. The molecular and neural basis of this age-related disparity in mating behavior is unknown. Here, we show that the Or47b odorant receptor is required for the copulation advantage of older males. Notably, the sensitivity of Or47b neurons to a stimulatory pheromone, palmitoleic acid, is low in young males but high in older ones, which accounts for older males' higher courtship intensity. Mechanistically, this age-related sensitization of Or47b neurons requires a reproductive hormone, juvenile hormone, as well as its binding protein Methoprene-tolerant in Or47b neurons. Together, our study identifies a direct neural substrate for juvenile hormone that permits coordination of courtship activity with reproductive maturity to maximize male reproductive fitness. Copyright © 2016 Elsevier Inc. All rights reserved.

Limited functional recovery can be achieved with rehabilitation after incomplete spinal cord injury. Eliminating the function of a repulsive Wnt receptor, Ryk, by either conditional knockout in the motor cortex or monoclonal antibody infusion, resulted in increased corticospinal axon collateral branches with pre-synaptic puncta in the spinal cord and enhanced recovery of forelimb reaching and grasping function following a cervical dorsal column lesion. Using optical stimulation, we observed that motor cortical output maps underwent massive changes after injury and the hindlimb cortical areas were recruited to control the forelimb over time. Furthermore, a greater cortical area was dedicated to control the forelimb in Ryk cKO. In the absence of weekly task-specific training, recruitment of ectopic cortical areas was greatly reduced without significant functional recovery even in Ryk cKO. Our study provides evidence that maximal circuit reorganization and functional recovery can be achieved by combining molecular manipulation and task-specific training.

Whisking and sniffing are predominant aspects of exploratory behaviour in rodents. Yet the neural mechanisms that generate and coordinate these and other orofacial motor patterns remain largely uncharacterized. Here we use anatomical, behavioural, electrophysiological and pharmacological tools to show that whisking and sniffing are coordinated by respiratory centres in the ventral medulla. We delineate a distinct region in the ventral medulla that provides rhythmic input to the facial motor neurons that drive protraction of the vibrissae. Neuronal output from this region is reset at each inspiration by direct input from the pre-Bötzinger complex, such that high-frequency sniffing has a one-to-one relationship with whisking, whereas basal respiration is accompanied by intervening whisks that occur between breaths. We conjecture that the respiratory nuclei, which project to other premotor regions for oral and facial control, function as a master clock for behaviours that coordinate with breathing.

How are sensory representations in the brain influenced by the state of an animal? Here we use chronic two-photon calcium imaging to explore how wakefulness and experience shape odor representations in the mouse olfactory bulb. Comparing the awake and anesthetized state, we show that wakefulness greatly enhances the activity of inhibitory granule cells and makes principal mitral cell odor responses more sparse and temporally dynamic. In awake mice, brief repeated odor experience leads to a gradual and long-lasting (months) weakening of mitral cell odor representations. This mitral cell plasticity is odor specific, recovers gradually over months, and can be repeated with different odors. Furthermore, the expression of this experience-dependent plasticity is prevented by anesthesia. Together, our results demonstrate the dynamic nature of mitral cell odor representations in awake animals, which is constantly shaped by recent odor experience.

Copyright © 2012 Elsevier Inc. All rights reserved.

MAPKKK dual leucine zipper-bearing kinases (DLKs) are regulators of synaptic development and axon regeneration. The mechanisms underlying their activation are not fully understood. Here, we show that C. elegans DLK-1 is activated by a Ca(2+)-dependent switch from inactive heteromeric to active homomeric protein complexes. We identify a DLK-1 isoform, DLK-1S, that shares identical kinase and leucine zipper domains with the previously described long isoform DLK-1L but acts to inhibit DLK-1 function by binding to DLK-1L. The switch between homo- or heteromeric DLK-1 complexes is influenced by Ca(2+) concentration. A conserved hexapeptide in the DLK-1L C terminus is essential for DLK-1 activity and is required for Ca(2+) regulation. The mammalian DLK-1 homolog MAP3K13 contains an identical C-terminal hexapeptide and can functionally complement dlk-1 mutants, suggesting that the DLK activation mechanism is conserved. The DLK activation mechanism is ideally suited for rapid and spatially controlled signal transduction in response to axonal injury and synaptic activity.

Copyright © 2012 Elsevier Inc. All rights reserved.

Grid cells in parahippocampal cortices fire at vertices of a periodic triangular grid that spans the entire recording environment. Such precise neural computations in space have been proposed to emerge from equally precise temporal oscillations within cells or within the local neural circuitry. We found that grid-like firing patterns in the entorhinal cortex vanished when theta oscillations were reduced after intraseptal lidocaine infusions in rats. Other spatially modulated cells in the same cortical region and place cells in the hippocampus retained their spatial firing patterns to a larger extent during these periods without well-organized oscillatory neuronal activity. Precisely timed neural activity within single cells or local networks is thus required for periodic spatial firing but not for single place fields.

PMID: 21458672 [PubMed - in process]

Internal physiological states influence behavioral decisions. We have investigated the underlying cellular and molecular mechanisms at the first olfactory synapse for starvation modulation of food-search behavior in Drosophila. We found that a local signal by short neuropeptide F (sNPF) and a global metabolic cue by insulin are integrated at specific odorant receptor neurons (ORNs) to modulate olfactory sensitivity. Results from two-photon calcium imaging show that starvation increases presynaptic activity via intraglomerular sNPF signaling. Expression of sNPF and its receptor (sNPFR1) in Or42b neurons is necessary for starvation-induced food-search behavior. Presynaptic facilitation in Or42b neurons is sufficient to mimic starvation-like behavior in fed flies. Furthermore, starvation elevates the transcription level of sNPFR1 but not that of sNPF, and insulin signaling suppresses sNPFR1 expression. Thus, starvation increases expression of sNPFR1 to change the odor map, resulting in more robust food-search behavior.

PMID: 21458672 [PubMed - in process]

Lateral competition for cortical space by layer-specific horizontal circuits. Adesnik H, Scanziani M. Nature. 2010 Apr 22;464(7292):1155-60.