Abstract: Sugars, fats, amino acids, and other nutrients cannot simply diffuse into the cell. Rather, they must be transported across the cell membrane by specific proteins that stretch from one side of the cell membrane to the other. One such ‘transporter’—system xc−—is of special interest. This transporter imports one molecule of cystine from outside the cell in exchange for one molecule of glutamate from inside the cell. Cystine, a variant of the amino acid cysteine, is essential for synthesizing new proteins and for preventing the accumulation of toxic species inside the cell. Not surprisingly, many cancer cells are dependent upon the transport activity of system xc− for growth and survival. Drugs that can inhibit system xc− could therefore be part of potential treatments for cancer and other diseases. Dixon, Patel, et al. have found that the compound erastin is a very effective inhibitor of system xc− function. Certain versions of erastin are over 1000 times more potent than the previously known best inhibitor of system xc−, sulfasalazine. Dixon, Patel et al. found that using erastin and sulfasalazine to inhibit system xc− in cancer cells grown in a petri dish results in an unusual type of iron-dependent cell death called ferroptosis. By inhibiting the uptake of cystine, erastin and other system xc− inhibitors interfere with the cellular machinery that folds proteins into their final, three-dimensional shape. The accumulation of these partially-folded proteins in the cell causes a specific kind of cellular stress that can be used as a readout, or biomarker, for the inhibition of system xc−. Such a biomarker will be essential for identifying cells in the body that have been exposed to agents that inhibit system xc− and that are undergoing ferroptosis. Unexpectedly, Dixon, Patel et al. also found that the FDA-approved anti-cancer drug sorafenib inhibits system xc−. Other drugs in the same class as sorafenib do not share this unusual property. Dixon, Patel, et al. synthesized variants of sorafenib and identified sites on the drug that are necessary for it to be able to interfere with system xc−. Alongside the erastin derivatives, these new molecules may help to develop new drugs that can inhibit this important transporter in a clinical setting.

Abstract: We identified the neurons comprising the Drosophila mushroom body (MB), an associative center in invertebrate brains, and provide a comprehensive map describing their potential connections. Each of the 21 MB output neuron (MBON) types elaborates segregated dendritic arbors along the parallel axons of ∼2000 Kenyon cells, forming 15 compartments that collectively tile the MB lobes. MBON axons project to five discrete neuropils outside of the MB and three MBON types form a feedforward network in the lobes. Each of the 20 dopaminergic neuron (DAN) types projects axons to one, or at most two, of the MBON compartments. Convergence of DAN axons on compartmentalized Kenyon cell-MBON synapses creates a highly ordered unit that can support learning to impose valence on sensory representations. The elucidation of the complement of neurons of the MB provides a comprehensive anatomical substrate from which one can infer a functional logic of associative olfactory learning and memory.

Abstract: Metformin is widely used to reduce the high blood sugar levels caused by diabetes. Recently, several studies have suggested that patients taking metformin who also develop cancer have tumors that grow more slowly than average. As clinical trials have already started to investigate if metformin is an effective anti-cancer treatment, it is important to understand how it might restrict tumor growth. Researchers have proposed two ways that metformin could affect tumors. First, insulin is known to prompt cancer cells to divide, so the slower rate of tumor growth could just be a side-effect of the metformin reducing the amount of insulin in the blood. Alternatively, metformin could target cancer cells more directly by cutting the energy supply produced by their mitochondria. Metformin has been shown to disrupt complex I of the electron transport chain that is used by cells to generate energy. However, it is not known if disrupting complex I would actually stop cells dividing because they can generate energy in other ways. Wheaton, Weinberg et al. have now demonstrated that metformin does target complex I in cancer cells, and that its effects depend on the amount of glucose available for cells to convert, without involving mitochondria, into energy. When there is plenty of glucose, metformin slows down the rate at which cancer cells divide, which slows down tumor growth. When the cells are deprived of glucose, metformin kills the cells instead. Metformin also inhibits the pathways that regulate hypoxia inducible factors (HIFs), which are part of a system that helps cells to survive low-oxygen conditions, a prominent feature of many tumors. This means that metformin may combat cancer more effectively if used alongside other treatments that reduce the availability of both oxygen and glucose inside cells. Metformin could also potentially treat conditions that are linked to overactive HIFs, such as pulmonary hypertension.

Abstract: Gene drives may be capable of addressing ecological problems by altering entire populations of wild organisms, but their use has remained largely theoretical due to technical constraints. Here we consider the potential for RNA-guided gene drives based on the CRISPR nuclease Cas9 to serve as a general method for spreading altered traits through wild populations over many generations. We detail likely capabilities, discuss limitations, and provide novel precautionary strategies to control the spread of gene drives and reverse genomic changes. The ability to edit populations of sexual species would offer substantial benefits to humanity and the environment. For example, RNA-guided gene drives could potentially prevent the spread of disease, support agriculture by reversing pesticide and herbicide resistance in insects and weeds, and control damaging invasive species. However, the possibility of unwanted ecological effects and near-certainty of spread across political borders demand careful assessment of each potential application. We call for thoughtful, inclusive, and well-informed public discussions to explore the responsible use of this currently theoretical technology.

Abstract: An animal's survival depends on its ability to respond appropriately to its environment, approaching stimuli that signal rewards and avoiding any that warn of potential threats. In fruit flies, this behavior requires activity in a region of the brain called the mushroom body, which processes sensory information and uses that information to influence responses to stimuli. Aso et al. recently mapped the mushroom body of the fruit fly in its entirety. This work showed, among other things, that the mushroom body contained 21 different types of output neurons. Building on this work, Aso et al. have started to work out how this circuitry enables flies to learn to associate a stimulus, such as an odor, with an outcome, such as the presence of food. Two complementary techniques—the use of molecular genetics to block neuronal activity, and the use of light to activate neurons (a technique called optogenetics)—were employed to study the roles performed by the output neurons in the mushroom body. Results revealed that distinct groups of output cells must be activated for flies to avoid—as opposed to approach—odors. Moreover, the same output neurons are used to avoid both odors and colors that have been associated with punishment. Together, these results indicate that the output cells do not encode the identity of stimuli: rather, they signal whether a stimulus should be approached or avoided. The output cells also regulate the amount of sleep taken by the fly, which is consistent with the mushroom body having a broader role in regulating the fly's internal state. The results of these experiments—combined with new knowledge about the detailed structure of the mushroom body—lay the foundations for new studies that explore associative learning at the level of individual circuits and their component cells. Given that the organization of the mushroom body has much in common with that of the mammalian brain, these studies should provide insights into the fundamental principles that underpin learning and memory in other species, including humans.

Abstract: Do the amino acid sequence identities of residues that make contact across protein interfaces covary during evolution? If so, such covariance could be used to predict contacts across interfaces and assemble models of biological complexes. We find that residue pairs identified using a pseudo-likelihood-based method to covary across protein–protein interfaces in the 50S ribosomal unit and 28 additional bacterial protein complexes with known structure are almost always in contact in the complex, provided that the number of aligned sequences is greater than the average length of the two proteins. We use this method to make subunit contact predictions for an additional 36 protein complexes with unknown structures, and present models based on these predictions for the tripartite ATP-independent periplasmic (TRAP) transporter, the tripartite efflux system, the pyruvate formate lyase-activating enzyme complex, and the methionine ABC transporter. DOI: http://dx.doi.org/10.7554/eLife.02030.001

Abstract: To provide an effective substrate for cognitive processes, functional brain networks should be able to reorganize and coordinate on a sub-second temporal scale. We used magnetoencephalography recordings of spontaneous activity to characterize whole-brain functional connectivity dynamics at high temporal resolution. Using a novel approach that identifies the points in time at which unique patterns of activity recur, we reveal transient (100–200 ms) brain states with spatial topographies similar to those of well-known resting state networks. By assessing temporal changes in the occurrence of these states, we demonstrate that within-network functional connectivity is underpinned by coordinated neuronal dynamics that fluctuate much more rapidly than has previously been shown. We further evaluate cross-network interactions, and show that anticorrelation between the default mode network and parietal regions of the dorsal attention network is consistent with an inability of the system to transition directly between two transient brain states.

Abstract: Production of mRNA depends critically on the rate of RNA polymerase II (Pol II) elongation To dissect Pol II dynamics in mouse ES cells, we inhibited Pol II transcription at either initiation or promoter-proximal pause escape with Triptolide or Flavopiridol, and tracked Pol II kinetically using GRO-seq Both inhibitors block transcription of more than 95% of genes, showing that pause escape, like initiation, is a ubiquitous and crucial step within the transcription cycle Moreover, paused Pol II is relatively stable, as evidenced from half-life measurements at ∼3200 genes Finally, tracking the progression of Pol II after drug treatment establishes Pol II elongation rates at over 1000 genes Notably, Pol II accelerates dramatically while transcribing through genes, but slows at exons Furthermore, intergenic variance in elongation rates is substantial, and is influenced by a positive effect of H3K79me2 and negative effects of exon density and CG content within genesDOI: http://dxdoiorg/107554/eLife02407001

Abstract: Chitin is a fungal microbe-associated molecular pattern recognized in Arabidopsis by a lysin motif receptor kinase (LYK), AtCERK1. Previous research suggested that AtCERK1 is the major chitin receptor and mediates chitin-induced signaling through homodimerization and phosphorylation. However, the reported chitin binding affinity of AtCERK1 is quite low, suggesting another receptor with high chitin binding affinity might be present. Here, we propose that AtLYK5 is the primary chitin receptor in Arabidopsis. Mutations in AtLYK5 resulted in a significant reduction in chitin response. However, AtLYK5 shares overlapping function with AtLYK4 and, therefore, Atlyk4/Atlyk5-2 double mutants show a complete loss of chitin response. AtLYK5 interacts with AtCERK1 in a chitin-dependent manner. Chitin binding to AtLYK5 is indispensable for chitin-induced AtCERK1 phosphorylation. AtLYK5 binds chitin at a much higher affinity than AtCERK1. The data suggest that AtLYK5 is the primary receptor for chitin, forming a chitin inducible complex with AtCERK1 to induce plant immunity.

Abstract: As the major mechanism of plant growth and morphogenesis, cell elongation is controlled by many hormonal and environmental signals. How these signals are coordinated at the molecular level to ensure coherent cellular responses remains unclear. In this study, we illustrate a molecular circuit that integrates all major growth-regulating signals, including auxin, brassinosteroid, gibberellin, light, and temperature. Analyses of genome-wide targets, genetic and biochemical interactions demonstrate that the auxin-response factor ARF6, the light/temperature-regulated transcription factor PIF4, and the brassinosteroid-signaling transcription factor BZR1, interact with each other and cooperatively regulate large numbers of common target genes, but their DNA-binding activities are blocked by the gibberellin-inactivated repressor RGA. In addition, a tripartite HLH/bHLH module feedback regulates PIFs and additional bHLH factors that interact with ARF6, and thereby modulates auxin sensitivity according to developmental and environmental cues. Our results demonstrate a central growth-regulation circuit that integrates hormonal, environmental, and developmental controls of cell elongation in Arabidopsis hypocotyl.

Abstract: Deep transcriptome sequencing has revealed the existence of many transcripts that lack long or conserved open reading frames (ORFs) and which have been termed long non-coding RNAs (lncRNAs). The vast majority of lncRNAs are lineage-specific and do not yet have a known function. In this study, we test the hypothesis that they may act as a repository for the synthesis of new peptides. We find that a large fraction of the lncRNAs expressed in cells from six different species is associated with ribosomes. The patterns of ribosome protection are consistent with the translation of short peptides. lncRNAs show similar coding potential and sequence constraints than evolutionary young protein coding sequences, indicating that they play an important role in de novo protein evolution.

Abstract: Recent sequencing studies have extensively explored the somatic alterations present in the nuclear genomes of cancers. Although mitochondria control energy metabolism and apoptosis, the origins and impact of cancer-associated mutations in mtDNA are unclear. In this study, we analyzed somatic alterations in mtDNA from 1675 tumors. We identified 1907 somatic substitutions, which exhibited dramatic replicative strand bias, predominantly C > T and A > G on the mitochondrial heavy strand. This strand-asymmetric signature differs from those found in nuclear cancer genomes but matches the inferred germline process shaping primate mtDNA sequence content. A number of mtDNA mutations showed considerable heterogeneity across tumor types. Missense mutations were selectively neutral and often gradually drifted towards homoplasmy over time. In contrast, mutations resulting in protein truncation undergo negative selection and were almost exclusively heteroplasmic. Our findings indicate that the endogenous mutational mechanism has far greater impact than any other external mutagens in mitochondria and is fundamentally linked to mtDNA replication.

Abstract: Though every cell in the body is surrounded by a membrane, there are a number of ways that molecules can pass through this membrane to either enter or leave the cell. Proteins from the GIRK family form channels in the membranes of mammalian cells, and when open these channels allow potassium ions to flow through the membrane to control the membrane's voltage. GIRK channels are found in the heart and in the central nervous system, and can be activated in a variety of ways. Sodium ions and molecules called ‘signaling lipids’ can regulate the activation of GIRK channels. These channels can also be caused to open by G proteins: proteins that are found inside cells and that help to transmit signals from the outside of a cell to the inside. Three G proteins—called Gα, Gβ, and Gγ—work together in a complex that functions a bit like a switch. When switched on, the Gα subunit is separated from the other two subunits (called Gβγ); and both parts can then activate different signaling pathways inside the cell. The Gβγ subunits and a signaling lipid have been known to regulate the opening of GIRK channels for a number of years, but these events have only been studied in the context of living cells. The specific role of each molecule, and whether the Gα subunit can also regulate the GIRK channels, remains unknown. Now Wang et al. have produced one type of mouse GIRK channel, called GIRK2, in yeast cells, purified this protein, and added it into an artificial membrane. This ‘reconstituted system’ allowed the regulation of a GIRK channel to be investigated under more controlled conditions than in previous experiments. Wang et al. found that the Gβγ subunits and the signaling lipid both need to be present to activate the GIRK2 channel. Sodium ions were not essential, but promoted further opening when Gβγ and the signaling lipid were already present. When locked in its ‘on’ state, the Gα subunit had no effect on GIRK2, but adding Gα locked in the ‘off’ state closed these channels by removing the Gβγ proteins. The findings of Wang et al. suggest that it should be possible to use a similar reconstituted system to investigate what allows different G proteins to activate specific signaling pathways.

Abstract: Mineral malnutrition stemming from undiversified plant-based diets is a top global challenge. In C3 plants (e.g., rice, wheat), elevated concentrations of atmospheric carbon dioxide (eCO2) reduce protein and nitrogen concentrations, and can increase the total non-structural carbohydrates (TNC; mainly starch, sugars). However, contradictory findings have obscured the effect of eCO2 on the ionome—the mineral and trace-element composition—of plants. Consequently, CO2-induced shifts in plant quality have been ignored in the estimation of the impact of global change on humans. This study shows that eCO2 reduces the overall mineral concentrations (−8%, 95% confidence interval: −9.1 to −6.9, p carbon:minerals in C 3 plants. The meta-analysis of 7761 observations, including 2264 observations at state of the art FACE centers, covers 130 species/cultivars. The attained statistical power reveals that the shift is systemic and global. Its potential to exacerbate the prevalence of 'hidden hunger' and obesity is discussed.

Abstract: The widely accepted model of G1 cell cycle progression proposes that cyclin D:Cdk4/6 inactivates the Rb tumor suppressor during early G1 phase by progressive multi-phosphorylation, termed hypo-phosphorylation, to release E2F transcription factors. However, this model remains unproven biochemically and the biologically active form(s) of Rb remains unknown. In this study, we find that Rb is exclusively mono-phosphorylated in early G1 phase by cyclin D:Cdk4/6. Mono-phosphorylated Rb is composed of 14 independent isoforms that are all targeted by the E1a oncoprotein, but show preferential E2F binding patterns. At the late G1 Restriction Point, cyclin E:Cdk2 inactivates Rb by quantum hyper-phosphorylation. Cells undergoing a DNA damage response activate cyclin D:Cdk4/6 to generate mono-phosphorylated Rb that regulates global transcription, whereas cells undergoing differentiation utilize un-phosphorylated Rb. These observations fundamentally change our understanding of G1 cell cycle progression and show that mono-phosphorylated Rb, generated by cyclin D:Cdk4/6, is the only Rb isoform in early G1 phase.

Abstract: Every year, seasonal influenza, commonly called flu, infects up to one in five people around the world, and causes up to half a million deaths. Even though the human immune system can detect and destroy the virus that causes influenza, people can catch flu many times throughout their lifetimes because the virus keeps evolving in an effort to avoid the immune system. This antigenic drift—so-called because the antigens displayed by the virus keep changing—also explains why influenza vaccines become less effective over time and need to be reformulated every year. It is possible to determine which antigens are displayed by a new strain of the virus by observing how blood samples that respond to known strains respond to the new strain. This information about the “antigenic phenotype” of the virus can be plotted on an antigenic map in which strains with similar antigens cluster together. Gene sequencing has shown that there are four subtypes of the flu virus that commonly infect people; but the relationship between changes in antigenic phenotype and changes in gene sequences of the influenza virus is poorly understood. Bedford et al. have now developed an approach to combine antigenic maps with genetic information about the four subtypes of the human flu virus. This revealed that the antigenic phenotype of H3N2—a subtype that is becoming increasingly common—evolved faster than the other three subtypes. Further, a correlation was observed between antigenic drift and the number of new influenza cases per year for each flu strain. This suggests that knowing which antigenic phenotypes are present at the start of flu season could help predict which strains of the virus will predominate later on. The work of Bedford et al. provides a useful framework to study influenza, and could help to pinpoint which changes in viral genes cause the changes in antigens. This information could potentially speed up the development of new flu vaccines for each flu season.

Abstract: For a gene to be expressed as a protein, its DNA is first used as a template to make a molecule of RNA, which is then translated to make the protein. In most cells, RNA molecules concentrate into aggregates called RNA granules. These granules contain both RNA and proteins that bind to RNA and are used to transport, store, and regulate the translation and breakdown of RNA molecules. Unlike many other structures within cells, RNA granules are not surrounded by a membrane; and the molecules that hold RNA granules together are not known. P granules are a type of RNA granule that is found in the germ cells (the cells that go on to form eggs and sperm) of a microscopic worm called C. elegans. When a C. elegans embryo is still a single cell, P granules move throughout the cell and the P granules at the front of the cell dissolve, while those at the back condense. As such, when the single-celled embryo divides, the front forms a cell without P granules (that will go on to form the tissues of the worm's body) and the back becomes a P granule-containing germ cell. Two proteins called MBK-2 and PPTR-1 have opposite effects on P granules: MBK-2 causes P granules to dissolve, while PPTR-1 makes them form. MBK-2 is an enzyme that adds phosphate groups onto other proteins, whereas PPTR-1 is part of an enzyme that removes such groups. Wang et al. have now searched for proteins that interact with MBK-2 and PPTR-1 in order to identify the molecules that regulate the assembly of P granules. They found that a group of proteins, known as MEG proteins, are acted upon by both of these proteins. Wang et al. found that MBK-2 adds phosphate groups to MEG proteins, which encourages granules to disassemble, while PPTR-1 removes these groups to promote granule assembly. Wang et al. generated mutant worms that lacked each of the MEG proteins. These mutant worms had fewer and smaller P granules than normal worms. Without MEG proteins, P granules failed to assemble or disassemble normally and the worms were infertile. Using high resolution microscopy, Wang et al. observed that the MEG proteins wrap around the P granules and that one of the MEG proteins—called MEG-3—follows an almost ribbon-like path that surrounds and enters each granule. These observations suggest that the MEG proteins stabilize RNA granules by forming a cage-like scaffold around each granule. How the MEG proteins—which are predicted to lack a fixed or ordered three-dimensional structure and show no similarity to proteins with known functions—assemble into a scaffold will be the focus of future studies.

Abstract: Over the course of billions of years, natural evolution has produced new proteins and adapted existing ones so that they work better. Scientists have learned how to use the principles that underlie evolution to similarly engineer proteins in the laboratory. This process, known as directed evolution, is a powerful tool for improving how proteins function. Directed evolution normally involves mutating the gene that encodes the protein of interest, selecting the genes that produce the most promising proteins for another round of mutation, and repeating the process until the desired protein function is achieved. In the first step of directed evolution, a gene is usually mutated randomly in order to create a large ‘library’ of different forms of the gene. These are joined to circular pieces of DNA known as plasmids that can replicate themselves inside cells. However, the number of plasmids than can be taken up differs from cell to cell. This complicates experiments, and the ideal directed evolution experiment would have the same number of plasmids, or target genes, being delivered into each cell. Ryan et al. have developed a new method for performing directed evolution experiments that uses a recently developed technique called the CRISPR-Cas9 system. This can make direct changes to a DNA strand such as inserting or deleting specific sequences that code for proteins. Ryan et al. used the CRISPR-Cas9 system to create multiple DNA breaks simultaneously across the genome of yeast cells, and joined ‘barcoded’ DNA or DNA for intact genes to these breaks. This avoids the need to use plasmids to introduce foreign DNA into cells. Ryan et al. have named this method the Multiplex CRISPR (or CRISPRm) system. Having established CRISPRm, Ryan et al. tested whether it could be used to engineer improved proteins by attempting to modify a transporter protein called CDT-1. This protein transports the sugar cellobiose into yeast cells, where it can be converted into alcohol by fermentation. This is important for making biofuel from plants. After just one round of directed evolution using CRISPRm, Ryan et al. successfully isolated a form of the CDT-1 protein that increased the rate of fermentation over 10-fold; hence this CDT-1 variant could be used to increase biofuel production. In the future, it will be important to implement multiple selection rounds with CRISPRm, and to test how large the DNA libraries can be for directed evolution. In time, CRISPRm could find use in evolving and engineering different combinations of genes, metabolic pathways, and possibly entire genomes.

Abstract: In electron cryo-microscopy (cryo-EM), the electron beam that is used for imaging also causes the sample to move. This motion blurs the images and limits the resolution attainable by single-particle analysis. In a previous Research article (Bai et al., 2013) we showed that correcting for this motion by processing movies from fast direct-electron detectors allowed structure determination to near-atomic resolution from 35,000 ribosome particles. In this Research advance article, we show that an improved movie processing algorithm is applicable to a much wider range of specimens. The new algorithm estimates straight movement tracks by considering multiple particles that are close to each other in the field of view, and models the fall-off of high-resolution information content by radiation damage in a dose-dependent manner. Application of the new algorithm to four data sets illustrates its potential for significantly improving cryo-EM structures, even for particles that are smaller than 200 kDa.

Abstract: Malaria inflicts an enormous burden on global human health. The emergence of parasite resistance to front-line drugs has prompted a renewed focus on the repositioning of clinically approved drugs as potential anti-malarial therapies. Antibiotics that inhibit protein translation are promising candidates for repositioning. We have solved the cryo-EM structure of the cytoplasmic ribosome from the human malaria parasite, Plasmodium falciparum, in complex with emetine at 3.2 A resolution. Emetine is an anti-protozoan drug used in the treatment of ameobiasis that also displays potent anti-malarial activity. Emetine interacts with the E-site of the ribosomal small subunit and shares a similar binding site with the antibiotic pactamycin, thereby delivering its therapeutic effect by blocking mRNA/tRNA translocation. As the first cryo-EM structure that visualizes an antibiotic bound to any ribosome at atomic resolution, this establishes cryo-EM as a powerful tool for screening and guiding the design of drugs that target parasite translation machinery.

Abstract: The extracellular matrix (ECM) is a major component of tumors and a significant contributor to cancer progression. In this study, we use proteomics to investigate the ECM of human mammary carcinoma xenografts and show that primary tumors of differing metastatic potential differ in ECM composition. Both tumor cells and stromal cells contribute to the tumor matrix and tumors of differing metastatic ability differ in both tumor- and stroma-derived ECM components. We define ECM signatures of poorly and highly metastatic mammary carcinomas and these signatures reveal up-regulation of signaling pathways including TGFβ and VEGF. We further demonstrate that several proteins characteristic of highly metastatic tumors (LTBP3, SNED1, EGLN1, and S100A2) play causal roles in metastasis, albeit at different steps. Finally we show that high expression of LTBP3 and SNED1 correlates with poor outcome for ER(-)/PR(-)breast cancer patients. This study thus identifies novel biomarkers that may serve as prognostic and diagnostic tools. DOI: http://dx.doi.org/10.7554/eLife.01308.001.

Abstract: The surfaces of plants are covered in epithelial cells that come in many different shapes, suggesting that individual cells must have some control over their own shape. An unusually shaped epithelial cell is the pavement cell, which looks like a jigsaw puzzle piece and is found in the leaves of many flowering plants. Relatively little was known about the exact contribution of mechanical properties of the wall to this shape. Furthermore, although it was known that parts of pavement cells are rich in microtubules—tubes of protein that act as a scaffold inside the cell— the possibility that shape impacts the behavior of microtubules was not fully addressed. Now, using a combination of computer modelling and experiments, Sampathkumar et al. reveal that the shape of the pavement cells relies in part on the response of the microtubules to stress. In an individual cell, microtubules align along the direction of the largest stress, with a protein severing those microtubules that are not aligned in this direction. As the stress inside a cell is determined in part by the cell’s shape, this sets up a feedback loop: the stress resulting from the cell shape aligns the microtubules that reinforce the cell wall, thus maintaining the shape of the cell. An external stress applied to the epithelium can override this internal stress. Because all of the plant cells are under turgor pressure from the inside, pressure from the outside, like squeezing a balloon, changes the stress pattern, causing the realignment of the microtubules so as to resist the new stress. This shows that the microtubules respond to local stresses within a cell, and are continually responsive to stress changes.

Abstract: Although a small number of the vast array of animal long non-coding RNAs (lncRNAs) have known effects on cellular processes examined in vitro, the extent of their contributions to normal cell processes throughout development, differentiation and disease for the most part remains less clear. Phenotypes arising from deletion of an entire genomic locus cannot be unequivocally attributed either to the loss of the lncRNA per se or to the associated loss of other overlapping DNA regulatory elements. The distinction between cis- or trans-effects is also often problematic. We discuss the advantages and challenges associated with the current techniques for studying the in vivo function of lncRNAs in the light of different models of lncRNA molecular mechanism, and reflect on the design of experiments to mutate lncRNA loci. These considerations should assist in the further investigation of these transcriptional products of the genome.

Abstract: To produce a protein a cell must first transcribe the DNA in a gene to make a messenger RNA molecule, which is then translated to make the protein. However, cells also produce other types of RNA molecules which do not become proteins. MicroRNAs, for example, regulate the expression of genes as proteins, while the role of other RNA molecules called long non-coding RNAs (lncRNAs) is not well understood. Now Krawczyk and Emerson have found an lncRNA that controls a gene called COX-2 that is often implicated in breast, colon, prostate, and lung cancer. This RNA molecule, which is called PACER, originates near the start of the COX-2 gene, but it cannot be messenger RNA because it does not contain the instructions to make the COX-2 protein, and it is too long to be a microRNA. Further experiments showed that the newly discovered lncRNA activates the expression of the COX-2 gene. Krawczyk and Emerson found that PACER attracts enzymes that spotlight genes that need to be activated, thus increasing the transcription of these genes to make messenger RNA. Genes can also be switched on and off by various molecules binding to nearby DNA, and PACER encourages the activation of COX-2 by keeping away the molecules that might switch it off. In addition to shedding new light on the role of lncRNAs, these results suggest that PACER could be a suitable therapeutic target in cancers that involve the COX-2 gene.

Abstract: During translation elongation, the ribosome ratchets along its mRNA template, incorporating each new amino acid and translocating from one codon to the next. The elongation cycle requires dramatic structural rearrangements of the ribosome. We show here that deep sequencing of ribosome-protected mRNA fragments reveals not only the position of each ribosome but also, unexpectedly, its particular stage of the elongation cycle. Sequencing reveals two distinct populations of ribosome footprints, 28-30 nucleotides and 20-22 nucleotides long, representing translating ribosomes in distinct states, differentially stabilized by specific elongation inhibitors. We find that the balance of small and large footprints varies by codon and is correlated with translation speed. The ability to visualize conformational changes in the ribosome during elongation, at single-codon resolution, provides a new way to study the detailed kinetics of translation and a new probe with which to identify the factors that affect each step in the elongation cycle.DOI: http://dx.doi.org/10.7554/eLife.01257.001.

Abstract: Thousands of small Open Reading Frames (smORFs) with the potential to encode small peptides of fewer than 100 amino acids exist in our genomes. However, the number of smORFs actually translated, and their molecular and functional roles are still unclear. In this study, we present a genome-wide assessment of smORF translation by ribosomal profiling of polysomal fractions in Drosophila. We detect two types of smORFs bound by multiple ribosomes and thus undergoing productive translation. The 'longer' smORFs of around 80 amino acids resemble canonical proteins in translational metrics and conservation, and display a propensity to contain transmembrane motifs. The 'dwarf' smORFs are in general shorter (around 20 amino-acid long), are mostly found in 5'-UTRs and non-coding RNAs, are less well conserved, and have no bioinformatic indicators of peptide function. Our findings indicate that thousands of smORFs are translated in metazoan genomes, reinforcing the idea that smORFs are an abundant and fundamental genome component.

Abstract: Cells are enclosed by a plasma membrane that is made of lipid molecules and proteins. Almost half of the lipid molecules in the plasma membranes of animal cells (including human cells) are cholesterol molecules. Since cholesterol helps to keep the membrane stable, its level in the plasma membrane is tightly regulated. Cholesterol is produced within animal cells, but it can also be taken up from outside the cell, primarily from low density lipoprotein (or LDL for short). When ingested LDL is broken down inside a cell, most of the cholesterol molecules are added to the plasma membrane, but some end up in the membrane of an organelle inside the cell called the endoplasmic reticulum. The amount of cholesterol in the membrane of the endoplasmic reticulum regulates the activation of a protein called SREBP, a transcription factor that is attached to this membrane. If the level of cholesterol becomes too low, this transcription factor travels to the cell nucleus, where it switches on the genes that cause the cell to produce more cholesterol and also to take up more LDL from the environment. When the amount of cholesterol in the membrane is high enough, the SREBP protein remains attached to the endoplasmic reticulum, which reduces the production of cholesterol and the uptake of LDL. Here, Das et al. study the movement of cholesterol molecules between the plasma membrane and the membrane of the endoplasmic reticulum by using a toxin that binds to membranes that are rich in cholesterol. These experiments showed that the plasma membrane contains three different types or ‘pools’ of cholesterol. Das et al. found that only one of these pools is ‘labile’: it grows when there is an excess of LDL, and shrinks when cholesterol is running low in the cell. Furthermore, excess cholesterol is first added to this labile pool in the plasma membrane before it is added to the pool in the endoplasmic reticulum. This suggests that the production, uptake, and breakdown of cholesterol are all controlled by partitioning this molecule between the labile pool in the plasma membrane and the endoplasmic reticulum. The next challenge is to determine how the three pools of cholesterol in the plasma membrane are maintained, and what regulates the distribution of cholesterol between the endoplasmic reticulum and the labile pool in the plasma membrane.

Abstract: A single, low dose of the NMDA receptor antagonist ketamine produces rapid antidepressant actions in treatment-resistant depressed patients. Understanding the cellular mechanisms underlying this will lead to new therapies for treating major depression. NMDARs are heteromultimeric complexes formed through association of two GluN1 and two GluN2 subunits. We show that in vivo deletion of GluN2B, only from principal cortical neurons, mimics and occludes ketamine's actions on depression-like behavior and excitatory synaptic transmission. Furthermore, ketamine-induced increases in mTOR activation and synaptic protein synthesis were mimicked and occluded in 2BΔCtx mice. We show here that cortical GluN2B-containing NMDARs are uniquely activated by ambient glutamate to regulate levels of excitatory synaptic transmission. Together these data predict a novel cellular mechanism that explains ketamine's rapid antidepressant actions. In this model, basal glutamatergic neurotransmission sensed by cortical GluN2B-containing NMDARs regulates excitatory synaptic strength in PFC determining basal levels of depression-like behavior.

Abstract: During infection, CD8(+) T cells initially expand then contract, leaving a small memory pool providing long lasting immunity. While it has been described that CD8(+) T cell memory formation becomes defective in old age, the cellular mechanism is largely unknown. Autophagy is a major cellular lysosomal degradation pathway of bulk material, and levels are known to fall with age. In this study, we describe a novel role for autophagy in CD8(+) T cell memory formation. Mice lacking the autophagy gene Atg7 in T cells failed to establish CD8(+) T cell memory to influenza and MCMV infection. Interestingly, autophagy levels were diminished in CD8(+) T cells from aged mice. We could rejuvenate CD8(+) T cell responses in elderly mice in an autophagy dependent manner using the compound spermidine. This study reveals a cell intrinsic explanation for poor CD8(+) T cell memory in the elderly and potentially offers novel immune modulators to improve aged immunity.