Search

eLife is changing its editorial process to emphasize public reviews and assessments of preprints by eliminating accept/reject decisions after peer review.

The Membrane Attack Complex (MAC or C5b-9) is an important effector of the immune system to kill invading microbes. MAC is formed when complement enzymes on the bacterial surface convert complement component C5 into C5b. Although the MAC is a membrane-inserted complex, soluble forms of MAC (sMAC, or terminal complement complex (TCC)) are often detected in sera of patients suffering from infections. Consequently, sMAC has been proposed as a biomarker, but it remains unclear when and how it is formed during infections. Here, we studied mechanisms of MAC formation on bacteria and found that sMAC is primarily formed in human serum by bacteria resistant to MAC-dependent killing. Surprisingly, C5 was converted into C5b more potently by MAC-resistant compared to MAC-sensitiveEscherichia colistrains. Both the increase in C5 conversion and sMAC generation were linked to the expression of lipopolysaccharide (LPS) O-Antigen in the bacterial outer membrane. In addition, we found that MAC precursors are released from the surface of MAC-resistant bacteria during MAC assembly. Release of MAC precursors from bacteria induced lysis of bystander human erythrocytes in the absence of other serum components. However, serum regulators vitronectin (Vn) and clusterin (Clu) can prevent this bystander lysis. Combining size exclusion chromatography with mass spectrometry profiling, we show that sMAC released from bacteria in serum is a heterogeneous mixture of complexes composed of C5b-8, up to 3 copies of C9 and multiple copies of Vn and Clu. Altogether, our data provide molecular insight into how sMAC is generated during bacterial infections. This fundamental knowledge could form the basis for exploring the use of sMAC as biomarker.

The major microtubule-organizing center (MTOC) in animal cells, the centrosome, comprises a pair of centrioles surrounded by pericentriolar material (PCM), which nucleates and anchors microtubules. Centrosome assembly depends on PCM binding to centrioles, PCM self-association and dynein­mediated PCM transport, but the self-assembly properties of PCM in interphase cells are poorly understood. Here, we used experiments and modeling to study centriole-independent features of interphase PCM assembly. We showed that when centrioles are lost due to PLK4 depletion or inhibition, dynein-based PCM transport and PCM self-clustering are sufficient to form a single compact MTOC, which generates a dense radial microtubule array. Interphase PCM self-assembly depends on γ-tubulin, pericentrin, CDK5RAP2 and ninein, but not NEDD1, CEP152 or CEP192. Formation of a compact acentriolar MTOC is inhibited by AKAP450-dependent PCM recruitment to the Golgi or by randomly organized CAMSAP2-stabilized microtubules, which keep PCM mobile and prevent its coalescence. Linking of CAMSAP2 to a minus-end-directed motor leads to the formation of an MTOC, but MTOC compaction requires cooperation with pericentrin-containing self-clustering PCM. Our data reveal that interphase PCM contains a set of components that can self-assemble into a compact structure and organize microtubules, but PCM self-organization is sensitive to motor-and microtubule-based rearrangement.

Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy that can be applied in both tissues and cells. In ExM, samples are embedded in a swellable polymer gel to physically expand the sample and isotropically increase resolution in x, y and z. The maximum resolution increase is limited by the expansion factor of the gel, which is four-fold for the original ExM protocol. Variations on the original ExM method have been reported that allow for greater expansion factors but at the cost of ease of adoption or versatility. Here, we systematically explore the ExM recipe space and present a novel method termed Ten-fold Robust Expansion Microscopy (TREx) that, like the original ExM method, requires no specialized equipment or procedures. We demonstrate that TREx gels expand ten-fold, can be handled easily, and can be applied to both thick mouse brain tissue sections and cultured human cells enabling high-resolution subcellular imaging with a single expansion step. Furthermore, we show that TREx can provide ultrastructural context to subcellular protein localization by combining antibody-stained samples with off-the-shelf small molecule stains for both total protein and membranes.

Research in many different areas of medicine will benefit from new approaches to peer review and publishing.

Peptidoglycan is an essential component of the bacterial cell envelope that surrounds the cytoplasmic membrane to protect the cell from osmotic lysis. Important antibiotics such as β-lactams and glycopeptides target peptidoglycan biosynthesis. Class A penicillin-binding proteins (PBPs) are bifunctional membrane-bound peptidoglycan synthases that polymerize glycan chains and connect adjacent stem peptides by transpeptidation. How these enzymes work in their physiological membrane environment is poorly understood. Here, we developed a novel Förster resonance energy transfer-based assay to follow in real time both reactions of class A PBPs reconstituted in liposomes or supported lipid bilayers and applied this assay with PBP1B homologues from Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii in the presence or absence of their cognate lipoprotein activator. Our assay will allow unravelling the mechanisms of peptidoglycan synthesis in a lipid-bilayer environment and can be further developed to be used for high-throughput screening for new antimicrobials.

The human naive T-cell receptor (TCR) repertoire is extremely diverse and accurately estimating its distribution is challenging. We address this challenge by combining a quantitative sequencing protocol of TCRA and TCRB sequences with computational modelling. We observed the vast majority of TCR chains only once in our samples, confirming the enormous diversity of the naive repertoire. However, a substantial number of sequences were observed multiple times within samples, and we demonstrated that this is due to expression by many cells in the naive pool. We reason that α and β chains are frequently observed due to a combination of selective processes and summation over multiple clones expressing these chains. We test the contribution of both mechanisms by predicting samples from phenomenological and mechanistically modelled repertoire distributions. By comparing these with sequencing data, we show that frequently observed chains are likely to be derived from multiple clones. Still, a neutral model of T-cell homeostasis cannot account for the observed distributions. We conclude that the data are only compatible with distributions of many small clones in combination with a sufficient number of very large naive T-cell clones, the latter most likely as a result of peripheral selection.

Linking interindividual differences in psychological phenotype to variations in brain structure is an old dream for psychology and a crucial question for cognitive neurosciences. Yet, replicability of the previously-reported ‘structural brain behavior’ (SBB)-associations has been questioned, recently. Here, we conducted an empirical investigation, assessing replicability of SBB among heathy adults. For a wide range of psychological measures, the replicability of associations with gray matter volume was assessed. Our results revealed that among healthy individuals 1) finding an association between performance at standard psychological tests and brain morphology is relatively unlikely 2) significant associations, found using an exploratory approach, have overestimated effect sizes and 3) can hardly be replicated in an independent sample. After considering factors such as sample size and comparing our findings with more replicable SBB-associations in a clinical cohort and replicable associations between brain structure and non-psychological phenotype, we discuss the potential causes and consequences of these findings., eLife digest All human brains share the same basic structure. But no two brains are exactly alike. Brain scans can reveal differences between people in the organization and activity of individual brain regions. Studies have suggested that these differences give rise to variation in personality, intelligence and even political preferences. But recent attempts to replicate some of these findings have failed, questioning the existence of such a direct link, specifically between brain structure and human behavior. This had led some disagreements whether there is a general replication crisis in psychology, or if the replication studies themselves are flawed. Kharabian Masouleh et al. have now used brain scans from hundreds of healthy volunteers from an already available dataset to try to resolve the issue. The volunteers had previously completed several psychological tests. These measured cognitive and behavioral aspects such as attention, memory, anxiety and personality traits. Kharabian Masouleh et al. performed more than 10,000 analyzes on their dataset to look for relationships between brain structure and psychological traits. But the results revealed very few statistically significant relationships. Moreover, the relationships that were identified proved difficult to replicate in independent samples. By contrast, the same analyzes demonstrated robust links between brain structure and memory in patients with Alzheimer's disease. They also showed connections between brain structure and non-psychological traits, such as age. This confirms that the analysis techniques do work. So why did the new study find so few relationships between brain structure and psychological traits, when so many links have been reported previously? One possibility is publication bias. Researchers and journals may be more likely to publish positive findings than negative ones. Another factor could be that that most studies use too few participants to be able to reliably detect relationships between brain structure and behavior, and that studies with 200 to 300 participants are still too small. Therefore, future studies should use samples with many hundreds of participants, or more. This will be possible if more groups make their data available for others to analyze. Researchers and journals must also be more willing to publish negative findings. This will help provide an accurate view of relationships between brain structure and behavior.

Theoretical and empirical advances have revealed the importance of biodiversity for stabilizing ecosystem functions through time. Despite the global degradation of soils, whether the loss of soil microbial diversity can destabilize ecosystem functioning is poorly understood. Here, we experimentally quantified the contribution of soil fungal and bacterial communities to the temporal stability of four key ecosystem functions related to biogeochemical cycling. Microbial diversity enhanced the temporal stability of all ecosystem functions and this pattern was particularly strong in plant-soil mesocosms with reduced microbial richness where over 50% of microbial taxa were lost. The stabilizing effect of soil biodiversity was linked to asynchrony among microbial taxa whereby different soil fungi and bacteria promoted different ecosystem functions at different times. Our results emphasize the need to conserve soil biodiversity for the provisioning of multiple ecosystem functions that soils provide to the society.

VRC01 broadly neutralizing antibodies (bnAbs) target the CD4-binding site (CD4BS) of the human immunodeficiency virus-1 (HIV-1) envelope glycoprotein (Env). Unlike mature antibodies, corresponding VRC01 germline precursors poorly bind to Env. Immunogen design has mostly relied on glycan removal from trimeric Env constructs and has had limited success in eliciting mature VRC01 bnAbs. To better understand elicitation of such bnAbs, we characterized the inferred germline precursor of VRC01 in complex with a modified trimeric 426c Env by cryo-electron microscopy and a 426c gp120 core by X-ray crystallography, biolayer interferometry, immunoprecipitation, and glycoproteomics. Our results show VRC01 germline antibodies interacted with a wild-type 426c core lacking variable loops 1–3 in the presence and absence of a glycan at position Asn276, with the latter form binding with higher affinity than the former. Interactions in the presence of an Asn276 oligosaccharide could be enhanced upon carbohydrate shortening, which should be considered for immunogen design.

The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas, including a commitment to open data, code, and community.

Computational modelling of the heart tube during development reveals the interplay between tissue asymmetry and growth that helps our hearts take shape.

The neural crest is a dynamic progenitor cell population that arises at the border of neural and non-neural ectoderm. The inductive roles of FGF, Wnt, and BMP at the neural plate border are well established, but the signals required for subsequent neural crest development remain poorly characterized. Here, we conducted a screen in primary zebrafish embryo cultures for chemicals that disrupt neural crest development, as read out by crestin:EGFP expression. We found that the natural product caffeic acid phenethyl ester (CAPE) disrupts neural crest gene expression, migration, and melanocytic differentiation by reducing Sox10 activity. CAPE inhibits FGF-stimulated PI3K/Akt signaling, and neural crest defects in CAPE-treated embryos are suppressed by constitutively active Akt1. Inhibition of Akt activity by constitutively active PTEN similarly decreases crestin expression and Sox10 activity. Our study has identified Akt as a novel intracellular pathway required for neural crest differentiation.

Site-specific histone ubiquitylation plays a central role in orchestrating the response to DNA double-strand breaks (DSBs). DSBs elicit a cascade of events controlled by the ubiquitin ligase RNF168, which promotes the accumulation of repair factors such as 53BP1 and BRCA1 on the chromatin flanking the break site. RNF168 also promotes its own accumulation, and that of its paralog RNF169, but how they recognize ubiquitylated chromatin is unknown. Using methyl-TROSY solution NMR spectroscopy and molecular dynamics simulations, we present an atomic resolution model of human RNF169 binding to a ubiquitylated nucleosome, and validate it by electron cryomicroscopy. We establish that RNF169 binds to ubiquitylated H2A-Lys13/Lys15 in a manner that involves its canonical ubiquitin-binding helix and a pair of arginine-rich motifs that interact with the nucleosome acidic patch. This three-pronged interaction mechanism is distinct from that by which 53BP1 binds to ubiquitylated H2A-Lys15 highlighting the diversity in site-specific recognition of ubiquitylated nucleosomes. DOI: http://dx.doi.org/10.7554/eLife.23872.001, eLife digest Inside cells, genetic information is encoded by molecules of DNA. It is important for a cell to quickly identify and repair any damage to DNA to prevent harmful changes in the genetic information. In humans and other animals failures in DNA repair can lead to cancer and other diseases. A molecule of DNA is made of two strands that twist together to form a double helix. Most of the DNA in an animal cell is organised by proteins called histones. Groups of eight histones are wrapped with DNA to form structures called nucleosomes. If both strands of a DNA double helix break in the same place, this leads to a molecule called ubiquitin being attached to a histone called H2A within a nucleosome to mark the position of the damage. This promotes DNA repair by attracting another protein called RNF169 to bind to the nucleosome. The precise location of the ubiquitin molecule on histone H2A is important because ubiquitin molecules act as signals for a variety of different processes when attached to specific positions on histones and other proteins. For example, ubiquitin molecules attached to some sites on histones can alter how the cell uses the genetic information contained within the nucleosome. However, it is not clear how the number and precise locations of ubiquitins on histones can produce such different signals. Kitevski-LeBlanc, Fradet-Turcotte et al. investigated why RNF169 is only attracted to nucleosomes when ubiquitin is attached to a particular site on histone H2A following damage to DNA. The experiments reveal that two regions of RNF169 known as arginine motifs play an important role in controlling when the protein binds to nucleosomes. These arginine motifs – which are next to the region of the protein that binds to ubiquitin – identify the position of the ubiquitin on H2A by making contact with an “acidic” patch on the surface of the nucleosome. These findings show that the combination of RNF169 binding to both the ubiquitin on H2A and an acidic patch on the nucleosome ensure that this protein only promotes DNA repair when and where it is needed. This acidic patch is involved in regulating the binding of various other proteins to nucleosomes. Understanding how cells interpret the signals produced by ubiquitin binding to proteins will help us to understand how disrupting these signals can contribute to cancer and other diseases. DOI: http://dx.doi.org/10.7554/eLife.23872.002

Almost all critical functions in cells rely on specific protein–protein interactions. Understanding these is therefore crucial in the investigation of biological systems. Despite all past efforts, we still lack a thorough understanding of the energetics of association of proteins. Here, we introduce a new and simple approach to predict binding affinity based on functional and structural features of the biological system, namely the network of interfacial contacts. We assess its performance against a protein–protein binding affinity benchmark and show that both experimental methods used for affinity measurements and conformational changes have a strong impact on prediction accuracy. Using a subset of complexes with reliable experimental binding affinities and combining our contacts and contact-types-based model with recent observations on the role of the non-interacting surface in protein–protein interactions, we reach a high prediction accuracy for such a diverse dataset outperforming all other tested methods. DOI: http://dx.doi.org/10.7554/eLife.07454.001, eLife digest Proteins help to copy DNA, transport nutrients and perform many other important roles in cells. To perform these tasks, proteins often interact with other proteins and work together. These interactions can be very complex because each protein has a three-dimensional shape that may change when it binds to other proteins. Also, two proteins may form several connections with each other. It is possible to carry out experiments to calculate how likely it is that two proteins will physically interact with each other and how strong their connections will be. However, these measurements are time consuming and costly to do. Some researchers have developed computer models to help predict the interactions between proteins, but these models are often incorrect because they leave out some of the chemical or physical properties that influence the ability of proteins to interact. With the aim of making a better model, Vangone and Bonvin examined 122 different combinations of proteins whose abilities to interact had previously been experimentally measured. Vangone and Bonvin found that the number of connections between each pair of proteins was a strong predictor of how tightly the proteins connect to each other. Particular features of the surface of the proteins—specifically, the region defined as the non-interacting surface—can also influence how strong the interaction is. Vangone and Bonvin used this information to develop a new model that predicts how tightly proteins interact with each other based on the number of connections between the two proteins and the characteristics of the non-interacting surface. The model is simple, and Vangone and Bonvin show that it is more accurate than previous models. Defects in the interactions between proteins can lead to many diseases in humans, so this model may be useful for the development of new drugs to treat these conditions. DOI: http://dx.doi.org/10.7554/eLife.07454.002

Regenerative therapy for degenerative spine disorders requires the identification of cells that can slow down and possibly reverse degenerative processes. Here, we identify an unanticipated wound-specific notochord sheath cell subpopulation that expresses Wilms Tumor (WT) 1b following injury in zebrafish. We show that localized damage leads to Wt1b expression in sheath cells, and that wt1b+cells migrate into the wound to form a stopper-like structure, likely to maintain structural integrity. Wt1b+sheath cells are distinct in expressing cartilage and vacuolar genes, and in repressing a Wt1b-p53 transcriptional programme. At the wound, wt1b+and entpd5+ cells constitute separate, tightly-associated subpopulations. Surprisingly, wt1b expression at the site of injury is maintained even into adult stages in developing vertebrae, which form in an untypical manner via a cartilage intermediate. Given that notochord cells are retained in adult intervertebral discs, the identification of novel subpopulations may have important implications for regenerative spine disorder treatments.