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We have combined chemical biology and genetic modification approaches to investigate the importance of protein myristoylation in the human malaria parasite, Plasmodium falciparum. Parasite treatment during schizogony in the last 10 to 15 hours of the erythrocytic cycle with IMP-1002, an inhibitor of N-myristoyl transferase (NMT), led to a significant blockade in parasite egress from the infected erythrocyte. Two rhoptry proteins were mislocalized in the cell, suggesting that rhoptry function is disrupted. We identified 16 NMT substrates for which myristoylation was significantly reduced by NMT inhibitor (NMTi) treatment, and, of these, 6 proteins were substantially reduced in abundance. In a viability screen, we showed that for 4 of these proteins replacement of the N-terminal glycine with alanine to prevent myristoylation had a substantial effect on parasite fitness. In detailed studies of one NMT substrate, glideosome-associated protein 45 (GAP45), loss of myristoylation had no impact on protein location or glideosome assembly, in contrast to the disruption caused by GAP45 gene deletion, but GAP45 myristoylation was essential for erythrocyte invasion. Therefore, there are at least 3 mechanisms by which inhibition of NMT can disrupt parasite development and growth: early in parasite development, leading to the inhibition of schizogony and formation of “pseudoschizonts,” which has been described previously; at the end of schizogony, with disruption of rhoptry formation, merozoite development and egress from the infected erythrocyte; and at invasion, when impairment of motor complex function prevents invasion of new erythrocytes. These results underline the importance of P. falciparum NMT as a drug target because of the pleiotropic effect of its inhibition., Understanding the essential factors needed for malaria parasite development could help us find new therapeutic targets. This study reveals that N-myristoylation is a posttranslational modification of proteins essential for the parasites’ growth and their invasion of red blood cells.

Mitigating the threat of insecticide resistance in African malaria vector populations requires comprehensive information about where resistance occurs, to what degree, and how this has changed over time. Estimating these trends is complicated by the sparse, heterogeneous distribution of observations of resistance phenotypes in field populations. We use 6,423 observations of the prevalence of resistance to the most important vector control insecticides to inform a Bayesian geostatistical ensemble modelling approach, generating fine-scale predictive maps of resistance phenotypes in mosquitoes from the Anopheles gambiae complex across Africa. Our models are informed by a suite of 111 predictor variables describing potential drivers of selection for resistance. Our maps show alarming increases in the prevalence of resistance to pyrethroids and DDT across sub-Saharan Africa from 2005 to 2017, with mean mortality following insecticide exposure declining from almost 100% to less than 30% in some areas, as well as substantial spatial variation in resistance trends., Malaria control efforts are threatened by the emergence of insecticide resistance in mosquito vector populations. This study provides fine-resolution maps of insecticide resistance levels in malaria vectors for large parts of Sub-Saharan Africa; the maps quantify recent spatial trends in resistance and can assist in the implementation of resistance management practices.

The Amazon is Brazil’s greatest natural resource and invaluable to the rest of the world as a buffer against climate change. The recent election of Brazil’s president brought disputes over development plans for the region back into the spotlight. Historically, the development model for the Amazon has focused on exploitation of natural resources, resulting in environmental degradation, particularly deforestation. Although considerable attention has focused on the long-term global cost of “losing the Amazon,” too little attention has focused on the emergence and reemergence of vector-borne diseases that directly impact the local population, with spillover effects to other neighboring areas. We discuss the impact of Amazon development models on human health, with a focus on vector-borne disease risk. We outline policy actions that could mitigate these negative impacts while creating opportunities for environmentally sensitive economic activities., This Perspective article maintains that a development model for the Amazon must be bold, creative, carefully planned, and sustainable, explicitly acknowledging the importance of public health in reinforcing economic development, environmental protection, and land use change.

Autophagy-related protein 8 (ATG8) is a highly conserved ubiquitin-like protein that modulates autophagy pathways by binding autophagic membranes and a number of proteins, including cargo receptors and core autophagy components. Throughout plant evolution, ATG8 has expanded from a single protein in algae to multiple isoforms in higher plants. However, the degree to which ATG8 isoforms have functionally specialized to bind distinct proteins remains unclear. Here, we describe a comprehensive protein–protein interaction resource, obtained using in planta immunoprecipitation (IP) followed by mass spectrometry (MS), to define the potato ATG8 interactome. We discovered that ATG8 isoforms bind distinct sets of plant proteins with varying degrees of overlap. This prompted us to define the biochemical basis of ATG8 specialization by comparing two potato ATG8 isoforms using both in vivo protein interaction assays and in vitro quantitative binding affinity analyses. These experiments revealed that the N-terminal β-strand—and, in particular, a single amino acid polymorphism—underpins binding specificity to the substrate PexRD54 by shaping the hydrophobic pocket that accommodates this protein’s ATG8-interacting motif (AIM). Additional proteomics experiments indicated that the N-terminal β-strand shapes the broader ATG8 interactor profiles, defining interaction specificity with about 80 plant proteins. Our findings are consistent with the view that ATG8 isoforms comprise a layer of specificity in the regulation of selective autophagy pathways in plants., ATG8 is a ubiquitin-like protein involved in autophagy pathways. Biochemical characterization of the expanded ATG8 gene family in plants revealed an additional layer of specificity in selective autophagy, determined by ATG8’s N-terminal β-strand.

Human immunity influences the evolution and impact of influenza strains. Because individuals are infected with multiple influenza strains during their lifetime, and each virus can generate a cross-reactive antibody response, it is challenging to quantify the processes that shape observed immune responses or to reliably detect recent infection from serological samples. Using a Bayesian model of antibody dynamics at multiple timescales, we explain complex cross-reactive antibody landscapes by inferring participants’ histories of infection with serological data from cross-sectional and longitudinal studies of influenza A/H3N2 in southern China and Vietnam. We find that individual-level influenza antibody profiles can be explained by a short-lived, broadly cross-reactive response that decays within a year to leave a smaller long-term response acting against a narrower range of strains. We also demonstrate that accounting for dynamic immune responses alongside infection history can provide a more accurate alternative to traditional definitions of seroconversion for the estimation of infection attack rates. Our work provides a general model for quantifying aspects of influenza immunity acting at multiple timescales based on contemporary serological data and suggests a two-armed immune response to influenza infection consistent with competitive dynamics between B cell populations. This approach to analysing multiple timescales for antigenic responses could also be applied to other multistrain pathogens such as dengue and related flaviviruses., Author summary It is challenging to determine the true extent of influenza infection and immunity within a population, because a person’s immune response to a specific influenza strain depends both on past infections with that strain as well as immunity generated by related influenza strains. To untangle these processes, we developed a mathematical model that considered individual histories of influenza infection and immune dynamics acting at multiple timescales. We combined this model with surveys of antibody levels in different individuals, showing how antibody dynamics are influenced by a short-lived, broadly cross-reactive response against a wide range of strains that wanes over time to leave a long-term response against a narrower collection of strains. By accounting for such short- and long-term responses, we also found that it was possible to obtain better estimates of the frequency of influenza infection. These methods could help to guide the design of studies to estimate key aspects of influenza immune dynamics or to estimate historical infection rates and would also be applicable to other pathogens with multiple strains.

Adaptive immunity relies on the generation and maintenance of memory T cells to provide protection against repeated antigen exposure. It has been hypothesised that a self-renewing population of T cells, named stem cell–like memory T (TSCM) cells, are responsible for maintaining memory. However, it is not clear if the dynamics of TSCM cells in vivo are compatible with this hypothesis. To address this issue, we investigated the dynamics of TSCM cells under physiological conditions in humans in vivo using a multidisciplinary approach that combines mathematical modelling, stable isotope labelling, telomere length analysis, and cross-sectional data from vaccine recipients. We show that, unexpectedly, the average longevity of a TSCM clone is very short (half-life < 1 year, degree of self-renewal = 430 days): far too short to constitute a stem cell population. However, we also find that the TSCM population is comprised of at least 2 kinetically distinct subpopulations that turn over at different rates. Whilst one subpopulation is rapidly replaced (half-life = 5 months) and explains the rapid average turnover of the bulk TSCM population, the half-life of the other TSCM subpopulation is approximately 9 years, consistent with the longevity of the recall response. We also show that this latter population exhibited a high degree of self-renewal, with a cell residing without dying or differentiating for 15% of our lifetime. Finally, although small, the population was not subject to excessive stochasticity. We conclude that the majority of TSCM cells are not stem cell–like but that there is a subpopulation of TSCM cells whose dynamics are compatible with their putative role in the maintenance of T cell memory., Author summary The human immune system remembers previously encountered pathogens so that, on meeting the same pathogen a second time, the response is quicker and more effective. This immune memory is the basis of all vaccinations. Immune memory persists for decades, but how memory is maintained is unclear. It has been hypothesised that there is a dedicated population of cells called stem cell–like memory T (TSCM) cells that have stem cell–like behaviour and are responsible for the persistence of T cell memory. Here, we show that a subset of TSCM cells, in healthy humans in vivo, have the dynamic properties of self-renewal and clonal longevity necessary to maintain long-lived immune memory.

The mechanical unfolding of proteins is a cellular mechanism for force transduction with potentially broad implications in cell fate. Despite this, the mechanism by which protein unfolding elicits differential downstream signalling pathways remains poorly understood. Here, we used protein engineering, atomic force microscopy, and biophysical tools to delineate how protein unfolding controls cell mechanics. Deleted in liver cancer 1 (DLC1) is a negative regulator of Ras homolog family member A (RhoA) and cell contractility that regulates cell behaviour when localised to focal adhesions bound to folded talin. Using a talin mutant resistant to force-induced unfolding of R8 domain, we show that talin unfolding determines DLC1 downstream signalling and, consequently, cell mechanics. We propose that this new mechanism of mechanotransduction may have implications for a wide variety of associated cellular processes., Author summary Mechano-induced conformational changes and the unfolding of protein domains are cornerstones of mechanotransduction and regulate the interaction of proteins with other molecules. Talin is a prominent molecule in focal adhesions and one of the few proteins that simultaneously connects integrin receptors in the cell membrane with the actin cytoskeleton. This bridging position, owing to the cytoskeleton’s contractile nature, exposes talin to forces along its length. In this work, we studied the implications of the R8 domain unfolding in the downstream activity of deleted in liver cancer 1 (DLC1), which binds the talin R8 domain and negatively regulates Ras homolog family member A (RhoA). We created a talin mutant with the R8 domain resistant to mechanical unfolding and observed that cells expressing these talin mutants have altered patterns of focal adhesion dynamics and lower levels of actomyosin contraction. This leads to decreased traction forces and diminished cell migration. We propose a novel force-controlled molecular switch that refines the mechanism of talin-mediated focal adhesion activation, providing negative feedback during focal adhesion maturation. The broader effects of this talin-mediated mechanism need to be elucidated, as it might regulate multiple cellular events.

The use of consumer-grade wearables for purposes beyond fitness tracking has not been comprehensively explored. We generated and analyzed multidimensional data from 233 normal volunteers, integrating wearable data, lifestyle questionnaires, cardiac imaging, sphingolipid profiling, and multiple clinical-grade cardiovascular and metabolic disease markers. We show that subjects can be stratified into distinct clusters based on daily activity patterns and that these clusters are marked by distinct demographic and behavioral patterns. While resting heart rates (RHRs) performed better than step counts in being associated with cardiovascular and metabolic disease markers, step counts identified relationships between physical activity and cardiac remodeling, suggesting that wearable data may play a role in reducing overdiagnosis of cardiac hypertrophy or dilatation in active individuals. Wearable-derived activity levels can be used to identify known and novel activity-modulated sphingolipids that are in turn associated with insulin sensitivity. Our findings demonstrate the potential for wearables in biomedical research and personalized health.

We present ethoscopes, machines for high-throughput analysis of behaviour inDrosophilaand other animals. Ethoscopes provide a software and hardware solution that is reproducible and easily scalable; they perform, in real-time, tracking and profiling of behaviour using a supervised machine learning algorithm; they can deliver behaviourally-triggered stimuli to flies in a feedback-loop mode; and they are highly customisable and open source. Ethoscopes can be built easily using 3D printing technology and rely on Raspberry Pi microcomputers and Arduino boards to provide affordable and flexible hardware. All software and construction specifications are available athttp://lab.gilest.ro/ethoscope.

The transition from single-cell to multicellular behavior is important in early development but rarely studied. The starvation-induced aggregation of the social amoeba Dictyostelium discoideum into a multicellular slug is known to result from single-cell chemotaxis towards emitted pulses of cyclic adenosine monophosphate (cAMP). However, how exactly do transient, short-range chemical gradients lead to coherent collective movement at a macroscopic scale? Here, we developed a multiscale model verified by quantitative microscopy to describe behaviors ranging widely from chemotaxis and excitability of individual cells to aggregation of thousands of cells. To better understand the mechanism of long-range cell—cell communication and hence aggregation, we analyzed cell—cell correlations, showing evidence of self-organization at the onset of aggregation (as opposed to following a leader cell). Surprisingly, cell collectives, despite their finite size, show features of criticality known from phase transitions in physical systems. By comparing wild-type and mutant cells with impaired aggregation, we found the longest cell—cell communication distance in wild-type cells, suggesting that criticality provides an adaptive advantage and optimally sized aggregates for the dispersal of spores., A multiscale model and imaging data show that cells of the slime mold Dictyostelium discoideum maximize their cell—cell communication range during aggregation by a critical-like state known from phase transitions in physical systems., Author summary Cells are often coupled to each other in cell collectives, such as aggregates during early development, tissues in the developed organism, and tumors in disease. How do cells communicate over macroscopic distances much larger than the typical cell—cell distance to decide how they should behave? Here, we developed a multiscale model of social amoeba, spanning behavior from individuals to thousands of cells. We show that local cell—cell coupling via secreted chemicals may be tuned to a critical value, resulting in emergent long-range communication and heightened sensitivity. Hence, these aggregates are remarkably similar to bacterial biofilms and neuronal networks, all communicating in a pulselike fashion. Similar organizing principles may also aid our understanding of the remarkable robustness in cancer development.

Biological systems are subject to inherent stochasticity. Nevertheless, development is remarkably robust, ensuring the consistency of key phenotypic traits such as correct cell numbers in a certain tissue. It is currently unclear which genes modulate phenotypic variability, what their relationship is to core components of developmental gene networks, and what is the developmental basis of variable phenotypes. Here, we start addressing these questions using the robust number of Caenorhabditis elegans epidermal stem cells, known as seam cells, as a readout. We employ genetics, cell lineage tracing, and single molecule imaging to show that mutations in lin-22, a Hes-related basic helix-loop-helix (bHLH) transcription factor, increase seam cell number variability. We show that the increase in phenotypic variability is due to stochastic conversion of normally symmetric cell divisions to asymmetric and vice versa during development, which affect the terminal seam cell number in opposing directions. We demonstrate that LIN-22 acts within the epidermal gene network to antagonise the Wnt signalling pathway. However, lin-22 mutants exhibit cell-to-cell variability in Wnt pathway activation, which correlates with and may drive phenotypic variability. Our study demonstrates the feasibility to study phenotypic trait variance in tractable model organisms using unbiased mutagenesis screens., Author summary Organisms are exposed to both internal and external perturbations in every molecular process they go through, and robustness—the ability to maintain their systems unchanged—is crucial for their development and survival. However, the processes that keep the variability of cells as low as possible are barely known. The nematode C. elegans is notable for its highly reproducible development, showing an almost invariant pattern of cell division and differentiation during development; it is thus an ideal model organism in which to search for genes that regulate phenotypic consistency among genetically identical individuals. We focus on a group of lateral epidermal cells—the seam cells—which undergo stem cell-like divisions during postembryonic development. These divisions can either be symmetric towards the seam cell fate, acting to increase the total number of cells, or asymmetric, giving rise to one daughter cell that differentiates into its final fate and another one that serves to keep the number of seam cells constant. We show here that mutations in the transcription factor lin-22 increase seam cell number variability due to stochastic conversion of symmetric divisions into asymmetric ones and vice versa during development, thereby altering the number of terminal seam cell number in opposing directions. We also show that the observed phenotypic variability correlates with the stochastic activation of the conserved Wnt signaling pathway. Our work suggests that core components of developmental gene networks modulate phenotypic variability in multicellular animals.

The glucagon-like peptide-1 receptor (GLP-1R), a key pharmacological target in type 2 diabetes (T2D) and obesity, undergoes rapid endocytosis after stimulation by endogenous and therapeutic agonists. We have previously highlighted the relevance of this process in fine-tuning GLP-1R responses in pancreatic beta cells to control insulin secretion. In the present study, we demonstrate an important role for the translocation of active GLP-1Rs into liquid-ordered plasma membrane nanodomains, which act as hotspots for optimal coordination of intracellular signaling and clathrin-mediated endocytosis. This process is dynamically regulated by agonist binding through palmitoylation of the GLP-1R at its carboxyl-terminal tail. Biased GLP-1R agonists and small molecule allosteric modulation both influence GLP-1R palmitoylation, clustering, nanodomain signaling, and internalization. Downstream effects on insulin secretion from pancreatic beta cells indicate that these processes are relevant to GLP-1R physiological actions and might be therapeutically targetable., Nanodomain segregation and clustering of the glucagon-like peptide-1 receptor, a key target for type 2 diabetes therapy, is regulated by agonist binding, leading to compartmentalization of downstream signaling and clathrin-dependent internalization and impacting pancreatic beta cell responses.

© The Author(s)., Posttranscriptional modifications in transfer RNA (tRNA) are often critical for normal development because they adapt protein synthesis rates to a dynamically changing microenvironment. However, the precise cellular mechanisms linking the extrinsic stimulus to the intrinsic RNA modification pathways remain largely unclear. Here, we identified the cytosine-5 RNA methyltransferase NSUN2 as a sensor for external stress stimuli. Exposure to oxidative stress efficiently repressed NSUN2, causing a reduction of methylation at specific tRNA sites. Using metabolic profiling, we showed that loss of tRNA methylation captured cells in a distinct catabolic state. Mechanistically, loss of NSUN2 altered the biogenesis of tRNA-derived noncoding fragments (tRFs) in response to stress, leading to impaired regulation of protein synthesis. The intracellular accumulation of a specific subset of tRFs correlated with the dynamic repression of global protein synthesis. Finally, NSUN2-driven RNA methylation was functionally required to adapt cell cycle progression to the early stress response. In summary, we revealed that changes in tRNA methylation profiles were sufficient to specify cellular metabolic states and efficiently adapt protein synthesis rates to cell stress., Authors received funding from Cancer Research UK - Senior Cancer Research Fellowship (C10701/A15181) and Cancer Research UK - PhD student stipend (C10701/A16134), and European Research Council - Consolidator Grant (m5C; 310360)

Despite the broad-spectrum antimicrobial activities of silver, its internal usage is restricted, owing to the toxicity. Strategies to enhance its efficacy are highly desirable but rely heavily on the understanding of its molecular mechanism of action. However, up to now, no direct silver-targeting proteins have been mined at a proteome-wide scale, which hinders systemic studies on the biological pathways interrupted by silver. Herein, we build up a unique system, namely liquid chromatography gel electrophoresis inductively coupled plasma mass spectrometry (LC-GE-ICP-MS), allowing 34 proteins directly bound by silver ions to be identified in Escherichia coli. By using integrated omic approaches, including metalloproteomics, metabolomics, bioinformatics, and systemic biology, we delineated the first dynamic antimicrobial actions of silver (Ag+) in E. coli, i.e., it primarily damages multiple enzymes in glycolysis and tricarboxylic acid (TCA) cycle, leading to the stalling of the oxidative branch of the TCA cycle and an adaptive metabolic divergence to the reductive glyoxylate pathway. It then further damages the adaptive glyoxylate pathway and suppresses the cellular oxidative stress responses, causing systemic damages and death of the bacterium. To harness these novel findings, we coadministrated metabolites involved in the Krebs cycles with Ag+ and found that they can significantly potentiate the efficacy of silver both in vitro and in an animal model. Our study reveals the comprehensive and dynamic mechanisms of Ag+ toxicity in E. coli cells and offers a novel and general approach for deciphering molecular mechanisms of metallodrugs in various pathogens and cells to facilitate the development of new therapeutics., The antimicrobial properties of silver are poorly understood. Tracking of the silver(I) proteome in Escherichia coli, in combination with metabolomics, bioinformatics and bioassays, delineates the dynamic antimicrobial actions of silver at a molecular level.

The association between species richness and ecosystem energy availability is one of the major geographic trends in biodiversity. It is often explained in terms of energetic constraints, such that coexistence among competing species is limited in low productivity environments. However, it has proven challenging to reject alternative views, including the null hypothesis that species richness has simply had more time to accumulate in productive regions, and thus the role of energetic constraints in limiting coexistence remains largely unknown. We use the phylogenetic relationships and geographic ranges of sister species (pairs of lineages who are each other’s closest extant relatives) to examine the association between energy availability and coexistence across an entire vertebrate class (Aves). We show that the incidence of coexistence among sister species increases with overall species richness and is elevated in more productive ecosystems, even when accounting for differences in the evolutionary time available for coexistence to occur. Our results indicate that energy availability promotes species coexistence in closely related lineages, providing a key step toward a more mechanistic understanding of the productivity–richness relationship underlying global gradients in biodiversity., An analysis of phylogenetic and geographic data across the world's bird species reveals that the probability of species coexistence is constrained by the availability of energy in the environment., Author Summary The increase in the number of species with the availability of energy in the environment is one of the most general but least understood patterns in global biodiversity. The finite amount of energy flowing through an ecosystem has long been suspected to place a fundamental constraint on the ability of species to subdivide ecological resources, with greater energy availability—and, thus, ecosystem productivity—in the tropics, potentially facilitating increased levels of coexistence. However, empirical support for this hypothesis has been lacking, raising the possibility that richness is higher in productive ecosystems for largely historical reasons, including the greater geological age and area of tropical biomes, which increases the period of time available for diversity to accumulate. By combining phylogenetic and geographic data from across the world’s bird species, we show that greater ecosystem productivity is associated with an increased probability of coexistence among closely related lineages and that this pattern contributes to the higher species richness in the tropics. Our results confirm that contemporary gradients in species richness are fundamentally shaped by energetic constraints on coexistence.

The cell envelope of gram-negative bacteria, a structure comprising an outer (OM) and an inner (IM) membrane, is essential for life. The OM and the IM are separated by the periplasm, a compartment that contains the peptidoglycan. The OM is tethered to the peptidoglycan via the lipoprotein, Lpp. However, the importance of the envelope’s multilayered architecture remains unknown. Here, when we removed physical coupling between the OM and the peptidoglycan, cells lost the ability to sense defects in envelope integrity. Further experiments revealed that the critical parameter for the transmission of stress signals from the envelope to the cytoplasm, where cellular behaviour is controlled, is the IM-to-OM distance. Augmenting this distance by increasing the length of the lipoprotein Lpp destroyed signalling, whereas simultaneously increasing the length of the stress-sensing lipoprotein RcsF restored signalling. Our results demonstrate the physiological importance of the size of the periplasm. They also reveal that strict control over the IM-to-OM distance is required for effective envelope surveillance and protection, suggesting that cellular architecture and the structure of transenvelope protein complexes have been evolutionarily co-optimised for correct function. Similar strategies are likely at play in cellular compartments surrounded by 2 concentric membranes, such as chloroplasts and mitochondria., Author summary Antibiotic resistance is one of the greatest threats that humanity faces today. In particular, the emergence of multidrug-resistant gram-negative bacteria has become a pressing issue. Infections caused by resistant gram-negative bacteria are indeed difficult to treat because of the presence of a double-membraned envelope that renders these bacteria less permeable to many antibiotics. Bacteria invest a great deal in protecting their envelope. They have evolved complex stress-sensing systems that allow them to monitor envelope integrity in order to detect and fix damage when it occurs. Here, using the model bacterium Escherichia coli, we show that the ability to sense and respond to stress depends on the architecture of the cell envelope, in general, and on the distance between the 2 membranes, in particular. Thus, searching for drugs targeting envelope architecture is an attractive strategy for new antibacterials. In addition, our work reveals the physiological function of the Braun’s lipoprotein (Lpp), the numerically most abundant protein in E. coli and the first lipoprotein that was discovered several decades ago.

Coastal marine ecosystems can be managed by actions undertaken both on the land and in the ocean. Quantifying and comparing the costs and benefits of actions in both realms is therefore necessary for efficient management. Here, we quantify the link between terrestrial sediment runoff and a downstream coastal marine ecosystem and contrast the cost-effectiveness of marine- and land-based conservation actions. We use a dynamic land- and sea-scape model to determine whether limited funds should be directed to 1 of 4 alternative conservation actions—protection on land, protection in the ocean, restoration on land, or restoration in the ocean—to maximise the extent of light-dependent marine benthic habitats across decadal timescales. We apply the model to a case study for a seagrass meadow in Australia. We find that marine restoration is the most cost-effective action over decadal timescales in this system, based on a conservative estimate of the rate at which seagrass can expand into a new habitat. The optimal decision will vary in different social–ecological contexts, but some basic information can guide optimal investments to counteract land- and ocean-based stressors: (1) marine restoration should be prioritised if the rates of marine ecosystem decline and expansion are similar and low; (2) marine protection should take precedence if the rate of marine ecosystem decline is high or if the adjacent catchment is relatively intact and has a low rate of vegetation decline; (3) land-based actions are optimal when the ratio of marine ecosystem expansion to decline is greater than 1:1.4, with terrestrial restoration typically the most cost-effective action; and (4) land protection should be prioritised if the catchment is relatively intact but the rate of vegetation decline is high. These rules of thumb illustrate how cost-effective conservation outcomes for connected land–ocean systems can proceed without complex modelling., Author summary Many coastal marine ecosystems are threatened by anthropogenic activities, but often, the best way to restore and protect these important ecosystems is unclear. Conventional wisdom suggests that the 2 most effective conservation actions to benefit coastal marine ecosystems are implementation of marine protected areas or, alternatively, reduction of land-based threats. Active marine restoration is typically considered a low-priority option, in part due to high costs and low success rates. But does this conventional wisdom hold up to closer scrutiny? We developed a model to ask: should we restore or protect, on either the land or in the ocean, to maximise the extent of coastal marine ecosystems? We based the model on seagrass meadows and adjacent catchments in Queensland, Australia. Surprisingly, we found that direct, active marine restoration can be the most cost-effective approach to maximising extent of marine ecosystems over longer (decades-long) timescales. There is, however, substantial uncertainty in our understanding of the dynamics of complex linked land–sea ecosystems. Further, geomorphological and ecological conditions vary geographically. Therefore, we also used the model to investigate how uncertainty in key parameters affects decision-making outcomes. Our results can be used to guide investment into coastal marine conservation in the absence of complex, region-specific modelling.

[This corrects the article DOI: 10.1371/journal.pbio.2001855.].

Super-resolution 3D imaging reveals remodeling of the cortical actin meshwork at the natural killer cell immune synapse, which is likely to be important for secretion of lytic granules., Natural Killer (NK) cells are innate immune cells that secrete lytic granules to directly kill virus-infected or transformed cells across an immune synapse. However, a major gap in understanding this process is in establishing how lytic granules pass through the mesh of cortical actin known to underlie the NK cell membrane. Research has been hampered by the resolution of conventional light microscopy, which is too low to resolve cortical actin during lytic granule secretion. Here we use two high-resolution imaging techniques to probe the synaptic organisation of NK cell receptors and filamentous (F)-actin. A combination of optical tweezers and live cell confocal microscopy reveals that microclusters of NKG2D assemble into a ring-shaped structure at the centre of intercellular synapses, where Vav1 and Grb2 also accumulate. Within this ring-shaped organisation of NK cell proteins, lytic granules accumulate for secretion. Using 3D-structured illumination microscopy (3D-SIM) to gain super-resolution of ∼100 nm, cortical actin was detected in a central region of the NK cell synapse irrespective of whether activating or inhibitory signals dominate. Strikingly, the periodicity of the cortical actin mesh increased in specific domains at the synapse when the NK cell was activated. Two-colour super-resolution imaging revealed that lytic granules docked precisely in these domains which were also proximal to where the microtubule-organising centre (MTOC) polarised. Together, these data demonstrate that remodelling of the cortical actin mesh occurs at the central region of the cytolytic NK cell immune synapse. This is likely to occur for other types of cell secretion and also emphasises the importance of emerging super-resolution imaging technology for revealing new biology., Author Summary Natural Killer (NK) cells are immune cells that can recognise and kill virus-infected and cancerous cells. This killing requires an intercellular contact —termed an immune synapse—between the NK cell and its target cell through which molecules can be delivered to trigger lysis. Reorganisation of the NK cell cytoskeleton is essential for the delivery and release at the synapse of granules containing the cytolytic molecules. Understanding precisely how the cytoskeleton is involved in these cytolytic events has been hampered by our inability to resolve cytoskeletal structure at immune synapses by conventional light microscopy. Very recent advances in imaging technology have now provided the resolving power to see previously undetectable cellular structures. Here, we have used 3D super-resolution imaging to observe the structure of the actin cytoskeleton at the NK immune synapse. We found that a dense mesh of actin underlies the immune synapse and that it is remodelled upon NK cell activation. Domains within the actin meshwork open up specifying where the lytic granules dock and also where the microtubule-organising centre moves towards. Thus, actin remodelling occurs at the immune synapse during secretion and this may be important for the regulation of lytic granule secretion.

In frog and fish embryos, SNW1 is a protein required for the spatio-temporal activity of BMP signaling necessary for neural plate border formation and specification of neural crest tissue., Bone morphogenetic protein (BMP) gradients provide positional information to direct cell fate specification, such as patterning of the vertebrate ectoderm into neural, neural crest, and epidermal tissues, with precise borders segregating these domains. However, little is known about how BMP activity is regulated spatially and temporally during vertebrate development to contribute to embryonic patterning, and more specifically to neural crest formation. Through a large-scale in vivo functional screen in Xenopus for neural crest fate, we identified an essential regulator of BMP activity, SNW1. SNW1 is a nuclear protein known to regulate gene expression. Using antisense morpholinos to deplete SNW1 protein in both Xenopus and zebrafish embryos, we demonstrate that dorsally expressed SNW1 is required for neural crest specification, and this is independent of mesoderm formation and gastrulation morphogenetic movements. By exploiting a combination of immunostaining for phosphorylated Smad1 in Xenopus embryos and a BMP-dependent reporter transgenic zebrafish line, we show that SNW1 regulates a specific domain of BMP activity in the dorsal ectoderm at the neural plate border at post-gastrula stages. We use double in situ hybridizations and immunofluorescence to show how this domain of BMP activity is spatially positioned relative to the neural crest domain and that of SNW1 expression. Further in vivo and in vitro assays using cell culture and tissue explants allow us to conclude that SNW1 acts upstream of the BMP receptors. Finally, we show that the requirement of SNW1 for neural crest specification is through its ability to regulate BMP activity, as we demonstrate that targeted overexpression of BMP to the neural plate border is sufficient to restore neural crest formation in Xenopus SNW1 morphants. We conclude that through its ability to regulate a specific domain of BMP activity in the vertebrate embryo, SNW1 is a critical regulator of neural plate border formation and thus neural crest specification., Author Summary A subset of cells in the ectoderm of vertebrate embryos becomes the neural crest, which contributes to the bones and cartilage of the adult face. The neural crest arises in a location between the epidermis, which becomes the future skin, and the neural plate, which becomes the future central nervous system. Through our studies in both frog and fish embryos, we have discovered that the protein SNW-domain containing protein 1 (SNW1) is absolutely essential for defining the edge of the neural plate, where neural crest forms. SNW-domain containing proteins have been implicated in a variety of nuclear activities ranging from transcriptional regulation and elongation to RNA splicing. We show that SNW1 functions upstream of bone morphogenetic protein (BMP) receptors to regulate BMP activity, and is necessary for the activity of the BMP signaling pathway at the neural plate border where the neural crest is specified. In the absence of SNW1, BMP activity is reduced in this region and neural crest cells are lost. Given that SNW1 family proteins are highly conserved from nematodes to humans, SNW1's BMP regulatory function is likely conserved in other animals.

The high-resolution structure of the rotor ring from alkaliphilic Bacillus pseudofirmus OF4 reveals a new type of ion binding in F1Fo-ATP synthases., We solved the crystal structure of a novel type of c-ring isolated from Bacillus pseudofirmus OF4 at 2.5 Å, revealing a cylinder with a tridecameric stoichiometry, a central pore, and an overall shape that is distinct from those reported thus far. Within the groove of two neighboring c-subunits, the conserved glutamate of the outer helix shares the proton with a bound water molecule which itself is coordinated by three other amino acids of outer helices. Although none of the inner helices contributes to ion binding and the glutamate has no other hydrogen bonding partner than the water oxygen, the site remains in a stable, ion-locked conformation that represents the functional state present at the c-ring/membrane interface during rotation. This structure reveals a new, third type of ion coordination in ATP synthases. It appears in the ion binding site of an alkaliphile in which it represents a finely tuned adaptation of the proton affinity during the reaction cycle., Author Summary Like the wind turbines that generate electricity, the F1Fo-ATP synthases are natural “ion turbines” each made up of a stator and a rotor that turns, when driven by a flow of ions, to generate the cell's energy supply of ATP. The Fo motor rotates by reversible binding and release of coupling ions that flow down the electrochemical ion gradient across the cytoplasmic cell membrane (in the case of bacteria) or intracellular organelle membranes (in the case of eukaryotic cells). Here, we present the structure of a rotor (c-)ring from a Bacillus species (B. pseudofirmus OF4) determined at high-resolution by X-ray crystallography. This bacterium prefers alkaline environments where the concentration of protons (H+) is lower outside than inside the cell – the inverse of the situation usually found in organisms that prefer neutral or acidic environments. The amino acid sequence of the protein subunits in this rotor, nevertheless, has features common to an important group of ATP synthases in organisms from bacteria to man. The structure reveals a new type of ion binding in which a protonated glutamate residue in the protein associates with a water molecule. This finding raises the possibility considered by Nobel laureate Paul Boyer several decades ago that a hydronium ion (a protonated water molecule, H3O+), rather than a proton alone, might be the coupling species that energizes ATP synthesis. Also, it demonstrates the finely tuned adaptation of ATP synthase rotor rings and their ion-binding sites to the specific requirements of different organisms.

Neuronal specification is often seen as a multistep process: earlier regulators confer broad neuronal identity and are followed by combinatorial codes specifying neuronal properties unique to specific subtypes. However, it is still unclear whether early regulators are re-deployed in subtype-specific combinatorial codes, and whether early patterning events act to restrict the developmental potential of postmitotic cells. Here, we use the differential peptidergic fate of two lineage-related peptidergic neurons in the Drosophila ventral nerve cord to show how, in a feedforward mechanism, earlier determinants become critical players in later combinatorial codes. Amongst the progeny of neuroblast 5–6 are two peptidergic neurons: one expresses FMRFamide and the other one expresses Nplp1 and the dopamine receptor DopR. We show the HLH gene collier functions at three different levels to progressively restrict neuronal identity in the 5–6 lineage. At the final step, collier is the critical combinatorial factor that differentiates two partially overlapping combinatorial codes that define FMRFamide versus Nplp1/DopR identity. Misexpression experiments reveal that both codes can activate neuropeptide gene expression in vast numbers of neurons. Despite their partially overlapping composition, we find that the codes are remarkably specific, with each code activating only the proper neuropeptide gene. These results indicate that a limited number of regulators may constitute a potent combinatorial code that dictates unique neuronal cell fate, and that such codes show a surprising disregard for many global instructive cues., By studying the differential peptidergic fate of two lineage-related neurons in theDrosophila ventral nerve cord, the authors provide deeper insights into how, in a feedforward mechanism, earlier developmental determinants become critical players in later combinatorial codes defining cell identity., Author Summary The nervous system contains a daunting number of different cell types, perhaps as many as 10,000 in mammals, far outnumbering regulatory genes in many animal species. Studies of the determinants of cell fate in many systems during the last decade have supported the conclusion that cell fate is not determined by any one regulatory gene, but results from the combinatorial action of several regulators. Many questions about the nature of such codes, however, remain. It is not known, for example, how complex such codes are or how they are established. It is also unclear whether they are confined in their action or if they act outside of their normal context. To address these outstanding issues, we have used two unique subsets of Drosophila neurons, identifiable by their specific expression of two different neuropeptide genes. We have identified two partially overlapping and relatively simple codes, consisting of four to seven regulators that act to specify these two cell types. Intriguingly, specification is achieved in a feedforward manner such that A activates B, followed by A/B activating C, and A/B/C activating D. Each code is surprisingly potent, and can ectopically activate neuropeptide gene expression in a variety of neurons, with a surprising disregard for many early patterning events.

Cohesion between sister chromatids is essential for their bi-orientation on mitotic spindles. It is mediated by a multisubunit complex called cohesin. In yeast, proteolytic cleavage of cohesin's alpha kleisin subunit at the onset of anaphase removes cohesin from both centromeres and chromosome arms and thus triggers sister chromatid separation. In animal cells, most cohesin is removed from chromosome arms during prophase via a separase-independent pathway involving phosphorylation of its Scc3-SA1/2 subunits. Cohesin at centromeres is refractory to this process and persists until metaphase, whereupon its alpha kleisin subunit is cleaved by separase, which is thought to trigger anaphase. What protects centromeric cohesin from the prophase pathway? Potential candidates are proteins, known as shugoshins, that are homologous to Drosophila MEI-S332 and yeast Sgo1 proteins, which prevent removal of meiotic cohesin complexes from centromeres at the first meiotic division. A vertebrate shugoshin-like protein associates with centromeres during prophase and disappears at the onset of anaphase. Its depletion by RNA interference causes HeLa cells to arrest in mitosis. Most chromosomes bi-orient on a metaphase plate, but precocious loss of centromeric cohesin from chromosomes is accompanied by loss of all sister chromatid cohesion, the departure of individual chromatids from the metaphase plate, and a permanent cell cycle arrest, presumably due to activation of the spindle checkpoint. Remarkably, expression of a version of Scc3-SA2 whose mitotic phosphorylation sites have been mutated to alanine alleviates the precocious loss of sister chromatid cohesion and the mitotic arrest of cells lacking shugoshin. These data suggest that shugoshin prevents phosphorylation of cohesin's Scc3-SA2 subunit at centromeres during mitosis. This ensures that cohesin persists at centromeres until activation of separase causes cleavage of its alpha kleisin subunit. Centromeric cohesion is one of the hallmarks of mitotic chromosomes. Our results imply that it is not an intrinsically stable property, because it can easily be destroyed by mitotic kinases, which are kept in check by shugoshin.

Pattern formation is essential in the development of higher eukaryotes. For example, in the Drosophila embryo, maternal morphogen gradients establish gap gene expression domain patterning along the anterior-posterior axis, through linkage with an elaborate gene network. To understand the evolution and behaviour of such systems better, it is important to establish the minimal determinants required for patterning. We have therefore engineered artificial transcription-translation networks that generate simple patterns, crudely analogous to the Drosophila gap gene system. The Drosophila syncytium was modelled using DNA-coated paramagnetic beads fixed by magnets in an artificial chamber, forming a gene expression network. Transient expression domain patterns were generated using various levels of network connectivity. Generally, adding more transcription repression interactions increased the “sharpness” of the pattern while reducing overall expression levels. An accompanying computer model for our system allowed us to search for parameter sets compatible with patterning. While it is clear that the Drosophila embryo is far more complex than our simplified model, several features of interest emerge. For example, the model suggests that simple diffusion may be too rapid for Drosophila-scale patterning, implying that sublocalisation, or “trapping,” is required. Second, we find that for pattern formation to occur under the conditions of our in vitro reaction-diffusion system, the activator molecules must propagate faster than the inhibitors. Third, adding controlled protease degradation to the system stabilizes pattern formation over time. We have reconstituted transcriptional pattern formation from purified substances, including phage RNA polymerases, ribonucleotides, and an eukaryotic translation extract. We anticipate that the system described here will be generally applicable to the study of any biological network with a spatial component., To understand how patterns are established during early development, these authors have created an artificial system to mimic aspects of the early Drosophila embryo

The evaluation of 160 meta-analyses of animal studies on potential treatments for neurological disorders reveals that the number of statistically significant results was too large to be true, suggesting biases., Animal studies generate valuable hypotheses that lead to the conduct of preventive or therapeutic clinical trials. We assessed whether there is evidence for excess statistical significance in results of animal studies on neurological disorders, suggesting biases. We used data from meta-analyses of interventions deposited in Collaborative Approach to Meta-Analysis and Review of Animal Data in Experimental Studies (CAMARADES). The number of observed studies with statistically significant results (O) was compared with the expected number (E), based on the statistical power of each study under different assumptions for the plausible effect size. We assessed 4,445 datasets synthesized in 160 meta-analyses on Alzheimer disease (n = 2), experimental autoimmune encephalomyelitis (n = 34), focal ischemia (n = 16), intracerebral hemorrhage (n = 61), Parkinson disease (n = 45), and spinal cord injury (n = 2). 112 meta-analyses (70%) found nominally (p≤0.05) statistically significant summary fixed effects. Assuming the effect size in the most precise study to be a plausible effect, 919 out of 4,445 nominally significant results were expected versus 1,719 observed (p, Author Summary Studies have shown that the results of animal biomedical experiments fail to translate into human clinical trials; this could be attributed either to real differences in the underlying biology between humans and animals, to shortcomings in the experimental design, or to bias in the reporting of results from the animal studies. We use a statistical technique to evaluate whether the number of published animal studies with “positive” (statistically significant) results is too large to be true. We assess 4,445 animal studies for 160 candidate treatments of neurological disorders, and observe that 1,719 of them have a “positive” result, whereas only 919 studies would a priori be expected to have such a result. According to our methodology, only eight of the 160 evaluated treatments should have been subsequently tested in humans. In summary, we judge that there are too many animal studies with “positive” results in the neurological disorder literature, and we discuss the reasons and potential remedies for this phenomenon.

Efficient extension of motor axons into the limb during development requires enhancement of BMP signaling responses by the ubiquitin ligase Ark2C., Little is known about extrinsic signals required for the advancement of motor neuron (MN) axons, which extend over long distances in the periphery to form precise connections with target muscles. Here we present that Rnf165 (Arkadia-like; Arkadia2; Ark2C) is expressed specifically in the nervous system and that its loss in mice causes motor innervation defects that originate during development and lead to wasting and death before weaning. The defects range from severe reduction of motor axon extension as observed in the dorsal forelimb to shortening of presynaptic branches of the phrenic nerve, as observed in the diaphragm. Molecular functional analysis showed that in the context of the spinal cord Ark2C enhances transcriptional responses of the Smad1/5/8 effectors, which are activated (phosphorylated) downstream of Bone Morphogenetic Protein (BMP) signals. Consistent with Ark2C-modulated BMP signaling influencing motor axons, motor pools in the spinal cord were found to harbor phosphorylated Smad1/5/8 (pSmad) and treatment of primary MN with BMP inhibitor diminished axon length. In addition, genetic reduction of BMP-Smad signaling in Ark2C +/− mice caused the emergence of Ark2C −/−-like dorsal forelimb innervation deficits confirming that enhancement of BMP-Smad responses by Ark2C mediates efficient innervation. Together the above data reveal an involvement of BMP-Smad signaling in motor axon advancement., Author Summary Motor neurons control movement via long axons that extend from the spinal cord to muscles as far as in distant limbs. Little is known about factors that regulate this extensive axonal growth in the periphery. Here we report that the ubiquitin ligase Ark2C (Arkadia2) is expressed in neurons and can serve to amplify neuronal responses to specific signals. We find that these signals belong to the Bone Morphogenetic Protein (BMP) family of secreted factors, which are highly expressed in the periphery and known to regulate the development of the limbs. Loss of Ark2C gene function in mice results in inefficient growth of motor axons to distant muscles, and we show that this process is regulated by BMP signaling. Ark2C targets BMP inhibitors for destruction, and therefore the presence of Ark2C helps to enhance BMP signaling, which in turn is necessary for the innervation of distal muscles. Our experiments reveal a previously unknown function of BMP in motor axon growth and describe a molecular mechanism for how axons and limbs coordinate their growth.

The authors demonstrate a connection between variability in the rate of transcription and differences in cellular mitochondrial content., Populations of genetically identical eukaryotic cells show significant cell-to-cell variability in gene expression. However, we lack a good understanding of the origins of this variation. We have found marked cell-to-cell variability in average cellular rates of transcription. We also found marked cell-to-cell variability in the amount of cellular mitochondrial mass. We undertook fusion studies that suggested that variability in transcription rate depends on small diffusible factors. Following this, in vitro studies showed that transcription rate has a sensitive dependence on [ATP] but not on the concentration of other nucleotide triphosphates (NTPs). Further experiments that perturbed populations by changing nutrient levels and available [ATP] suggested this connection holds in vivo. We found evidence that cells with higher mitochondrial mass, or higher total membrane potential, have a faster rate of transcription per unit volume of nuclear material. We also found evidence that transcription rate variability is substantially modulated by the presence of anti- or prooxidants. Daughter studies showed that a cause of variability in mitochondrial content is apparently stochastic segregation of mitochondria at division. We conclude by noting that daughters that stochastically inherit a lower mitochondrial mass than their sisters have relatively longer cell cycles. Our findings reveal a link between variability in energy metabolism and variability in transcription rate., Author Summary Though pairs of cells may have identical genes, they still show behavioural differences. These cell-to-cell differences may arise from variations in how genes are transcribed and translated by the cellular machinery. Identifying the origins of this variation is important as it helps us understand why genetically identical cells can show a range of responses to the environment. In this work, we measured the rate at which the genes yield transcripts in cultured human cells. We found marked cell-to-cell variability in average rates of transcription. This variability is related to mitochondrial content. Cells with a higher mitochondrial mass have a faster rate of transcription, and we show that part of this variability is due to the unequal distribution of mitochondria to daughter cells when cells divide. Additionally, we find that cells that inherit more mitochondria divide earlier. These findings make a connection between variability in transcript production and variability in cellular mitochondrial content.

Little is known about the molecular mechanisms and intrinsic factors that are responsible for the emergence of neuronal subtype identity. Several transcription factors that are expressed mainly in precursors of the ventral telencephalon have been shown to control neuronal specification, but it has been unclear whether subtype identity is also specified in these precursors, or if this happens in postmitotic neurons, and whether it involves the same or different factors. SOX1, an HMG box transcription factor, is expressed widely in neural precursors along with the two other SOXB1 subfamily members, SOX2 and SOX3, and all three have been implicated in neurogenesis. SOX1 is also uniquely expressed at a high level in the majority of telencephalic neurons that constitute the ventral striatum (VS). These neurons are missing in Sox1-null mutant mice. In the present study, we have addressed the requirement for SOX1 at a cellular level, revealing both the nature and timing of the defect. By generating a novel Sox1-null allele expressing β-galactosidase, we found that the VS precursors and their early neuronal differentiation are unaffected in the absence of SOX1, but the prospective neurons fail to migrate to their appropriate position. Furthermore, the migration of non-Sox1-expressing VS neurons (such as those expressing Pax6) was also affected in the absence of SOX1, suggesting that Sox1-expressing neurons play a role in structuring the area of the VS. To test whether SOX1 is required in postmitotic cells for the emergence of VS neuronal identity, we generated mice in which Sox1 expression was directed to all ventral telencephalic precursors, but to only a very few VS neurons. These mice again lacked most of the VS, indicating that SOX1 expression in precursors is not sufficient for VS development. Conversely, the few neurons in which Sox1 expression was maintained were able to migrate to the VS. In conclusion, Sox1 expression in precursors is not sufficient for VS neuronal identity and migration, but this is accomplished in postmitotic cells, which require the continued presence of SOX1. Our data also suggest that other SOXB1 members showing expression in specific neuronal populations are likely to play continuous roles from the establishment of precursors to their final differentiation., The mechanisms whereby neurons establish their final identity are largely unknown. This study shows that the transcription factor SOX1 plays an important part in this process.

How did evolution generate the extraordinary diversity of vertebrates on land? Zero species are known prior to ~380 million years ago, and more than 30,000 are present today. An expansionist model suggests this was achieved by large and unbounded increases, leading to substantially greater diversity in the present than at any time in the geological past. This model contrasts starkly with empirical support for constrained diversification in marine animals, suggesting different macroevolutionary processes on land and in the sea. We quantify patterns of vertebrate standing diversity on land during the Mesozoic–early Paleogene interval, applying sample-standardization to a global fossil dataset containing 27,260 occurrences of 4,898 non-marine tetrapod species. Our results show a highly stable pattern of Mesozoic tetrapod diversity at regional and local levels, underpinned by a weakly positive, but near-zero, long-term net diversification rate over 190 million years. Species diversity of non-flying terrestrial tetrapods less than doubled over this interval, despite the origins of exceptionally diverse extant groups within mammals, squamates, amphibians, and dinosaurs. Therefore, although speciose groups of modern tetrapods have Mesozoic origins, rates of Mesozoic diversification inferred from the fossil record are slow compared to those inferred from molecular phylogenies. If high speciation rates did occur in the Mesozoic, then they seem to have been balanced by extinctions among older clades. An apparent 4-fold expansion of species richness after the Cretaceous/Paleogene (K/Pg) boundary deserves further examination in light of potential taxonomic biases, but is consistent with the hypothesis that global environmental disturbances such as mass extinction events can rapidly adjust limits to diversity by restructuring ecosystems, and suggests that the gradualistic evolutionary diversification of tetrapods was punctuated by brief but dramatic episodes of radiation., A large-scale examination of the fossil record reveals slow diversification rates in tetrapods across the 190-million-year Mesozoic era, followed by a possible rapid and abrupt 4-fold increase in species richness after the Cretaceous-Paleogene mass extinction event., Author Summary Vertebrates invaded the land more than 360 million years ago. Since then, they diversified to more than 30,000 tetrapod species today, including birds, mammals, squamates, and amphibians. The fossil record provides our best window onto diversification across such long spans of time, but is unevenly sampled. Previous studies counted observed families of fossil tetrapods and supported an expansionist model, entailing large and unbounded diversity increases through time. We applied methods that correct for differences in sampling through time and space to a comprehensive species-level database of Mesozoic to early Cenozoic fossil tetrapods. We find strong evidence that tetrapod diversity increased during the Mesozoic, but that the long-term net rate of diversification was low; species richness only doubled or tripled over 190 million years. This is enigmatic because today’s high biodiversity could not have been realised at such a slow rate. Diversification rates must have been much higher during other intervals, or rapid diversification might have been concentrated during brief episodes such as the earliest Cenozoic. Patterns of diversification on geological timescales and their relationships to hypothesised drivers such as ecological opportunity and environmental volatility must receive renewed scrutiny if we are to understand how land vertebrates and other animals attained the high biodiversity seen today.