Discovery of highly neutralizing human antibodies targeting Pseudomonas aeruginosa.

Drug-resistant Pseudomonas aeruginosa (PA) poses an emerging threat to human health with urgent need for alternative therapeutic approaches. Here, we deciphered the B cell and antibody response to the virulence-associated type III secretion system (T3SS) in a cohort of patients chronically infected with PA. Single-cell analytics revealed a diverse B cell receptor repertoire directed against the T3SS needle-tip protein PcrV, enabling the production of monoclonal antibodies (mAbs) abrogating T3SS-mediated cytotoxicity. Mechanistic studies involving cryoelectron microscopy identified a surface-exposed C-terminal PcrV epitope as the target of highly neutralizing mAbs with broad activity against drug-resistant PA isolates. These anti-PcrV mAbs were as effective as treatment with conventional antibiotics in vivo. Our study reveals that chronically infected patients represent a source of neutralizing antibodies, which can be exploited as therapeutics against PA.

Filopodia In Vitro and In Vivo.

Filopodia are dynamic cell surface protrusions used for cell motility, pathogen infection, and tissue development. The molecular mechanisms determining how and where filopodia grow and retract need to integrate mechanical forces and membrane curvature with extracellular signaling and the broader state of the cytoskeleton. The involved actin regulatory machinery nucleates, elongates, and bundles actin filaments separately from the underlying actin cortex. The refined membrane and actin geometry of filopodia, importance of tissue context, high spatiotemporal resolution required, and high degree of redundancy all limit current models. New technologies are improving opportunities for functional insight, with reconstitution of filopodia in vitro from purified components, endogenous genetic modification, inducible perturbation systems, and the study of filopodia in multicellular environments. In this review, we explore recent advances in conceptual models of how filopodia form, the molecules involved in this process, and our latest understanding of filopodia in vitro and in vivo.

Thioredoxin is a metabolic rheostat controlling regulatory B cells.

Metabolic programming is important for B cell fate, but the bioenergetic requirement for regulatory B (Breg) cell differentiation and function is unknown. Here we show that Breg cell differentiation, unlike non-Breg cells, relies on mitochondrial electron transport and homeostatic levels of reactive oxygen species (ROS). Single-cell RNA sequencing analysis revealed that TXN, encoding the metabolic redox protein thioredoxin (Trx), is highly expressed by Breg cells, unlike Trx inhibitor TXNIP which was downregulated. Pharmacological inhibition or gene silencing of TXN resulted in mitochondrial membrane depolarization and increased ROS levels, selectively suppressing Breg cell differentiation and function while favoring pro-inflammatory B cell differentiation. Patients with systemic lupus erythematosus (SLE), characterized by Breg cell deficiencies, present with B cell mitochondrial membrane depolarization, elevated ROS and fewer Trx+ B cells. Exogenous Trx stimulation restored Breg cells and mitochondrial membrane polarization in SLE B cells to healthy B cell levels, indicating Trx insufficiency underlies Breg cell impairment in patients with SLE.

RTN4/NoGo-receptor binding to BAI adhesion-GPCRs regulates neuronal development.

RTN4-binding proteins were widely studied as "NoGo" receptors, but their physiological interactors and roles remain elusive. Similarly, BAI adhesion-GPCRs were associated with numerous activities, but their ligands and functions remain unclear. Using unbiased approaches, we observed an unexpected convergence: RTN4 receptors are high-affinity ligands for BAI adhesion-GPCRs. A single thrombospondin type 1-repeat (TSR) domain of BAIs binds to the leucine-rich repeat domain of all three RTN4-receptor isoforms with nanomolar affinity. In the 1.65 Å crystal structure of the BAI1/RTN4-receptor complex, C-mannosylation of tryptophan and O-fucosylation of threonine in the BAI TSR-domains creates a RTN4-receptor/BAI interface shaped by unusual glycoconjugates that enables high-affinity interactions. In human neurons, RTN4 receptors regulate dendritic arborization, axonal elongation, and synapse formation by differential binding to glial versus neuronal BAIs, thereby controlling neural network activity. Thus, BAI binding to RTN4/NoGo receptors represents a receptor-ligand axis that, enabled by rare post-translational modifications, controls development of synaptic circuits.

Neuromodulator Signaling Bidirectionally Controls Vesicle Numbers in Human Synapses.

Neuromodulators bind to pre- and postsynaptic G protein-coupled receptors (GPCRs), are able to quickly change intracellular cyclic AMP (cAMP) and Ca2+ levels, and are thought to play important roles in neuropsychiatric and neurodegenerative diseases. Here, we discovered in human neurons an unanticipated presynaptic mechanism that acutely changes synaptic ultrastructure and regulates synaptic communication. Activation of neuromodulator receptors bidirectionally controlled synaptic vesicle numbers within nerve terminals. This control correlated with changes in the levels of cAMP-dependent protein kinase A-mediated phosphorylation of synapsin-1. Using a conditional deletion approach, we reveal that the neuromodulator-induced control of synaptic vesicle numbers was largely dependent on synapsin-1. We propose a mechanism whereby non-phosphorylated synapsin-1 "latches" synaptic vesicles to presynaptic clusters at the active zone. cAMP-dependent phosphorylation of synapsin-1 then removes the vesicles. cAMP-independent dephosphorylation of synapsin-1 in turn recruits vesicles. Synapsin-1 thereby bidirectionally regulates synaptic vesicle numbers and modifies presynaptic neurotransmitter release as an effector of neuromodulator signaling in human neurons.

Structural Basis for Teneurin Function in Circuit-Wiring: A Toxin Motif at the Synapse.

Teneurins (TENs) are cell-surface adhesion proteins with critical roles in tissue development and axon guidance. Here, we report the 3.1-Å cryoelectron microscopy structure of the human TEN2 extracellular region (ECR), revealing a striking similarity to bacterial Tc-toxins. The ECR includes a large ß barrel that partially encapsulates a C-terminal domain, which emerges to the solvent through an opening in the mid-barrel region. An immunoglobulin (Ig)-like domain seals the bottom of the barrel while a ß propeller is attached in a perpendicular orientation. We further show that an alternatively spliced region within the ß propeller acts as a switch to regulate trans-cellular adhesion of TEN2 to latrophilin (LPHN), a transmembrane receptor known to mediate critical functions in the central nervous system. One splice variant activates trans-cellular signaling in a LPHN-dependent manner, whereas the other induces inhibitory postsynaptic differentiation. These results highlight the unusual structural organization of TENs giving rise to their multifarious functions.

Synaptic Neurexin Complexes: A Molecular Code for the Logic of Neural Circuits.

Synapses are specialized junctions between neurons in brain that transmit and compute information, thereby connecting neurons into millions of overlapping and interdigitated neural circuits. Here, we posit that the establishment, properties, and dynamics of synapses are governed by a molecular logic that is controlled by diverse trans-synaptic signaling molecules. Neurexins, expressed in thousands of alternatively spliced isoforms, are central components of this dynamic code. Presynaptic neurexins regulate synapse properties via differential binding to multifarious postsynaptic ligands, such as neuroligins, cerebellin/GluD complexes, and latrophilins, thereby shaping the input/output relations of their resident neural circuits. Mutations in genes encoding neurexins and their ligands are associated with diverse neuropsychiatric disorders, especially schizophrenia, autism, and Tourette syndrome. Thus, neurexins nucleate an overall trans-synaptic signaling network that controls synapse properties, which thereby determines the precise responses of synapses to spike patterns in a neuron and circuit and which is vulnerable to impairments in neuropsychiatric disorders.

ApoE2, ApoE3, and ApoE4 Differentially Stimulate APP Transcription and Aß Secretion.

Human apolipoprotein E (ApoE) apolipoprotein is primarily expressed in three isoforms (ApoE2, ApoE3, and ApoE4) that differ only by two residues. ApoE4 constitutes the most important genetic risk factor for Alzheimer's disease (AD), ApoE3 is neutral, and ApoE2 is protective. How ApoE isoforms influence AD pathogenesis, however, remains unclear. Using ES-cell-derived human neurons, we show that ApoE secreted by glia stimulates neuronal Aß production with an ApoE4 > ApoE3 > ApoE2 potency rank order. We demonstrate that ApoE binding to ApoE receptors activates dual leucine-zipper kinase (DLK), a MAP-kinase kinase kinase that then activates MKK7 and ERK1/2 MAP kinases. Activated ERK1/2 induces cFos phosphorylation, stimulating the transcription factor AP-1, which in turn enhances transcription of amyloid-ß precursor protein (APP) and thereby increases amyloid-ß levels. This molecular mechanism also regulates APP transcription in mice in vivo. Our data describe a novel signal transduction pathway in neurons whereby ApoE activates a non-canonical MAP kinase cascade that enhances APP transcription and amyloid-ß synthesis.

ß-Neurexins Control Neural Circuits by Regulating Synaptic Endocannabinoid Signaling.

α- and ß-neurexins are presynaptic cell-adhesion molecules implicated in autism and schizophrenia. We find that, although ß-neurexins are expressed at much lower levels than α-neurexins, conditional knockout of ß-neurexins with continued expression of α-neurexins dramatically decreased neurotransmitter release at excitatory synapses in cultured cortical neurons. The ß-neurexin knockout phenotype was attenuated by CB1-receptor inhibition, which blocks presynaptic endocannabinoid signaling, or by 2-arachidonoylglycerol synthesis inhibition, which impairs postsynaptic endocannabinoid release. In synapses formed by CA1-region pyramidal neurons onto burst-firing subiculum neurons, presynaptic in vivo knockout of ß-neurexins aggravated endocannabinoid-mediated inhibition of synaptic transmission and blocked LTP; presynaptic CB1-receptor antagonists or postsynaptic 2-arachidonoylglycerol synthesis inhibition again reversed this block. Moreover, conditional knockout of ß-neurexins in CA1-region neurons impaired contextual fear memories. Thus, our data suggest that presynaptic ß-neurexins control synaptic strength in excitatory synapses by regulating postsynaptic 2-arachidonoylglycerol synthesis, revealing an unexpected role for ß-neurexins in the endocannabinoid-dependent regulation of neural circuits.

Structural Basis of Vesicle Formation at the Inner Nuclear Membrane.

Vesicular nucleo-cytoplasmic transport is becoming recognized as a general cellular mechanism for translocation of large cargoes across the nuclear envelope. Cargo is recruited, enveloped at the inner nuclear membrane (INM), and delivered by membrane fusion at the outer nuclear membrane. To understand the structural underpinning for this trafficking, we investigated nuclear egress of progeny herpesvirus capsids where capsid envelopment is mediated by two viral proteins, forming the nuclear egress complex (NEC). Using a multi-modal imaging approach, we visualized the NEC in situ forming coated vesicles of defined size. Cellular electron cryo-tomography revealed a protein layer showing two distinct hexagonal lattices at its membrane-proximal and membrane-distant faces, respectively. NEC coat architecture was determined by combining this information with integrative modeling using small-angle X-ray scattering data. The molecular arrangement of the NEC establishes the basic mechanism for budding and scission of tailored vesicles at the INM.

Alternative splicing of latrophilin-3 controls synapse formation.

The assembly and specification of synapses in the brain is incompletely understood1-3. Latrophilin-3 (encoded by Adgrl3, also known as Lphn3)-a postsynaptic adhesion G-protein-coupled receptor-mediates synapse formation in the hippocampus4 but the mechanisms involved remain unclear. Here we show in mice that LPHN3 organizes synapses through a convergent dual-pathway mechanism: activation of Gαs signalling and recruitment of phase-separated postsynaptic protein scaffolds. We found that cell-type-specific alternative splicing of Lphn3 controls the LPHN3 G-protein-coupling mode, resulting in LPHN3 variants that predominantly signal through Gαs or Gα12/13. CRISPR-mediated manipulation of Lphn3 alternative splicing that shifts LPHN3 from a Gαs- to a Gα12/13-coupled mode impaired synaptic connectivity as severely as the overall deletion of Lphn3, suggesting that Gαs signalling by LPHN3 splice variants mediates synapse formation. Notably, Gαs-coupled, but not Gα12/13-coupled, splice variants of LPHN3 also recruit phase-transitioned postsynaptic protein scaffold condensates, such that these condensates are clustered by binding of presynaptic teneurin and FLRT ligands to LPHN3. Moreover, neuronal activity promotes alternative splicing of the synaptogenic Gαs-coupled variant of LPHN3. Together, these data suggest that activity-dependent alternative splicing of a key synaptic adhesion molecule controls synapse formation by parallel activation of two convergent pathways: Gαs signalling and clustered phase separation of postsynaptic protein scaffolds.

The cortical amygdala consolidates a socially transmitted long-term memory.

Social communication guides decision-making, which is essential for survival. Social transmission of food preference (STFP) is an ecologically relevant memory paradigm in which an animal learns a desirable food odour from another animal in a social context, creating a long-term memory1,2. How food-preference memory is acquired, consolidated and stored is unclear. Here we show that the posteromedial nucleus of the cortical amygdala (COApm) serves as a computational centre in long-term STFP memory consolidation by integrating social and sensory olfactory inputs. Blocking synaptic signalling by the COApm-based circuit selectively abolished STFP memory consolidation without impairing memory acquisition, storage or recall. COApm-mediated STFP memory consolidation depends on synaptic inputs from the accessory olfactory bulb and on synaptic outputs to the anterior olfactory nucleus. STFP memory consolidation requires protein synthesis, suggesting a gene-expression mechanism. Deep single-cell and spatially resolved transcriptomics revealed robust but distinct gene-expression signatures induced by STFP memory formation in the COApm that are consistent with synapse restructuring. Our data thus define a neural circuit for the consolidation of a socially communicated long-term memory, thereby mechanistically distinguishing protein-synthesis-dependent memory consolidation from memory acquisition, storage or retrieval.

Spatial transcriptomics reveal neuron-astrocyte synergy in long-term memory.

Memory encodes past experiences, thereby enabling future plans. The basolateral amygdala is a centre of salience networks that underlie emotional experiences and thus has a key role in long-term fear memory formation1. Here we used spatial and single-cell transcriptomics to illuminate the cellular and molecular architecture of the role of the basolateral amygdala in long-term memory. We identified transcriptional signatures in subpopulations of neurons and astrocytes that were memory-specific and persisted for weeks. These transcriptional signatures implicate neuropeptide and BDNF signalling, MAPK and CREB activation, ubiquitination pathways, and synaptic connectivity as key components of long-term memory. Notably, upon long-term memory formation, a neuronal subpopulation defined by increased Penk and decreased Tac expression constituted the most prominent component of the memory engram of the basolateral amygdala. These transcriptional changes were observed both with single-cell RNA sequencing and with single-molecule spatial transcriptomics in intact slices, thereby providing a rich spatial map of a memory engram. The spatial data enabled us to determine that this neuronal subpopulation interacts with adjacent astrocytes, and functional experiments show that neurons require interactions with astrocytes to encode long-term memory.

The UFM1 E3 ligase recognizes and releases 60S ribosomes from ER translocons.

Stalled ribosomes at the endoplasmic reticulum (ER) are covalently modified with the ubiquitin-like protein UFM1 on the 60S ribosomal subunit protein RPL26 (also known as uL24)1,2. This modification, which is known as UFMylation, is orchestrated by the UFM1 ribosome E3 ligase (UREL) complex, comprising UFL1, UFBP1 and CDK5RAP3 (ref. 3). However, the catalytic mechanism of UREL and the functional consequences of UFMylation are unclear. Here we present cryo-electron microscopy structures of UREL bound to 60S ribosomes, revealing the basis of its substrate specificity. UREL wraps around the 60S subunit to form a C-shaped clamp architecture that blocks the tRNA-binding sites at one end, and the peptide exit tunnel at the other. A UFL1 loop inserts into and remodels the peptidyl transferase centre. These features of UREL suggest a crucial function for UFMylation in the release and recycling of stalled or terminated ribosomes from the ER membrane. In the absence of functional UREL, 60S-SEC61 translocon complexes accumulate at the ER membrane, demonstrating that UFMylation is necessary for releasing SEC61 from 60S subunits. Notably, this release is facilitated by a functional switch of UREL from a 'writer' to a 'reader' module that recognizes its product-UFMylated 60S ribosomes. Collectively, we identify a fundamental role for UREL in dissociating 60S subunits from the SEC61 translocon and the basis for UFMylation in regulating protein homeostasis at the ER.

Autism-associated neuroligin-3 mutations commonly impair striatal circuits to boost repetitive behaviors.

In humans, neuroligin-3 mutations are associated with autism, whereas in mice, the corresponding mutations produce robust synaptic and behavioral changes. However, different neuroligin-3 mutations cause largely distinct phenotypes in mice, and no causal relationship links a specific synaptic dysfunction to a behavioral change. Using rotarod motor learning as a proxy for acquired repetitive behaviors in mice, we found that different neuroligin-3 mutations uniformly enhanced formation of repetitive motor routines. Surprisingly, neuroligin-3 mutations caused this phenotype not via changes in the cerebellum or dorsal striatum but via a selective synaptic impairment in the nucleus accumbens/ventral striatum. Here, neuroligin-3 mutations increased rotarod learning by specifically impeding synaptic inhibition onto D1-dopamine receptor-expressing but not D2-dopamine receptor-expressing medium spiny neurons. Our data thus suggest that different autism-associated neuroligin-3 mutations cause a common increase in acquired repetitive behaviors by impairing a specific striatal synapse and thereby provide a plausible circuit substrate for autism pathophysiology.

De novo evolution of macroscopic multicellularity.

While early multicellular lineages necessarily started out as relatively simple groups of cells, little is known about how they became Darwinian entities capable of sustained multicellular evolution1-3. Here we investigate this with a multicellularity long-term evolution experiment, selecting for larger group size in the snowflake yeast (Saccharomyces cerevisiae) model system. Given the historical importance of oxygen limitation4, our ongoing experiment consists of three metabolic treatments5-anaerobic, obligately aerobic and mixotrophic yeast. After 600 rounds of selection, snowflake yeast in the anaerobic treatment group evolved to be macroscopic, becoming around 2 × 104 times larger (approximately mm scale) and about 104-fold more biophysically tough, while retaining a clonal multicellular life cycle. This occurred through biophysical adaptation-evolution of increasingly elongate cells that initially reduced the strain of cellular packing and then facilitated branch entanglements that enabled groups of cells to stay together even after many cellular bonds fracture. By contrast, snowflake yeast competing for low oxygen5 remained microscopic, evolving to be only around sixfold larger, underscoring the critical role of oxygen levels in the evolution of multicellular size. Together, this research provides unique insights into an ongoing evolutionary transition in individuality, showing how simple groups of cells overcome fundamental biophysical limitations through gradual, yet sustained, multicellular evolution.

Heterogeneous melting near the Thwaites Glacier grounding line.

Thwaites Glacier represents 15% of the ice discharge from the West Antarctic Ice Sheet and influences a wider catchment1-3. Because it is grounded below sea level4,5, Thwaites Glacier is thought to be susceptible to runaway retreat triggered at the grounding line (GL) at which the glacier reaches the ocean6,7. Recent ice-flow acceleration2,8 and retreat of the ice front8-10 and GL11,12 indicate that ice loss will continue. The relative impacts of mechanisms underlying recent retreat are however uncertain. Here we show sustained GL retreat from at least 2011 to 2020 and resolve mechanisms of ice-shelf melt at the submetre scale. Our conclusions are based on observations of the Thwaites Eastern Ice Shelf (TEIS) from an underwater vehicle, extending from the GL to 3 km oceanward and from the ice-ocean interface to the sea floor. These observations show a rough ice base above a sea floor sloping upward towards the GL and an ocean cavity in which the warmest water exceeds 2 °C above freezing. Data closest to the ice base show that enhanced melting occurs along sloped surfaces that initiate near the GL and evolve into steep-sided terraces. This pronounced melting along steep ice faces, including in crevasses, produces stratification that suppresses melt along flat interfaces. These data imply that slope-dependent melting sculpts the ice base and acts as an important response to ocean warming.

The recovery of European freshwater biodiversity has come to a halt.

Owing to a long history of anthropogenic pressures, freshwater ecosystems are among the most vulnerable to biodiversity loss1. Mitigation measures, including wastewater treatment and hydromorphological restoration, have aimed to improve environmental quality and foster the recovery of freshwater biodiversity2. Here, using 1,816 time series of freshwater invertebrate communities collected across 22 European countries between 1968 and 2020, we quantified temporal trends in taxonomic and functional diversity and their responses to environmental pressures and gradients. We observed overall increases in taxon richness (0.73% per year), functional richness (2.4% per year) and abundance (1.17% per year). However, these increases primarily occurred before the 2010s, and have since plateaued. Freshwater communities downstream of dams, urban areas and cropland were less likely to experience recovery. Communities at sites with faster rates of warming had fewer gains in taxon richness, functional richness and abundance. Although biodiversity gains in the 1990s and 2000s probably reflect the effectiveness of water-quality improvements and restoration projects, the decelerating trajectory in the 2010s suggests that the current measures offer diminishing returns. Given new and persistent pressures on freshwater ecosystems, including emerging pollutants, climate change and the spread of invasive species, we call for additional mitigation to revive the recovery of freshwater biodiversity.

Presynaptic neurexin-3 alternative splicing trans-synaptically controls postsynaptic AMPA receptor trafficking.

Neurexins are essential presynaptic cell adhesion molecules that are linked to schizophrenia and autism and are subject to extensive alternative splicing. Here, we used a genetic approach to test the physiological significance of neurexin alternative splicing. We generated knockin mice in which alternatively spliced sequence #4 (SS4) of neuexin-3 is constitutively included but can be selectively excised by cre-recombination. SS4 of neurexin-3 was chosen because it is highly regulated and controls neurexin binding to neuroligins, LRRTMs, and other ligands. Unexpectedly, constitutive inclusion of SS4 in presynaptic neurexin-3 decreased postsynaptic AMPA, but not NMDA receptor levels, and enhanced postsynaptic AMPA receptor endocytosis. Moreover, constitutive inclusion of SS4 in presynaptic neurexin-3 abrogated postsynaptic AMPA receptor recruitment during NMDA receptor-dependent LTP. These phenotypes were fully rescued by constitutive excision of SS4 in neurexin-3. Thus, alternative splicing of presynaptic neurexin-3 controls postsynaptic AMPA receptor trafficking, revealing an unanticipated alternative splicing mechanism for trans-synaptic regulation of synaptic strength and long-term plasticity.