Inter-domain dynamics drive cholesterol transport by NPC1 and NPC1L1 proteins
Transport of LDL-derived cholesterol from lysosomes into the cytoplasm requires NPC1 protein; NPC1L1 mediates uptake of dietary cholesterol. We introduced single disulfide bonds into NPC1 and NPC1L1 to explore the importance of inter-domain dynamics in cholesterol transport. Using a sensitive method to monitor lysosomal cholesterol efflux, we found that NPC1 ’s N-terminal domain need not release from the rest of the protein for efficient cholesterol export. Either introducing single disulfide bonds to constrain lumenal/extracellular domains or shortening a cytoplasmic loop abolishes transport activity by both NPC1 an...
Source: eLife - May 15, 2020 Category: Biomedical Science Tags: Biochemistry and Chemical Biology Cell Biology Source Type: research

Connectomic analysis reveals an interneuron with an integral role in the retinal circuit for night vision
Night vision in mammals depends fundamentally on rod photoreceptors and the well-studied rod bipolar (RB) cell pathway. The central neuron in this pathway, the AII amacrine cell (AC), exhibits a spatially tuned receptive field, composed of an excitatory center and an inhibitory surround, that propagates to ganglion cells, the retina ’s projection neurons. The circuitry underlying the surround of the AII, however, remains unresolved. Here, we combined structural, functional and optogenetic analyses of the mouse retina to discover that surround inhibition of the AII depends primarily on a single interneuron type, the N...
Source: eLife - May 15, 2020 Category: Biomedical Science Tags: Neuroscience Source Type: research

Efficient targeted integration directed by short homology in zebrafish and mammalian cells
Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Here, we describe a set of resources to streamline reporter gene knock-ins in zebrafish and demonstrate the broader utility of the method in mammalian cells. Our approach uses short homology of 24 –48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at high frequency at a double strand break in the targeted gene. Our vector series, pGTag (plasmids for Gene Tagging), contains reporters flanked by a universal CRISPR sgRNA sequence which enable s in v...
Source: eLife - May 15, 2020 Category: Biomedical Science Tags: Developmental Biology Genetics and Genomics Source Type: research

Identification of novel, clinically correlated autoantigens in the monogenic autoimmune syndrome APS1 by proteome-wide PhIP-Seq
The identification of autoantigens remains a critical challenge for understanding and treating autoimmune diseases. Autoimmune polyendocrine syndrome type 1 (APS1), a rare monogenic form of autoimmunity, presents as widespread autoimmunity with T and B cell responses to multiple organs. Importantly, autoantibody discovery in APS1 can illuminate fundamental disease pathogenesis, and many of the antigens found in APS1 extend to more common autoimmune diseases. Here, we performed proteome-wide programmable phage-display (PhIP-Seq) on sera from a cohort of people with APS1 and discovered multiple common antibody targets. These...
Source: eLife - May 15, 2020 Category: Biomedical Science Tags: Human Biology and Medicine Immunology and Inflammation Source Type: research

Stimulation strength controls the rate of initiation but not the molecular organization of TCR-induced signalling
Millions of na ïve T cells with different TCRs may interact with a peptide-MHC ligand, but very few will activate. Remarkably, this fine control is orchestrated using a limited set of intracellular machinery. It remains unclear whether changes in stimulation strength alter the programme of signalling events leadi ng to T cell activation. Using mass cytometry to simultaneously measure multiple signalling pathways during activation of murine CD8+ T cells, we found a programme of distal signalling events that is shared, regardless of the strength of TCR stimulation. Moreover, the relationship between transcription of ear...
Source: eLife - May 15, 2020 Category: Biomedical Science Tags: Computational and Systems Biology Immunology and Inflammation Source Type: research

Finding the mutations that drive resistance
Mutations that allow tumors to evolve and become resistant to treatment can be readily identified with a new sequencing approach. (Source: eLife)
Source: eLife - May 15, 2020 Category: Biomedical Science Tags: Cancer Biology Genetics and Genomics Source Type: research

Untangling the immune basis of disease susceptibility
Interactions between immune cell receptors and proteins that determine disease susceptibility shed light on how different arms of the immune system are involved in three viral infections and Crohn's disease. (Source: eLife)
Source: eLife - May 14, 2020 Category: Biomedical Science Tags: Computational and Systems Biology Immunology and Inflammation Source Type: research

Reconstitution reveals two paths of force transmission through the kinetochore
Partitioning duplicated chromosomes equally between daughter cells is a microtubule-mediated process essential to eukaryotic life. A multi-protein machine, the kinetochore, drives chromosome segregation by coupling the chromosomes to dynamic microtubule tips, even as the tips grow and shrink through the gain and loss of subunits. The kinetochore must harness, transmit, and sense mitotic forces, as a lack of tension signals incorrect chromosome-microtubule attachment and precipitates error correction mechanisms. But though the field has arrived at a 'parts list' of dozens of kinetochore proteins organized into subcomplexes,...
Source: eLife - May 14, 2020 Category: Biomedical Science Tags: Biochemistry and Chemical Biology Cell Biology Source Type: research

Single-cell transcriptome reveals the novel role of T-bet in suppressing the immature NK gene signature
The transcriptional activation and repression during NK cell ontology are poorly understood. Here, using single-cell RNA-sequencing, we reveal a novel role for T-bet in suppressing the immature gene signature during murine NK cell development. Based on transcriptome, we identified five distinct NK cell clusters and define their relative developmental maturity in the bone marrow. Transcriptome-based machine-learning classifiers revealed that half of the mTORC2-deficient NK cells belongs to the least mature NK cluster. Mechanistically, loss of mTORC2 results in an increased expression of signature genes representing immature...
Source: eLife - May 14, 2020 Category: Biomedical Science Tags: Immunology and Inflammation Source Type: research

Novel insights into breast cancer copy number genetic heterogeneity revealed by single-cell genome sequencing
Copy number alterations (CNAs) play an important role in molding the genomes of breast cancers and have been shown to be clinically useful for prognostic and therapeutic purposes. However, our knowledge of intra-tumoral genetic heterogeneity of this important class of somatic alterations is limited. Here, using single-cell sequencing, we comprehensively map out the facets of copy number alteration heterogeneity in a cohort of breast cancer tumors. Ou/var/www/html/elife/12-05-2020/backup/r analyses reveal: genetic heterogeneity of non-tumor cells (i.e. stroma) within the tumor mass; the extent to which copy number heterogen...
Source: eLife - May 13, 2020 Category: Biomedical Science Tags: Cancer Biology Genetics and Genomics Source Type: research

A role for fat precursors in the marrow
A group of cells that can become adipocytes controls the formation of blood vessels in the bone marrow, and also regulates the differentiation of resident mesenchymal progenitor cells. (Source: eLife)
Source: eLife - May 13, 2020 Category: Biomedical Science Tags: Cell Biology Source Type: research

A critical re-evaluation of fMRI signatures of motor sequence learning
Despite numerous studies, there is little agreement about what brain changes accompany motor sequence learning, partly because of a general publication bias that favors novel results. We therefore decided to systematically reinvestigate proposed functional magnetic resonance imaging correlates of motor learning in a preregistered longitudinal study with four scanning sessions over 5 weeks of training. Activation decreased more for trained than untrained sequences in premotor and parietal areas, without any evidence of learning-related activation increases. Premotor and parietal regions also exhibited changes in the fine-gr...
Source: eLife - May 13, 2020 Category: Biomedical Science Tags: Neuroscience Source Type: research

Synergistic roles of Synaptotagmin-1 and complexin in calcium-regulated neuronal exocytosis
Calcium (Ca2+)-evoked release of neurotransmitters from synaptic vesicles requires mechanisms both to prevent un-initiated fusion of vesicles (clamping) and to trigger fusion following Ca2+-influx. The principal components involved in these processes are the vesicular fusion machinery (SNARE proteins) and the regulatory proteins, Synaptotagmin-1 and Complexin. Here, we use a reconstituted single-vesicle fusion assay under physiologically-relevant conditions to delineate a novel mechanism by which Synaptotagmin-1 and Complexin act synergistically to establish Ca2+-regulated fusion. We find that under each vesicle, Synaptota...
Source: eLife - May 13, 2020 Category: Biomedical Science Tags: Structural Biology and Molecular Biophysics Source Type: research

Spontaneous and evoked neurotransmission are partially segregated at inhibitory synapses
We report picrotoxin, a GABAAR antagonist, blocks neurotransmission in a use-dependent manner at rat hippocampal synapses and therefore can be used to interrogate synaptic properties. Using this tool, we uncovered partial segregation of inhibitory spontaneous and evoked neurotransmission. We found up to 40% of the evoked response is mediated through GABAARs which are only activated by evoked neurotransmission. These data indicate GABAergic spontaneous and evoked neurotransmission processes are partially non-overlapping, suggesting they may serve divergent roles in neuronal signaling. (Source: eLife)
Source: eLife - May 13, 2020 Category: Biomedical Science Tags: Neuroscience Source Type: research

ODELAM Rapid sequence-independent detection of drug resistance in isolates of < i > Mycobacterium tuberculosis < /i >
We present ODELAM (One-cell Doubling Evaluation of Living Arrays of Mycobacterium), a time-lapse microscopy-based method that observes individual cells growing into microcolonies. ODELAM enables rapid quantitative measures of growth kinetics in as little as 30 hours under a wide variety of environmental conditions. We demonstrate ODELAM's utility by identifying ofloxacin resistance in cultured clinical isolates of Mtb and benchmark its performance with standard minimum inhibitory concentration (MIC) assays. In Mtb isolate, ODELAM identified ofloxacin heteroresistance and identifies the presence of drug resistant colony for...
Source: eLife - May 13, 2020 Category: Biomedical Science Tags: Microbiology and Infectious Disease Source Type: research

Synergism of type 1 metabotropic and ionotropic glutamate receptors in cerebellar molecular layer interneurons in vivo
Type 1 metabotropic glutamate receptors (mGluR1s) are key elements in neuronal signaling. While their function is well documented in slices, requirements for their activation in vivo are poorly understood. We examine this question in adult mice in vivo using 2-photon imaging of cerebellar molecular layer interneurons (MLIs) expressing GCaMP. In anesthetized mice, parallel fiber activation evokes beam-like Cai rises in postsynaptic MLIs which depend on co-activation of mGluR1s and ionotropic glutamate receptors (iGluRs). In awake mice, blocking mGluR1 decreases Cai rises associated with locomotion. In vitro studies and free...
Source: eLife - May 13, 2020 Category: Biomedical Science Tags: Neuroscience Source Type: research

Dissecting the phenotypic and functional heterogeneity of mouse inflammatory osteoclasts by the expression of < i > Cx3cr1 < /i >
Bone destruction relies on interactions between bone and immune cells. Bone-resorbing osteoclasts (OCLs) were recently identified as innate immune cells activating T cells toward tolerance or inflammation. Thus, pathological bone destruction not only relies on increased osteoclast differentiation, but also on the presence of inflammatory OCLs (i-OCLs), part of which expressCx3cr1. Here, we investigated the contribution of mouse Cx3cr1+ and Cx3cr1neg i-OCLs to bone loss. We showed that Cx3cr1+ and Cx3cr1neg i-OCLs differ considerably in transcriptional and functional aspects. Cx3cr1neg i-OCLs have a high ability to resorb b...
Source: eLife - May 13, 2020 Category: Biomedical Science Tags: Cell Biology Immunology and Inflammation Source Type: research

Cohesin and condensin extrude DNA loops in a cell-cycle dependent manner
Loop extrusion by structural maintenance of chromosomes complexes (SMCs) has been proposed as a mechanism to organize chromatin in interphase and metaphase. However, the requirements for chromatin organization in these cell cycle phases are different, and it is unknown whether loop extrusion dynamics and the complexes that extrude DNA also differ. Here, we usedXenopus egg extracts to reconstitute and image loop extrusion of single DNA molecules during the cell cycle. We show that loops form in both metaphase and interphase, but with distinct dynamic properties. Condensin extrudes DNA loops non-symmetrically in metaphase, w...
Source: eLife - May 12, 2020 Category: Biomedical Science Tags: Cell Biology Chromosomes and Gene Expression Source Type: research

Ant collective cognition allows for efficient navigation through disordered environments
The cognitive abilities of biological organisms only make sense in the context of their environment. Here, we study longhorn crazy ant collective navigation skills within the context of a semi-natural, randomized environment. Mapping this biological setting into the 'Ant-in-a-Labyrinth' framework which studies physical transport through disordered media allows us to formulate precise links between the statistics of environmental challenges and the ants' collective navigation abilities. We show that, in this environment, the ants use their numbers to collectively extend their sensing range. Although this extension is modera...
Source: eLife - May 12, 2020 Category: Biomedical Science Tags: Ecology Physics of Living Systems Source Type: research

IP < sub > 3 < /sub > mediated global Ca < sup > 2+ < /sup > signals arise through two temporally and spatially distinct modes of Ca < sup > 2+ < /sup > release
The 'building-block' model of inositol trisphosphate (IP3)-mediated Ca2+ liberation posits that cell-wide cytosolic Ca2+ signals arise through coordinated activation of localized Ca2+ puffs generated by stationary clusters of IP3 receptors (IP3Rs). Here, we revise this hypothesis, applying fluctuation analysis to resolve Ca2+ signals otherwise obscured during large Ca2+ elevations. We find the rising phase of global Ca2+ signals is punctuated by a flurry of puffs, which terminate before the peak by a mechanism involving partial ER Ca2+ depletion. The continuing rise in Ca2+, and persistence of global signals even when puff...
Source: eLife - May 12, 2020 Category: Biomedical Science Tags: Cell Biology Structural Biology and Molecular Biophysics Source Type: research

Intelligent classification of platelet aggregates by agonist type
Platelets are anucleate cells in blood whose principal function is to stop bleeding by forming aggregates for hemostatic reactions. In addition to their participation in physiological hemostasis, platelet aggregates are also involved in pathological thrombosis and play an important role in inflammation, atherosclerosis, and cancer metastasis. The aggregation of platelets is elicited by various agonists, but these platelet aggregates have long been considered indistinguishable and impossible to classify. Here we present an intelligent method for classifying them by agonist type. It is based on a convolutional neural network...
Source: eLife - May 12, 2020 Category: Biomedical Science Tags: Cell Biology Human Biology and Medicine Source Type: research

Local emergence in Amazonia of < i > Plasmodium falciparum k13 < /i > C580Y mutants associated with < i > in vitro < /i > artemisinin resistance
We report that mutantpfk13 has emerged independently in Guyana, with genome analysis indicating an evolutionary origin distinct from Southeast Asia.Pfk13 C580Y parasites were observed in 1.6% (14/854) of samples collected in Guyana in 2016 –2017. Introducingpfk13 C580Y or R539T mutations by gene editing into local parasites conferred high levels ofin vitro artemisinin resistance.In vitro growth competition assays revealed a fitness cost associated with thesepfk13 variants, potentially explaining why these resistance alleles have not increased in frequency more quickly in South America. These data place local malaria ...
Source: eLife - May 12, 2020 Category: Biomedical Science Tags: Epidemiology and Global Health Microbiology and Infectious Disease Source Type: research

Bmal1 integrates mitochondrial metabolism and macrophage activation
Metabolic pathways and inflammatory processes are under circadian regulation. While rhythmic immune cell recruitment is known to impact infection outcomes, whether the circadian clock modulates immunometabolism remains unclear. We find the molecular clock Bmal1 is induced by inflammatory stimulants, including Ifn-g/lipopolysaccharide (M1) and tumor-conditioned medium, to maintain mitochondrial metabolism under these metabolically stressed conditions in mouse macrophages. Upon M1 stimulation, myeloid-specificBmal1 knockout (M-BKO) renders macrophages unable to sustain mitochondrial function, enhancing succinate dehydrogenas...
Source: eLife - May 12, 2020 Category: Biomedical Science Tags: Cell Biology Source Type: research

Adult chondrogenesis and spontaneous cartilage repair in the skate, < i > Leucoraja erinacea < /i >
Mammalian articular cartilage is an avascular tissue with poor capacity for spontaneous repair. Here, we show that embryonic development of cartilage in the skate (Leucoraja erinacea) mirrors that of mammals, with developing chondrocytes co-expressing genes encoding the transcription factors Sox5, Sox6 and Sox9. However, in skate, transcriptional features of developing cartilage persist into adulthood, both in peripheral chondrocytes and in cells of the fibrous perichondrium that ensheaths the skeleton. Using pulse-chase label retention experiments and multiplexed in situ hybridization, we identify a population of cyclingS...
Source: eLife - May 12, 2020 Category: Biomedical Science Tags: Developmental Biology Stem Cells and Regenerative Medicine Source Type: research

A single-cell survey of < i > Drosophila < /i > blood
Drosophila blood cells, called hemocytes, are classified into plasmatocytes, crystal cells, and lamellocytes based on the expression of a few marker genes and cell morphologies, which are inadequate to classify the complete hemocyte repertoire. Here, we used single-cell RNA sequencing (scRNA-seq) to map hemocytes across different inflammatory conditions in larvae. We resolved plasmatocytes into different states based on the expression of genes involved in cell cycle, antimicrobial response, and metabolism together with the identification of intermediate states. Further, we discovered rare subsets within crystal cells and l...
Source: eLife - May 12, 2020 Category: Biomedical Science Tags: Developmental Biology Immunology and Inflammation Source Type: research

Heterogeneity of proteome dynamics between connective tissue phases of adult tendon
Maintenance of connective tissue integrity is fundamental to sustain function, requiring protein turnover to repair damaged tissue. However, connective tissue proteome dynamics remain largely undefined, as do differences in turnover rates of individual proteins in the collagen and glycoprotein phases of connective tissue extracellular matrix (ECM). Here, we investigate proteome dynamics in the collagen and glycoprotein phases of connective tissues by exploiting the spatially distinct fascicular (collagen-rich) and interfascicular (glycoprotein-rich) ECM phases of tendon. Using isotope labelling, mass spectrometry and bioin...
Source: eLife - May 12, 2020 Category: Biomedical Science Tags: Biochemistry and Chemical Biology Structural Biology and Molecular Biophysics Source Type: research

Tgfb3 collaborates with PP2A and Notch signaling pathways to inhibit retina regeneration
Neuronal degeneration in the zebrafish retina stimulates M üller glia (MG) to proliferate and generate multipotent progenitors for retinal repair. Controlling this proliferation is critical to successful regeneration. Previous studies reported that retinal injury stimulates pSmad3 signaling in injury-responsive MG. Contrary to these findings, we report pSm ad3 expression is restricted to quiescent MG and suppressed in injury-responsive MG. Our data indicates that Tgfb3 is the ligand responsible for regulating pSmad3 expression. Remarkably, although overexpression of either Tgfb1b or Tgfb3 can stimulate pSmad3 expressi...
Source: eLife - May 12, 2020 Category: Biomedical Science Tags: Stem Cells and Regenerative Medicine Source Type: research

A mammalian < i > Wnt5a –Ror2–Vangl2 < /i > axis controls the cytoskeleton and confers cellular properties required for alveologenesis
Alveolar formation increases the surface area for gas-exchange and is key to the physiological function of the lung. Alveolar epithelial cells, myofibroblasts and endothelial cells undergo coordinated morphogenesis to generate epithelial folds (secondary septa) to form alveoli. A mechanistic understanding of alveologenesis remains incomplete. We found that the planar cell polarity (PCP) pathway is required in alveolar epithelial cells and myofibroblasts for alveologenesis in mammals. Our studies uncovered aWnt5a –Ror2–Vangl2 cascade that endows cellular properties and novel mechanisms of alveologenesis. This in...
Source: eLife - May 12, 2020 Category: Biomedical Science Tags: Developmental Biology Source Type: research

Mechanics and kinetics of dynamic instability
During dynamic instability, self-assembling microtubules (MTs) stochastically alternate between phases of growth and shrinkage. This process is driven by the presence of two distinct states of MT subunits, GTP- and GDP-bound tubulin dimers, that have different structural properties. Here, we use a combination of analysis and computer simulations to study the mechanical and kinetic regulation of dynamic instability in three-dimensional (3D) self-assembling MTs. Our model quantifies how the 3D structure and kinetics of the distinct states of tubulin dimers determine the mechanical stability of MTs. We further show that dynam...
Source: eLife - May 11, 2020 Category: Biomedical Science Tags: Physics of Living Systems Source Type: research

Genetic transformation of < i > Spizellomyces punctatus < /i > , a resource for studying chytrid biology and evolutionary cell biology
Chytrids are early-diverging fungi that share features with animals that have been lost in most other fungi. They hold promise as a system to study fungal and animal evolution, but we lack genetic tools for hypothesis testing. Here, we generated transgenic lines of the chytridSpizellomyces punctatus, and used fluorescence microscopy to explore chytrid cell biology and development during its life cycle. We show that the chytrid undergoes multiple rounds of synchronous nuclear division, followed by cellularization, to create and release many daughter ‘zoospores’. The zoospores, akin to animal cells, crawl using a...
Source: eLife - May 11, 2020 Category: Biomedical Science Tags: Evolutionary Biology Source Type: research

Screening of healthcare workers for SARS-CoV-2 highlights the role of asymptomatic carriage in COVID-19 transmission
Significant differences exist in the availability of healthcare worker (HCW) SARS-CoV-2 testing between countries, and existing programmes focus on screening symptomatic rather than asymptomatic staff. Over a 3-week period (April 2020), 1,032 asymptomatic HCWs were screened for SARS-CoV-2 in a large UK teaching hospital. Symptomatic staff and symptomatic household contacts were additionally tested. Real-time RT-PCR was used to detect viral RNA from a throat+nose self-swab. 3% of HCWs in theasymptomatic screening group tested positive for SARS-CoV-2. 17/30 (57%) were truly asymptomatic/pauci-symptomatic. 12/30 (40%) had exp...
Source: eLife - May 11, 2020 Category: Biomedical Science Tags: Epidemiology and Global Health Human Biology and Medicine Source Type: research

Temporal transcription factors determine circuit membership by permanently altering motor neuron-to-muscle synaptic partnerships
How circuit wiring is specified is a key question in developmental neurobiology. Previously, using the Drosophila motor system as a model, we found the classic temporal transcription factor, Hunchback acts in NB7-1 neuronal stem cells to control how many NB7-1 neuronal progeny form functional synapses on dorsal muscles (Meng et al., 2019). However, it is unknown to what extent control of motor neuron-to-muscle synaptic partnerships is a general feature of temporal transcription factors. Here, we perform additional temporal transcription factor manipulations —prolonging expression of Hunchback in NB3-1, as well as pre...
Source: eLife - May 11, 2020 Category: Biomedical Science Tags: Developmental Biology Neuroscience Source Type: research

A conserved and regulated mechanism drives endosomal Rab transition
Endosomes and lysosomes harbor Rab5 and Rab7 on their surface as key proteins involved in their identity, biogenesis, and fusion. Rab activation requires a guanine nucleotide exchange factor (GEF), which is Mon1-Ccz1 for Rab7. During endosome maturation, Rab5 is replaced by Rab7, though the underlying mechanism remains poorly understood. Here, we identify the molecular determinants for Rab conversionin vivo andin vitro, and reconstitute Rab7 activation with yeast and metazoan proteins. We show (i) that Mon1-Ccz1 is an effector of Rab5, (ii) that membrane-bound Rab5 is the key factor to directly promote Mon1-Ccz1 dependent ...
Source: eLife - May 11, 2020 Category: Biomedical Science Tags: Biochemistry and Chemical Biology Cell Biology Source Type: research

Insights into herpesvirus assembly from the structure of the pUL7:pUL51 complex
Herpesviruses acquire their membrane envelopes in the cytoplasm of infected cells via a molecular mechanism that remains unclear. Herpes simplex virus (HSV)-1 proteins pUL7 and pUL51 form a complex required for efficient virus envelopment. We show that interaction between homologues of pUL7 and pUL51 is conserved across human herpesviruses, as is their association withtrans-Golgi membranes. We characterized the HSV-1 pUL7:pUL51 complex by solution scattering and chemical crosslinking, revealing a 1:2 complex that can form higher-order oligomers in solution, and we solved the crystal structure of the core pUL7:pUL51 heterod...
Source: eLife - May 11, 2020 Category: Biomedical Science Tags: Microbiology and Infectious Disease Structural Biology and Molecular Biophysics Source Type: research

Linker histone H1.2 and H1.4 affect the neutrophil lineage determination
Neutrophils are important innate immune cells that tackle invading pathogens with different effector mechanisms. They acquire this antimicrobial potential during their maturation in the bone marrow, where they differentiate from hematopoietic stem cells in a process called granulopoiesis. Mature neutrophils are terminally differentiated and short-lived with a high turnover rate. Here, we show a critical role for linker histone H1 on the differentiation and function of neutrophils using a genome-wide CRISPR/Cas9 screen in the human cell line PLB-985. We systematically disrupted expression of somatic H1 subtypes to show that...
Source: eLife - May 11, 2020 Category: Biomedical Science Tags: Developmental Biology Immunology and Inflammation Source Type: research

Synergistic actions of v-SNARE transmembrane domains and membrane-curvature modifying lipids in neurotransmitter release
Vesicle fusion is mediated by assembly of SNARE proteins between opposing membranes. While previous work suggested an active role of SNARE transmembrane domains (TMDs) in promoting membrane merger (Dhara et al., 2016), the underlying mechanism remained elusive. Here, we show that naturally-occurring v-SNARE TMD variants differentially regulate fusion pore dynamics in mouse chromaffin cells, indicating TMD flexibility as a mechanistic determinant that facilitates transmitter release from differentially-sized vesicles. Membrane curvature-promoting phospholipids like lysophosphatidylcholine or oleic acid profoundly alter pore...
Source: eLife - May 11, 2020 Category: Biomedical Science Tags: Neuroscience Source Type: research

Loss of Ena/VASP interferes with lamellipodium architecture, motility and integrin-dependent adhesion
Cell migration entails networks and bundles of actin filaments termed lamellipodia and microspikes or filopodia, respectively, as well as focal adhesions, all of which recruit Ena/VASP family members hitherto thought to antagonize efficient cell motility. However, we find these proteins to act as positive regulators of migration in different murine cell lines. CRISPR/Cas9-mediated loss of Ena/VASP proteins reduced lamellipodial actin assembly and perturbed lamellipodial architecture, as evidenced by changed network geometry as well as reduction of filament length and number that was accompanied by abnormal Arp2/3 complex a...
Source: eLife - May 11, 2020 Category: Biomedical Science Tags: Cell Biology Source Type: research

Circulating myeloid cells invade the central nervous system to mediate cachexia during pancreatic cancer
Weight loss and anorexia are common symptoms in cancer patients that occur prior to initiation of cancer therapy. Inflammation in the brain is a driver of these symptoms, yet cellular sources of neuroinflammation during malignancy are unknown. In a mouse model of pancreatic ductal adenocarcinoma (PDAC), we observed early and robust myeloid cell infiltration into the brain. Infiltrating immune cells were predominately neutrophils, which accumulated at a unique central nervous system entry portal called the velum interpositum, where they expressed CCR2. Pharmacologic CCR2 blockade and genetic deletion ofCcr2 both resulted in...
Source: eLife - May 11, 2020 Category: Biomedical Science Tags: Immunology and Inflammation Neuroscience Source Type: research

AirID, a novel proximity biotinylation enzyme, for analysis of protein-protein interactions
Proximity biotinylation based onEscherichia coli BirA enzymes like BioID (BirA*) and TurboID is a key technology for identifying proteins interacting with a target protein in a cell or organism. However, there have been some improvements in the enzymes for that purpose. Here, we demonstrate a novel BirA enzyme, AirID (ancestral BirA for proximity-dependent biotin identification), which was designedde novo using an ancestral enzyme reconstruction algorithm and metagenome data. AirID-fusion proteins like AirID-p53 or AirID-I κBα indicated biotinylation of MDM2 or RelA, respectively,in vitro and in cells, respecti...
Source: eLife - May 11, 2020 Category: Biomedical Science Tags: Biochemistry and Chemical Biology Cell Biology Source Type: research

Driving polar growth
Profiling the phenotype of 200,000 mutants revealed a new cofactor that may help a group of rod-shaped bacteria elongate and grow. (Source: eLife)
Source: eLife - May 7, 2020 Category: Biomedical Science Tags: Microbiology and Infectious Disease Source Type: research

Temporal integration of auxin information for the regulation of patterning
Positional information is essential for coordinating the development of multicellular organisms. In plants, positional information provided by the hormone auxin regulates rhythmic organ production at the shoot apex, but the spatio-temporal dynamics of auxin gradients is unknown. We used quantitative imaging to demonstrate that auxin carries high-definition graded information not only in space but also in time. We show that, during organogenesis, temporal patterns of auxin arise from rhythmic centrifugal waves of high auxin travelling through the tissue faster than growth. We further demonstrate that temporal integration of...
Source: eLife - May 7, 2020 Category: Biomedical Science Tags: Developmental Biology Plant Biology Source Type: research

Spen links RNA-mediated endogenous retrovirus silencing and X chromosome inactivation
The Xist lncRNA mediates X chromosome inactivation (XCI)1,2. Here we show that Spen, an Xist-binding repressor protein essential for XCI3-9, binds to ancient retroviral RNA, performing a surveillance role to recruit chromatin silencing machinery to these parasitic loci. Spen inactivation activates a subset of endogenous retroviral (ERV) elements in mouse embryonic stem cells, with gain of chromatin accessibility, active histone modifications, and ERV RNA transcription. Spen binds directly to ERV RNAs that show structural similarity to the A-repeat of Xist, a region critical for Xist-mediated gene silencing10-11. ERV RNA an...
Source: eLife - May 7, 2020 Category: Biomedical Science Tags: Chromosomes and Gene Expression Source Type: research

Essential function of the alveolin network in the subpellicular microtubules and conoid assembly in < i > Toxoplasma gondii < /i >
The coccidian subgroup of Apicomplexa possesses an apical complex harboring a conoid, made of unique tubulin polymer fibers. This enigmatic organelle extrudes in extracellular invasive parasites and is associated to the apical polar ring (APR). The APR serves as microtubule-organizing center for the 22 subpellicular microtubules (SPMTs) that are linked to a patchwork of flattened vesicles, via an intricate network composed of alveolins. Here, we capitalize on ultrastructure expansion microscopy (U-ExM) to localize theToxoplasma gondii Apical Cap protein 9 (AC9) and its partner AC10, identified by BioID, to the alveolin net...
Source: eLife - May 7, 2020 Category: Biomedical Science Tags: Microbiology and Infectious Disease Source Type: research

Quantifying antibiotic impact on within-patient dynamics of extended-spectrum beta-lactamase resistance
Antibiotic-induced perturbation of the human gut flora is expected to play an important role in mediating the relationship between antibiotic use and the population prevalence of antibiotic resistance in bacteria, but little is known about how antibiotics affect within-host resistance dynamics. Here we develop a data-driven model of the within-host dynamics of extended-spectrum beta-lactamase (ESBL) producingEnterobacteriaceae. We use blaCTX-M (the most widespread ESBL gene family) and 16S rRNA (a proxy for bacterial load) abundance data from 833 rectal swabs from 133 ESBL-positive patients followed up in a prospective coh...
Source: eLife - May 7, 2020 Category: Biomedical Science Tags: Epidemiology and Global Health Microbiology and Infectious Disease Source Type: research

Recurrent circuit dynamics underlie persistent activity in the macaque frontoparietal network
During delayed oculomotor response tasks, neurons in the lateral intraparietal area (LIP) and the frontal eye fields (FEF) exhibit persistent activity that reflects the active maintenance of behaviorally relevant information. Despite many computational models of the mechanisms of persistent activity, there is a lack of circuit-level data from the primate to inform the theories. To fill this gap, we simultaneously recorded ensembles of neurons in both LIP and FEF while macaques performed a memory-guided saccade task. A population encoding model revealed strong and symmetric long-timescale recurrent excitation between LIP an...
Source: eLife - May 7, 2020 Category: Biomedical Science Tags: Neuroscience Source Type: research

LKB1 coordinates neurite remodeling to drive synapse layer emergence in the outer retina
Structural changes in pre and postsynaptic neurons that accompany synapse formation often temporally and spatially overlap. Thus, it has been difficult to resolve which processes drive patterned connectivity. To overcome this, we use the laminated outer murine retina. We identify the serine/threonine kinase LKB1 as a key driver of synapse layer emergence. The absence of LKB1 in the retina caused a marked mislocalization and delay in synapse layer formation. In parallel, LKB1 modulated postsynaptic horizontal cell refinement and presynaptic photoreceptor axon growth. Mislocalized horizontal cell processes contacted aberrant...
Source: eLife - May 7, 2020 Category: Biomedical Science Tags: Neuroscience Source Type: research

Auditory cortical alpha/beta desynchronization prioritizes the representation of memory items during a retention period
To-be-memorized information in working-memory could be protected against distracting influences by processes of functional inhibition or prioritization. Modulations of oscillations in the alpha to beta range in task-relevant sensory regions have been suggested to play an important role for both mechanisms. We adapted a Sternberg task variant to the auditory modality, with a strong or a weak distracting sound presented at a predictable time during the retention period. Using a time-generalized decoding approach relatively decreased strength of memorized information was found prior to strong distractors, paralleled by decrea...
Source: eLife - May 7, 2020 Category: Biomedical Science Tags: Neuroscience Source Type: research

Cryo-EM structures and functional properties of CALHM channels of the human placenta
The transport of substances across the placenta is essential for the development of the fetus. Here, we were interested in the role of channels of the calcium homeostasis modulator (CALHM) family in the human placenta. By transcript analysis, we found the paralogs CALHM2, 4, and 6 to be highly expressed in this organ and upregulated during trophoblast differentiation. Based on electrophysiology, we found that activation of these paralogs differs from the voltage- and calcium-gated channel CALHM1. Cryo-EM structures of CALHM4 display decameric and undecameric assemblies with large cylindrical pore, while in CALHM6 a conform...
Source: eLife - May 6, 2020 Category: Biomedical Science Tags: Structural Biology and Molecular Biophysics Source Type: research

Encouraging cartilage production
A long non-coding RNA calledGRASLND is essential to help stem cells create stable cartilage. (Source: eLife)
Source: eLife - May 6, 2020 Category: Biomedical Science Tags: Stem Cells and Regenerative Medicine Source Type: research

Modelling the climatic suitability of Chagas disease vectors on a global scale
The Triatominae are a vector species forTrypanosoma cruzi, the aetiological agent of the neglected tropical Chagas disease. Their distribution stretches across Latin America, with some species occurring outside of the Americas. In particular, the cosmopolitan vector,Triatoma rubrofasciata, has already been detected in many Asian and African countries. We applied an ensemble forecasting niche modelling approach to project the climatic suitability of 11 triatomine species under current climate conditions on a global scale. Our results revealed potential hotspots of triatomine species diversity in tropical and subtropical reg...
Source: eLife - May 6, 2020 Category: Biomedical Science Tags: Ecology Source Type: research