Biology subjects

Brown, A.

Publications and source records attributed to Brown, A..

At least 109 records · Page 6Linked to original sources

bioRxiv2022

iPheGWAS : an intelligent computational framework to integrate and visualise genome-phenome wide association studies

Estimating the genetic correlations by LDSC is computationally demanding and visualising multiple GWAS results along with their genetic relationships is restricted. This study developed iPheGWAS, a novel approach which applied hierarchical clustering to GWAS summary statistics to (i) calculate their genetic relatedness, and (ii) enable three-dimensional visualisation of multiple ordered GWAS plots. Simulation and real-world data analysis demonstrated that when investigating genetic relationships among multiple phenotypes, iPheGWAS can deliver comparable results with LDSC but with 8 times faster computational speed. It can also provide novel findings in studying genetically-correlated comorbidities, such as mental illness and rheumatoid arthritis.

bioinformatics↗

bioRxiv2021

Test-retest reproducibility of in vivo magnetization transfer ratio and saturation index in mice at 9.4 Tesla

BackgroundMagnetization transfer saturation (MTsat) imaging was developed to reduce T1 dependence and improve specificity to myelin compared to the widely used MT ratio (MTR), while maintaining a feasible scan time. Knowledge of MTsat reproducibility is necessary to apply MTsat in preclinical neuroimaging. PurposeTo assess the test-retest reproducibility of MTR and MTsat in the mouse brain at 9.4 T and calculate sample sizes required to detect various effect sizes. Study TypeProspective Animal ModelC57Bl/6 Mouse Model (6 females and 6 males, aged 12 - 14 weeks) Field Strength/SequenceMagnetization Transfer Imaging at 9.4 T AssessmentAll mice were scanned at two timepoints (5 days apart). MTR and MTsat maps were analyzed using mean region-of-interest (ROI), and whole brain voxel-wise analysis. Statistical TestsBland-Altman plots assessed biases between test and retest measurements. Test-retest reproducibility was evaluated via between and within-subject coefficients of variation (CV). Sample sizes required were calculated (at a 95 % significance level and power of 80 %), given various minimum detectable effect sizes, using both between and within-subject approaches. ResultsBland-Altman plots showed negligible biases between test and retest sessions. ROI-based and voxel-wise CVs revealed high reproducibility for both MTR (ROI: CVs < 8 %) and MTsat (ROI: CVs < 10 %). With a sample size of 6, changes on the order of 15% can be detected in MTR and MTsat, both between and within subjects, while smaller changes (6 - 8 %) require sample sizes of 10 - 15 for MTR, and 15 - 20 for MTsat. Data ConclusionMTsat exhibits comparable reproducibility to MTR, while providing sensitivity to myelin with less T1 dependence than MTR. Our findings suggest both MTR and MTsat can detect moderate changes, common in pathologies, with feasible preclinical sample sizes.

neuroscience↗

bioRxiv2021

Extensive sequence divergence of non-coding regions between Aspergillus fumigatus, a major fungal pathogen of humans, and its relatives

Invasive aspergillosis is a deadly fungal disease; more than 400,000 patients are infected worldwide each year and the mortality rate can be as high as 50-95%. Of the [~]450 species in the genus Aspergillus only a few are known to be clinically relevant, with the major pathogen Aspergillus fumigatus being responsible for [~]50% of all invasive mold infections. Genomic comparisons of A. fumigatus to other Aspergillus species have historically focused on protein-coding regions. However, most A. fumigatus genes, including those that modulate its virulence, are also present in non-pathogenic close relatives of A. fumigatus. Our hypothesis is that differential gene regulation - mediated through the non-coding regions upstream of genes transcription start sites - contributes to A. fumigatus pathogenicity. To begin testing this, we compared non-coding regions up to 500 base pairs upstream of the first codon of single-copy orthologous genes from the two A. fumigatus reference strains Af293 and A1163 and eight closely related Aspergillus section Fumigati species. We found that non-coding regions showed extensive sequence variation and lack of homology across species. By examining the evolutionary rates of both protein-coding and non-coding regions in a subset of orthologous genes with highly conserved non-coding regions across the phylogeny, we identified 418 genes, including 25 genes known to modulate A. fumigatus virulence, whose non-coding regions exhibit a different rate of evolution in A. fumigatus. Examination of sequence alignments of these non-coding regions revealed numerous instances of insertions, deletions, and other types of mutations of at least a few nucleotides in A. fumigatus compared to its close relatives. These results show that closely related Aspergillus species that vary greatly in their pathogenicity exhibit extensive non-coding sequence variation and identify numerous changes in non-coding regions of A. fumigatus genes known to contribute to virulence.

evolutionary biology↗

bioRxiv2021

Sciara coprophila larvae upregulate DNA repair pathways and downregulate developmental regulators in response to ionizing radiation

Robust DNA damage prevention and repair strategies are crucial to faithful reproduction and inheritance of the genetic material. Although many molecular pathways that respond to DNA damage are well conserved through evolution, the quality and effectiveness of these systems can vary between species. Studies dating back for nearly a century document that the dark-winged fungus gnat Sciara coprophila (Order: Diptera; sub-order: Nematocera) is relatively resistant to irradiation-induced mutations that cause visible phenotypes when compared to the fruit fly Drosophila melanogaster (Order: Diptera; sub-order: Brachycera). However, the molecular responses to irradiation for S. coprophila have yet to be analyzed. To address this gap, we first characterized the effects of ionizing radiation on S. coprophila throughout its life cycle. Our data show that developing S. coprophila embryos are highly sensitive to even low doses of gamma-irradiation, whereas larvae can tolerate up to 80 Gy and still retain their ability to develop to adulthood with a developmental delay of 5 to 8 extra days in the larval stage. To survey the genes involved in the early transcriptional response to irradiation, we compared RNA-seq profiles of larvae with and without radiation treatment. Our analysis showed that 327 genes are differentially expressed in irradiated larvae, with 232 genes upregulated and 95 genes downregulated relative to controls. The upregulated genes were enriched for DNA damage response genes, including those involved in DNA repair, cell cycle arrest, and apoptosis, whereas the down-regulated genes were enriched for developmental regulators, consistent with the developmental delay observed in irradiated larvae. Thus, our study has laid the groundwork to further dissect how Sciara copes with radiation-induced damage.

genomics↗

bioRxiv2021

A Developmentally Prometastatic Niche to Hepatoblastoma in Neonatal Liver mediated by the Cxcl1/Cxcr2 Axis

Background and RationaleHepatoblastoma (HB) is the most common pediatric liver cancer. Its predominant occurrence in very young children led us to investigating whether the neonatal liver provides a protumorigenic niche to HB development. MethodsHB development was compared between orthotopic transplantation models established in postnatal day 5 and 60 mice (P5Tx and P60Tx models). Single-cell RNA-sequencing was performed using tumor and liver tissues from both models and the top candidate cell types and genes identified are investigated for their roles in HB cell growth, migration, and survival. ResultsWe found that various HB cell lines including HepG2 cells were consistently and considerably more tumorigenic and metastatic in the P5Tx model than in the P60Tx models. Sc-RNAseq of the P5Tx and P60Tx HepG2 models revealed that the P5Tx tumor was more hypoxic and had a larger number of activated hepatic stellate cells (aHSCs) in the tumor-surrounding liver which express significantly higher levels of Cxcl1 than those from the P60Tx model. We found these differences were developmentally present in normal P5 and P60 liver. We showed that the Cxcl1/Cxcr2 axis mediated HB cell migration and was critical to HB cell survival under hypoxia. Treating HepG2 P60Tx model with recombinant CXCL1 protein induced intrahepatic and pulmonary metastasis and CXCR2 knockout in HepG2 cells abolished their metastatic potential in the P5Tx model. Lastly, we showed that in metastatic HB patient tumors there was a similar larger population of aHSCs in the tumor-surrounding liver than in localized tumors, and tumor hypoxia was uniquely associated with HB patient prognosis among pediatric cancers. ConclusionWe demonstrated that the neonatal liver provides a prometastatic niche to HB development via the Cxcl1/Cxcr2 axis.

cancer biology↗

bioRxiv2021

Test-retest reproducibility of in vivo oscillating gradient and microscopic anisotropy diffusion MRI in mice at 9.4 Tesla

Background and PurposeMicrostructure imaging with advanced diffusion MRI (dMRI) techniques have shown increased sensitivity and specificity to microstructural changes in various disease and injury models. Oscillating gradient spin echo (OGSE) dMRI, implemented by varying the oscillating gradient frequency, and microscopic anisotropy ({micro}A) dMRI, implemented via tensor valued diffusion encoding, may provide additional insight by increasing sensitivity to smaller spatial scales and disentangling fiber orientation dispersion from true microstructural changes, respectively. The aims of this study were to characterize the test-retest reproducibility of in vivo OGSE and {micro}A dMRI metrics in the mouse brain at 9.4 Tesla and provide estimates of required sample sizes for future investigations. MethodsEight adult C57Bl/6 mice were scanned twice (5 days apart). Each imaging session consisted of multifrequency OGSE and {micro}A dMRI protocols. Metrics investigated included {micro}A, isotropic and anisotropic kurtosis, and the diffusion dispersion rate ({Lambda}), which explores the power-law frequency dependence of mean diffusivity. The dMRI metric maps were analyzed with mean region-of-interest (ROI) and whole brain voxel-wise analysis. Bland-Altman plots and coefficients of variation (CV) were used to assess the reproducibility of OGSE and {micro}A metrics. Furthermore, we estimated sample sizes required to detect a variety of effect sizes. ResultsBland-Altman plots showed negligible biases between test and retest sessions. ROI-based CVs revealed high reproducibility for both {micro}A (CVs < 8 %) and {Lambda} (CVs < 15 %). Voxel-wise CV maps revealed high reproducibility for {micro}A (CVs [~] 10 %), but low reproducibility for OGSE metrics (CVs [~] 50 %). ConclusionMost of the {micro}A dMRI metrics are reproducible in both ROI-based and voxel-wise analysis, while the OGSE dMRI metrics are only reproducible in ROI-based analysis. {micro}A and {Lambda} may provide sensitivity to subtle microstructural changes (4 - 8 %) with feasible sample sizes (10 - 15).

neuroscience↗

bioRxiv2021

Heterogeneity in the transcriptional response of the human pathogen Aspergillus fumigatus to the antifungal agent caspofungin

Aspergillus fumigatus is the main causative agent of invasive pulmonary aspergillosis (IPA), a severe disease that affects immunosuppressed patients worldwide. The fungistatic drug caspofungin is the second line therapy against IPA but has increasingly been used against clinical strains that are resistant to azoles, the first line antifungal therapy. In high concentrations, caspofungin induces a tolerance phenotype with partial reestablishment of fungal growth called caspofungin paradoxical effect (CPE), resulting from a change in the composition of the cell wall. An increasing number of studies has shown that different isolates of A. fumigatus exhibit phenotypic heterogeneity, including heterogeneity in their CPE response. To gain insights into the underlying molecular mechanisms of CPE response heterogeneity, we analyzed the transcriptomes of two A. fumigatus reference strains, Af293 and CEA17, exposed to low and high caspofungin concentrations. We found that there is a core transcriptional response that involves genes related to cell wall remodeling processes, mitochondrial function, transmembrane transport, and amino acid and ergosterol metabolism, and a variable response related to secondary metabolite (SM) biosynthesis and iron homeostasis. Specifically, we show here that the overexpression of a SM pathway that works as an iron chelator extinguishes the CPE in both backgrounds, whereas iron depletion is detrimental for the CPE in Af293 but not in CEA17. We next investigated the function of the transcription factor CrzA, whose deletion was previously shown to result in heterogeneity in the CPE response of the Af293 and CEA17 strains. We found that CrzA constitutively binds to and modulates the expression of several genes related to processes involved in caspofungin tolerance, and that crzA deletion differentially impacts the SM production and growth of Af293 and CEA17. As opposed to the {Delta}crzACEA17 mutant, the {Delta}crzAAf293 mutant fails to activate cell wall remodeling genes upon caspofungin exposure, which most likely severely affects its macrostructure and extinguishes its CPE. This work describes how heterogeneity in the response to an antifungal agent between A. fumigatus strains stems from heterogeneity in the function of a transcription factor and its downstream target genes.

microbiology↗

bioRxiv2021

Define protein variant functions with high-complexity mutagenesis libraries and enhanced mutation detection software

Pooled variant expression libraries can test the phenotypes of thousands of variants of a gene in a single multiplexed experiment. In a library encoding all single-amino-acid substitutions of a protein, each variant differs from its reference only at a single codon-position located anywhere along the coding sequence. Consequently, accurately identifying these variants by sequencing is a major technical challenge. A popular but expensive brute-force approach is to divide the pool of variants into multiple smaller sub-libraries that each contains variants of a small region and that must each be constructed and screened individually, but that can then be PCR-amplified and fully sequenced with a single read to allow direct readout of variant abundance. Here we present an approach to screen very large variant libraries with mutations spanning a wide region in a single pool, including library design criteria and mutant-detection algorithms that permit reliable calling and counting of variants from large-scale sequencing data.

systems biology↗

bioRxiv2021

Endoplasmic Reticulum morphological regulation by RTN4/NOGO modulates neuronal regeneration by slowing luminal transport

Cell and tissue functions rely on an elaborate intracellular transport system responsible for distributing bioactive molecules with high spatiotemporal accuracy. The tubular network of the Endoplasmic Reticulum (ER) constitutes a system for the delivery of luminal solutes it stores, including Ca2+, across the cell periphery. The physical nature and factors underlying the ERs functioning as a fluidics system are unclear. Using an improved ER transport visualisation methodology combined with optogenetic Ca2+ dynamics imaging, we observed that ER luminal transport is modulated by natural ER tubule narrowing and dilation, directly proportional to the amount of an ER membrane morphogen, Reticulon 4 (RTN4). Consequently, the ER morphoregulatory effect of RTN4 defines ERs capacity for peripheral Ca2+ delivery and thus controls axonogenesis. Excess RTN4 limited ER luminal transport, Ca2+ release and iPSC-derived cortical neurons axonal extension, while RTN4 elimination reversed the effects. SummaryIntracellular transport through the lumen of the ER network is modulated through narrowing/dilation of ER tubules by a membrane morphogen - RTN4, a process controlling axonogenesis by limiting the delivery of ER-stored Ca2+.

cell biology↗

bioRxiv2021

ePlant in 2021: New Species, Viewers, Data Sets, and Widgets

ePlant was introduced in 2017 for exploring large Arabidopsis thaliana data sets from the kilometre to nanometre scales. In the past four years we have used the ePlant framework to develop ePlants for 15 agronomically-important species: maize, poplar, tomato, Camelina sativa, soybean, potato, barley, Medicago truncatula, eucalyptus, rice, willow, sunflower, Cannabis sativa, wheat and sugarcane. We also updated the interface to improve performance and accessibility, and added two new views to the Arabidopsis ePlant - the Navigator and Pathways viewers. The former shows phylogenetic relationships between homologs in other species and their expression pattern similarities, with links to view data for those genes in the respective ePlants. The latter shows Plant Reactome metabolic reactions. We also describe new Arabidopsis data sets including single cell RNA-seq data from roots, and how to embed ePlant eFP expression pictographs into any web page.

bioinformatics↗

bioRxiv2021

Paradoxical Roles of Peritumoral Hepatic Stellate Cells in Liver Tumorigenesis in Mice: Retarding Tumor Growth and Promoting Tumor Dissemination

Intrahepatic cholangiocarcinoma (iCCA) is an aggressive type of primary liver cancer characterized by its highly desmoplastic stroma. Compared to the ample knowledge on cancer-associated fibroblast residing within iCCA tumor mass, little is known about the fibrotic response from the tumor-surrounding liver and its role in iCCA development. In this study, we find a significant accumulation of peritumoral myofibroblasts (pMFs) in both patient iCCA and tumors from an iCCA orthotopic allograft mouse model. Using tumor and liver spheroid coculture we show that iCCA-liver interaction induces rapid pMF accumulation at the interface. We find pMFs placed around iCCA spheroids exert a strong suppressive effect on tumor cells growth, in contrast to the pro-proliferative effect of MFs mixed within tumor spheroids. However, prolonged iCCA-pMF interaction elicits tumor cell dissemination in vitro. We find an upregulation of Vcam1 in tumor cells in the early phase of iCCA-pMF interaction both in vitro and in vivo which is downregulated when tumor cells disseminate. Blocking Vcam1 activity in iCCA allograft mouse models slows primary tumor growth but lead to increased tumor metastasis. Our data suggest that pMFs are beyond simple pro- or anti-tumorigenic in iCCA, with the ability to suppress tumor growth but elicit tumor cell dissemination.

cancer biology↗

bioRxiv2021

Behavioral fingerprints predict insecticide and anthelmintic mode of action

Novel invertebrate-killing compounds are required in agriculture and medicine to overcome resistance to existing treatments. Because insecticides and anthelmintics are discovered in phenotypic screens, a crucial step in the discovery process is determining the mode of action of hits. Visible whole-organism symptoms are combined with molecular and physiological data to determine mode of action. However, manual symptomology is laborious and requires symptoms that are strong enough to see by eye. Here we use high-throughput imaging and quantitative phenotyping to measure C. elegans behavioral responses to compounds and train a classifier that predicts mode of action with an accuracy of 88% for a set of ten common modes of action. We also classify compounds within each mode of action to discover pharmacological relationships that are not captured in broad mode of action labels. High-throughput imaging and automated phenotyping could therefore accelerate mode of action discovery in invertebrate-targeting compound development and help to refine mode of action categories.

systems biology↗

bioRxiv2021

Regulation of neurofilament length and transport by a dynamic cycle of polymer severing and annealing

Neurofilaments are space-filling cytoskeletal polymers that are transported into axons where they accumulate during development to expand axon caliber. We previously described novel severing and end-to-end annealing mechanisms in neurons that alter neurofilament length. To explore the functional significance of neurofilament length, we developed a long-term multi-field time-lapse method to track the movement of fluorescently tagged neurofilaments in axons of cultured neurons for up to 30 minutes. All filaments moved rapidly, but long filaments paused and reversed more often, resulting in little net movement, whereas short filaments moved persistently for long distances, pausing and reversing less often. Long filaments severed more frequently, generating shorter filaments, and short filaments annealed more frequently, generating longer filaments. Thus, neurofilament length is regulated by a dynamic cycle of severing and annealing and this influences neurofilament transport. Site-directed mutagenesis to mimic phosphorylation at four known phosphorylation sites in the head domain of neurofilament protein L generated shorter neurofilaments that moved more frequently. A non-phosphorylatable mutant had the opposite effect. Treatment of cultured neurons with activators of protein kinase A, which phosphorylates three of these sites, increased neurofilament severing. This effect was blocked by the non-phosphorylatable mutant. We propose that focal destabilization of intermediate filaments by N-terminal phosphorylation of their constituent polypeptides at specific locations along their length may be a general enzymatic mechanism for severing this class of cytoskeletal polymers. Our data suggest a novel mechanism for the control of neurofilament transport and accumulation in axons based on regulation of neurofilament polymer length. SUMMARYNeurofilaments are space-filling cytoskeletal polymers that are transported into axons where they accumulate to expand axon caliber, which is an important determinant of axonal conduction velocity. We reported previously that neurofilaments can lengthen and shorten by novel end-to-end annealing and severing mechanisms. Here, we show that neurofilament annealing and severing are robust phenomena in cultured neurons that act antagonistically to dynamically regulate neurofilament length, which in turn regulates their transport. In addition, we present evidence for a novel enzymatic mechanism of intermediate filament severing based on site-directed phosphorylation of the neurofilament subunit proteins. We propose that modulation of neurofilament length by annealing and severing may be a mechanism for the regulation of neurofilament transport and accumulation in axons.

cell biology↗

bioRxiv2020

A β-catenin-driven switch in TCF/LEF transcription factor binding to DNA targets sites promotes commitment of mammalian nephron progenitor cells

The canonical Wnt pathway transcriptional co-activator {beta}-catenin regulates self-renewal and differentiation of mammalian nephron progenitor cells (NPCs). We modulated {beta}-catenin levels in NPC cultures using the GSK3 inhibitor CHIR9902 (CHIR) to examine opposing developmental actions of {beta}-catenin. Low CHIR-mediated maintenance and expansion of NPCs is independent of direct engagement of TCF/LEF/ {beta}-catenin transcriptional complexes at low-CHIR dependent cell-cycle targets. In contrast, in high CHIR, TCF7/LEF1/{beta}-catenin complexes replaced TCF7L1/TCF7L2 binding on enhancers of differentiation-promoting target genes. Chromosome confirmation studies showed pre-established promoter-enhancer connections to these target genes in NPCs. High CHIR-associated de novo looping was observed in positive transcriptional feedback regulation to the canonical Wnt pathway. Thus, {beta}-catenins direct transcriptional role is restricted to the induction of NPCs where rising {beta}-catenin levels switch inhibitory TCF7L1/TCF7L2 complexes to activating LEF1/TCF7 complexes at primed gene targets poised for rapid initiation of a nephrogenic program.

developmental biology↗

bioRxiv2020

Chromosome painting in cultivated banana and their wild relatives (Musa spp.) reveals differences in chromosome structure

Edible banana cultivars are diploid, triploid or tetraploid hybrids which originated by natural cross hybridization between subspecies of diploid Musa acuminata, or between M. acuminata and diploid M. balbisiana. Participation of two other wild diploid species M. schizocarpa and M. textilis was also indicated by molecular studies. Fusion of gametes with structurally different chromosome sets may give rise to progenies with structural chromosome heterozygosity and reduced fertility due to aberrant chromosome pairing and unbalanced chromosome segregation. Only a few translocations have been classified on the genomic level so far and a comprehensive molecular cytogenetic characterization of cultivars and species of the family Musaceae is still lacking. FISH with chromosome-arm specific oligo painting probes was used for comparative karyotype analysis in a set of wild Musa species and edible banana clones. The results revealed large differences in chromosome structure discriminating individual accessions. These results permitted identification of putative progenitors of cultivated clones and clarified genomic constitution and evolution of aneuploid banana clones, which seem to be common among the polyploid banana accessions. New insights into the chromosome organization and structural chromosome changes will be a valuable asset in breeding programs, particularly in selection of appropriate parents for cross hybridization. HighlightOligo painting FISH revealed chromosomal translocations in subspecies of Musa acuminata (A genome), their intra-specific hybrids as well as in M. balbisiana (B genome) and in interspecific hybrid clones originating from cross hybridization between M. acuminata and M. balbisiana

plant biology↗

bioRxiv2020

Purification of recombinant SARS-CoV-2 spike, its receptor binding domain, and CR3022 mAb for serological assay

Serology testing for COVID-19 is highly attractive because of the relatively short diagnosis time and the ability to test for an active immune response against the SARS-CoV-2. In many types of serology tests, the sensitivity and the specificity are directly influenced by the quality of the antigens manufactured. Protein purification of these recombinantly expressed viral antigens [e.g., spike and its receptor binding domain (RBD)] is an important step in the manufacturing process. Simple and high-capacity protein purification schemes for spike, RBD, and CR3022 mAb, recombinantly expressed in CHO and HEK293 cells, are reported in this article. The schemes consist of an affinity chromatography step and a desalting step. Purified proteins were validated in ELISA-based serological tests. Interestingly, extracellular matrix proteins [most notably heparan sulfate proteoglycan (HSPG)] were co-purified from spike-expressing CHO culture with a long cultivation time. HSPG-spike interaction could play a functional role in the pathology and the pathogenesis of SARS-CoV-2 and other coronaviruses.

biochemistry↗

bioRxiv2020

Intrauterine growth restriction causes cellular, molecular, and behavioral deficits consistent with abnormal dentate gyrus neurogenesis in mice

BackgroundChildren born with intrauterine growth restriction (IUGR) are at increased risk for cognitive impairment including learning and memory deficits. Dentate gyrus (DG) granule neurons relay cortical information into the hippocampus proper for memory formation, and their production is highly dependent on environmental signals. However, it is unknown whether IUGR affects DG neurogenesis, and thus provides a potential mechanism underlying abnormal learning and memory function. MethodsUsing a hypertensive disease of pregnancy mouse model of IUGR, we assessed multiple behaviors, quantified neural stem and progenitor cells (NSPCs) and developing neurons in the DG, and characterized transcriptional effects on molecular pathways in the hippocampus. ResultsWe found that the predominant behavioral phenotype in IUGR offspring, short-term implicit learning and memory deficits, was associated with accelerated DG neurogenesis and NSPC depletion. Consistent with known molecular regulators of DG neurogenesis, we also found strong evidence for decreased Wnt pathway activity following IUGR. ConclusionWe have discovered that postnatal memory deficits are associated with accelerated NSPC differentiation following IUGR, a phenotype that could be explained by decreased Wnt signaling.

neuroscience↗

bioRxiv2020

Cryo-EM structure of an activated GPCR-G protein complex in lipid nanodiscs

G protein coupled receptors (GPCRs) are the largest superfamily of transmembrane proteins and the targets of over 30% of currently marketed pharmaceuticals1,2. Although several structures have been solved for GPCR-G protein complexes3-17, structural studies of the complex in a physiological lipid membrane environment are lacking. Additionally, most previous studies required additional antibodies/nanobodies and/or engineered G proteins for complex stabilization. In the absence of a native complex structure, the underlying mechanism of G protein activation leading to GDP/GTP exchange remains unclear. Here, we report cryo-EM structures of lipid bilayer-bound complexes of neurotensin, neurotensin receptor 1, and Gi1{beta}1{gamma}1 protein in two conformational states, resolved to 4.1 and 4.2 [A] resolution. The structures were determined without any stabilizing antibodies/nanobodies, and thus provide a native-like platform for understanding the structural basis of GPCR-G protein complex formation. Our structures reveal an extended network of protein-protein interactions at the GPCR-G protein interface compared to in detergent micelles, defining roles for the lipid membrane in modulating the structure and dynamics of complex formation, and providing a molecular explanation for the stronger interaction between GPCR and G protein in lipid bilayers. We propose a detailed allosteric mechanism for GDP release, providing new insights into the activation of G proteins for downstream signaling under near native conditions.

molecular biology↗