Biology subjects

Johnson, A.

Publications and source records attributed to Johnson, A..

At least 73 records · Page 4Linked to original sources

bioRxiv2022

Lymphocyte networks are dynamic cellular communities in the immunoregulatory landscape of lung adenocarcinoma

Lymphocytes play a key role in immune surveillance of tumors, but our understanding of the spatial organization and physical interactions that facilitate lymphocyte anti-cancer functions is limited. Here, we used multiplexed imaging, quantitative spatial analysis, and machine learning to create high-definition maps of tumor-bearing lung tissues from a Kras/p53 (KP) mouse model and human resections. Networks of directly interacting lymphocytes ( lymphonets) emerge as a distinctive feature of the anti-cancer immune response. Lymphonets nucleate from small T-cell clusters and incorporate B cells with increasing size. CXCR3-mediated trafficking modulates lymphonet size and number, but neoantigen expression directs intratumoral localization. Lymphonets preferentially harbor TCF1+/PD1+ progenitor CD8 T cells involved in responses to immune checkpoint blockade (ICB). Upon treatment of mice with ICB therapy or a neoantigen-targeted vaccine, lymphonets retain progenitor and gain cytotoxic CD8 T-cell populations, likely via progenitor differentiation. These data show that lymphonets create a spatial environment supportive of CD8 T-cell anti-tumor responses.

cancer biology↗

bioRxiv2022

Global Protein-Turnover Quantification in Escherichia coli Reveals Cytoplasmic Recycling under Nitrogen-Limitation

Protein turnover is critical for proteostasis, but turnover quantification is challenging, and even in well-studied E. coli, proteome-wide measurements remain scarce. Here, we quantify the degradation rates of [~]3.2k E. coli proteins under 12 conditions by combining heavy isotope labeling with complement reporter ion quantification and find that cytoplasmic proteins are recycled when nitrogen is limited. We use knockout experiments to assign substrates to the known cytoplasmic ATP-dependent proteases. Surprisingly, none of these proteases are responsible for the observed cytoplasmic protein degradation in nitrogen limitation, suggesting that a major proteolysis pathway in E. coli remains to be discovered. Lastly, we show that protein degradation rates are generally independent of cell division rates. Thus, we introduce broadly applicable technology for protein turnover measurements and provide a rich resource for protein half-lives and protease substrates in E. coli, complementary to genomics data, that will allow researchers to decipher the control of proteostasis.

systems biology↗

bioRxiv2022

Super-resolution mass spectrometry enables rapid, accurate, and highly-multiplexed proteomics at the MS2-level

In tandem mass spectrometry (MS2)-based multiplexed quantitative proteomics, the complement reporter ion approaches (TMTc and TMTproC) were developed to eliminate the ratio-compression problem of conventional MS2 level approaches. Resolving all high m/z complement reporter ions ([~]6.32 mDa spaced) requires mass resolution and scan speeds above the performance levels of Orbitrap instruments. Therefore, complement reporter ion quantification with TMT/TMTpro reagents is currently limited to 5 out of 11 (TMT) or 9 out of 18 (TMTpro) channels ([~]1 Da spaced). We first demonstrate that a Fusion Lumos Orbitrap can resolve 6.32 mDa spaced complement reporter ions with standard acquisition modes extended with 3-second transients. We then implemented a super-resolution mass spectrometry approach using the least-squares fitting (LSF) method for processing Orbitrap transients to achieve shotgun proteomics-compatible scan rates. The LSF performance resolves the 6.32 mDa doublets for all TMTproC channels in the standard mass range with transients as short as [~]108 ms (Orbitrap resolution setting of 50 000 at m/z 200). However, we observe a slight decrease in measurement precision compared to 1 Da spacing with the 108 ms transients. With 256 ms transients (resolution of 120 000 at m/z 200), coefficients of variation are essentially indistinguishable from 1 Da samples. We thus demonstrate the feasibility of highly-multiplexed, accurate, and precise shotgun-proteomics at the MS2 level.

systems biology↗

bioRxiv2022

Socio-economic disadvantage is associated with alterations in brain wiring economy

The quality of a childs social and physical environment is a key influence on brain development, educational attainment and mental wellbeing. However, there still remains a mechanistic gap in our understanding of how environmental influences converge on changes in the brains developmental trajectory. In a sample of 145 children with structural diffusion tensor imaging data, we used generative network modelling to simulate the emergence of whole brain network organisation. We then applied data-driven clustering to stratify the sample according to socio-economic disadvantage, with one of the resulting clusters containing mostly children living below the poverty line. A formal comparison of the simulated networks from the generative model revealed that the computational principles governing network formation were subtly different for children experiencing socio-economic disadvantage, and that this resulted in significantly altered developmental timing of network modularity emergence. Children in the low socio-economic status (SES) group had a significantly slower time to peak modularity, relative to the higher SES group (t(69) = 3.02, P = 3.50 x 10-4, d = 0.491). In a subsequent simulation we showed that the alteration in generative properties increases the variability in wiring probabilities during network formation (KS test: D = 0.012, P < 0.001). One possibility is that multiple environmental influences such as stress, diet and environmental stimulation impact both the systematic coordination of neuronal activity and biological resource constraints, converging on a shift in the economic conditions under which networks form. Alternatively, it is possible that this stochasticity reflects an adaptive mechanism that creates "resilient" networks better suited to unpredictable environments. Author SummaryWe used generative network models to simulate macroscopic brain network development in a sample of 145 children. Within these models, network connections form probabilistically depending on the estimated "cost" of forming a connection, versus topological "value" that the connection would confer. Tracking the formation of the network across the simulation, we could establish the changes in global brain organisation measures such as integration and segregation. Simulations for children experiencing socio-economic disadvantage were associated with a shift in emergence of a topologically valuable network property, namely modularity.

neuroscience↗

bioRxiv2022

User-friendly electron microscopy protocols for the visualization of biological macromolecular complexes in three dimensions: Visualization of planta clathrin-coated vesicles at ultrastructural resolution

Biological systems are the sum of their dynamic 3-dimensional (3D) parts. Therefore, it is critical to study biological structures in 3D and at high resolutions to gain insights into their physiological functions. Electron microscopy of metal replicas of unroofed cells and isolated organelles has been a key technique to visualize intracellular structures at nanometer resolution. However, many of these protocols require specialized equipment and personnel to complete them. Here we present novel accessible protocols to analyze biological structures in unroofed cells and biochemically isolated organelles in 3D and at nanometer resolutions, focusing on Arabidopsis clathrin-coated vesicles (CCVs) - an essential trafficking organelle lacking detailed structural characterization due to their low preservation in classical electron microscopy techniques. First, we establish a protocol to visualize CCVs in unroofed cells using scanning-transmission electron microscopy (STEM) tomography, providing sufficient resolution to define the clathrin coat arrangements. Critically, the samples are prepared directly on electron microscopy grids, removing the requirement to use extremely corrosive acids, thereby enabling the use of this protocol in any electron microscopy lab. Secondly, we demonstrate this standardized sample preparation allows the direct comparison of isolated CCV samples with those visualized in cells. Finally, to facilitate the high-throughput and robust screening of metal replicated samples, we provide a deep learning analysis workflow to screen the pseudo 3D morphology of CCVs imaged with 2D modalities. Overall, we present accessible ways to examine the 3D structure of biological samples and provide novel insights into the structure of plant CCVs.

plant biology↗

bioRxiv2022

Morphogen Directed Coordination of GPCR Activity Promotes Primary Cilium Function for Downstream Signaling

Sonic Hedgehog (SHH) signaling is an essential driver of embryonic patterning that, when corrupted, leads to developmental disorders and cancers. SHH effector responses are organized through nonmotile primary cilia that grow and retract with the cell cycle and in response to distinct extracellular cues. Destabilization of primary cilium length corrupts SHH pathway regulation, which places significant pressure on SHH to maintain ciliary architecture. Herein, we investigate whether SHH signaling promotes ciliary length control. We reveal a signal crosstalk circuit induced by SHH activation of Phospholipase A2 (cPLA2) that drives ciliary EP4 signaling to stabilize primary cilium length. We demonstrate that chemical or genetic blockade of SHH-EP4 crosstalk leads to destabilized primary cilium cyclic AMP (cAMP) control, reduced ciliary length, and attenuated SHH pathway induction. Accordingly, we find that Ep -/- mice display shortened neuroepithelial primary cilia and altered SHH-dependent neuronal cell fate specification. Thus, SHH initiates a signaling crosstalk circuit that maintains primary cilium length for a robust downstream signaling response.

cell biology↗

bioRxiv2022

A single 2'-O-methylation of ribosomal RNA gates assembly of a functionalribosome

RNA modifications are widespread in biology, and particularly abundant in ribosomal RNA. However, the significance of these modifications is not well understood. We show that methylation of a single universally conserved nucleotide, in the catalytic center of the large subunit, gates ribosome assembly. Massively parallel mutational scanning of the essential nuclear GTPase Nog2 identified important interactions with ribosomal RNA helix 92, particularly with the methylated A-site base Gm2922. We found that 2-O-methylation of G2922 is needed for efficient nuclear export of the large subunit. Critically, we identified single amino acid changes in Nog2 that completely bypass its dependence on G2922 methylation. By solving the cryo-EM structure of the unmodified nascent subunit, we reveal how methylation flips Gm2922 into the active site channel of Nog2. This work demonstrates that a single RNA modification is a critical checkpoint in ribosome biogenesis, and suggests that RNA modifications can play an important role in regulation and assembly of macromolecular machines.

molecular biology↗

bioRxiv2021

The N-linker region of hERG1a upregulates hERG1b potassium channels

A major physiological role of hERG1 (human Ether-a-go-go-Related Gene) potassium channels is to repolarize cardiac action potentials. Two isoforms, hERG1a and hERG1b, associate to form the native cardiac IKr current in vivo. Inherited mutations in hERG1a or hERG1b cause prolonged cardiac repolarization, Long QT Syndrome and sudden death arrhythmia. hERG1a subunits assemble with and enhance the number of hERG1b subunits at the plasma membrane, but the mechanism for the increase in hERG1b by hERG1a is not well understood. Here, we report that the hERG1a N-terminal PAS (Per-Arnt-Sim) domain-N-linker region expressed in trans with hERG1b markedly increased hERG1b currents and increased biotin-labelled hERG1b protein at the membrane surface. hERG1b channels with a deletion of the 1b domain did not have a measurable increase in current or biotinylated protein when co-expressed with hERG1a PAS domain-N-linker regions indicating that the 1b domain was required for the increase in hERG1b. Using a biochemical pull-down interaction assay and a FRET hybridization experiment, we detected a direct interaction between the hERG1a PAS domain-N-linker region and the hERG1b N-terminal 1b domain. Using engineered deletions and alanine mutagenesis, we identified a short span of amino acids at positions 216-220 within the hERG1a N-linker region that were necessary for the upregulation of hERG1b. Taken together, we propose that direct structural interactions between the hERG1a N-linker region and the hERG1b N-terminal 1b domain increase hERG1b at the plasma membrane. Mechanisms that enhance hERG1b current would be anticipated to shorten action potentials, which could be anti-arrhythmic, and may point toward hERG1b or the hERG1a N-linker as molecular targets for therapy for Long QT syndrome. Significance StatementPotassium (K) ion channels help to control the membrane voltage in cardiac cells and are responsible for maintaining the normal rhythm of the heart. A decrease in the number of K channels causes cardiac arrhythmia and sudden death. One K channel (KCNH2b, hERG1b) weakly forms channels at the cell membrane. Here we describe a new mechanism for upregulation of hERG1b. An N-terminal domain of a related K channel (KCNH2a, hERG1a), expressed in trans as a separate piece, interacts with hERG1b and increases the number of hERG1b channels at the membrane. A loss of hERG channels is pro-arrhythmic and an increase in hERG channels is considered anti-arrhythmic, thus the hERG1a N-terminal domain is a potential therapeutic for cardiac arrhythmias.

physiology↗

bioRxiv2021

Brain endothelial antigen presentation detains CD8+ T cells at the blood-brain barrier leading to its breakdown

Blood-brain barrier (BBB) breakdown and immune cell infiltration into the central nervous system (CNS) are early hallmarks of multiple sclerosis (MS). High numbers of CD8+ T cells are found in MS lesions and antigen (Ag)-presentation at the BBB was proposed to promote CD8+ T-cell entry into the CNS. Employing live cell imaging and primary mouse brain microvascular endothelial cells (pMBMECs) as in vitro model of the BBB and a mouse model of CNS autoimmunity, we here show that pMBMECs process and present antigens leading to effector CD8+ T-cell differentiation. Under physiological flow, endothelial Ag-presentation prohibited CD8+ T-cell crawling and diapedesis leading to pMBMEC apoptosis. Reduced motility of Ag-specific CD8+ T cells was also observed in CNS microvessels in neuroinflammation in vivo. Luminal MHC class I Ag-presentation at the BBB thus prohibits CD8+ T-cell entry into the CNS and rather triggers CD8+ T cell mediated focal BBB breakdown.

immunology↗

bioRxiv2021

Proteomic Characterization of Isolated Arabidopsis Clathrin-Coated Vesicles Reveals Evolutionarily Conserved and Plant Specific Components

In eukaryotes, clathrin-coated vesicles (CCVs) facilitate the internalization of material from the cell surface as well as the movement of cargo in post-Golgi trafficking pathways. This diversity of functions is partially provided by multiple monomeric and multimeric clathrin adaptor complexes that provide compartment and cargo selectivity. The adaptor- protein AP-1 complex operates as part of the secretory pathway at the trans-Golgi network, while the AP-2 complex and the TPLATE complex (TPC) jointly operate at the plasma membrane to execute clathrin-mediated endocytosis. Key to our further understanding of clathrin-mediated trafficking in plants will be the comprehensive identification and characterization of the network of evolutionarily conserved and plant- specific core and accessory machinery involved in the formation and targeting of CCVs. To facilitate these studies, we have analyzed the proteome of enriched trans-Golgi network/early endosome-derived and endocytic CCVs isolated from dividing and expanding suspension-cultured Arabidopsis cells. Tandem mass spectrometry analysis results were validated by differential chemical labeling experiments to identify proteins co-enriching with CCVs. Proteins enriched in CCVs included previously characterized CCV components and cargos such as the vacuolar sorting receptors in addition to conserved and plant-specific components whose function in clathrin-mediated trafficking has not been previously defined. Notably, in addition to AP-1 and AP-2, all subunits of the AP-4 complex, but not AP-3 or AP-5, were found to be in high abundance in the CCV proteome. The association of AP-4 with suspension-cultured Arabidopsis CCVs is further supported via additional biochemical data.

cell biology↗

bioRxiv2021

Spike protein multiorgan tropism suppressed by antibodies targeting SARS-CoV-2

While there is clinical evidence of severe acute respiratory syndrome coronavirus 2 multiorgan tropism in severely infected coronavirus 19 patients, its unclear if there is differential multiorgan biodistribution and organ uptake in healthy young individuals, a group that usually has asymptomatic to moderate coronavirus 19 symptoms. In addition, for antibody therapies and vaccines that target the spike protein, its unclear if these reduce severe acute respiratory syndrome coronavirus 2 or spike protein multiorgan tropism equally. We used fluorescently labeled spike protein near infrared fluorescence to study viral behavior, using an in vivo dynamic imaging system, in young mice. We found a spike protein body-wide biodistribution followed by a slow regional elimination, except for the liver, which showed an accumulation. Spike protein uptake was highest for the lungs, and this was followed by kidney, heart and liver, but, unlike the choroid plexus, it was not detected in the brain parenchyma or cerebrospinal fluid. Thus, the brain vascular barriers were effective in restricting the entry of spike protein into brain parenchyma in young healthy mice. While both anti-angiotensin converting enzyme 2 and anti-spike protein antibodies suppressed spike protein biodistribution and organ uptake, anti-spike protein antibody was more effective. By extension, our data support the efficacy of these antibodies on severe acute respiratory syndrome coronavirus 2 biodistribution kinetics and multiorgan tropism that could determine coronavirus 19 organ-specific outcomes.

neuroscience↗

bioRxiv2021

The Genetic Architecture of Strawberry Yield and Fruit Quality Traits

Over the last two centuries breeders have drastically modified the fruit quality of strawberries through artificial selection. However, there remains significant variation in quality across germplasm with scope for further improvements to be made. We report extensive phenotyping of fruit quality and yield traits in a multi-parental strawberry population to allow genomic prediction and QTL identification, thereby enabling the description of genetic architecture to inform the efficacy of implementing advanced breeding strategies. A trade-off was observed between two essential traits: sugar content and class one yield. This result highlights an established dilemma for strawberry breeders and a need to uncouple the relationship, particularly under June-bearing, protected production systems comparable to this study. A large effect QTL was associated with perceived acidity and pH whereas multiple loci were associated with firmness, we therefore recommend the implementation of both MAS and genomic prediction to capture the observed variation respectively. Ultimately, our results suggest that the best method to improve strawberry yield is through selecting parental lines based upon the number of marketable fruit produced per plant. Strawberry number metrics were less influenced by environmental fluctuations and had a larger additive genetic component when compared to mass traits. As such, selecting using "number" traits should lead to faster genetic gain. Finally, we identify a large effect locus associated with an increase in class one fruit.

plant biology↗

bioRxiv2021

Gene regulatory networks underlying human microglia maturation

The fetal period is a critical time for brain development, characterized by neurogenesis, neural migration, and synaptogenesis1-3. Microglia, the tissue resident macrophages of the brain, are observed as early as the fourth week of gestation4 and are thought to engage in a variety of processes essential for brain development and homeostasis5-11. Conversely, microglia phenotypes are highly regulated by the brain environment12-14. Mechanisms by which human brain development influences the maturation of microglia and microglia potential contribution to neurodevelopmental disorders remain poorly understood. Here, we performed transcriptomic analysis of human fetal and postnatal microglia and corresponding cortical tissue to define age-specific brain environmental factors that may drive microglia phenotypes. Comparative analysis of open chromatin profiles using bulk and single-cell methods in conjunction with a new computational approach that integrates epigenomic and single-cell RNA-seq data allowed decoding of cellular heterogeneity with inference of subtype- and development stage-specific transcriptional regulators. Interrogation of in vivo and in vitro iPSC-derived microglia models provides evidence for roles of putative instructive signals and downstream gene regulatory networks which establish human-specific fetal and postnatal microglia gene expression programs and potentially contribute to neurodevelopmental disorders.

genomics↗

bioRxiv2021

Crowdsourcing biocuration: the Community Assessment of Community Annotation with Ontologies (CACAO)

Experimental data about known gene functions curated from the primary literature have enormous value for research scientists in understanding biology. Using the Gene Ontology (GO), manual curation by experts has provided an important resource for studying gene function, especially within model organisms. Unprecedented expansion of the scientific literature and validation of the predicted proteins have increased both data value and the challenges of keeping pace. Capturing literature-based functional annotations is limited by the ability of biocurators to handle the massive and rapidly growing scientific literature. Within the community-oriented wiki framework for GO annotation called the Gene Ontology Normal Usage Tracking System (GONUTS), we describe an approach to expand biocuration through crowdsourcing with undergraduates. This multiplies the number of high-quality annotations in international databases, enriches our coverage of the literature on normal gene function, and pushes the field in new directions. From an intercollegiate competition judged by experienced biocurators, Community Assessment of Community Annotation with Ontologies (CACAO), we have contributed nearly 5000 literature-based annotations. Many of those annotations are to organisms not currently well-represented within GO. Over a ten-year history, our community contributors have spurred changes to the ontology not traditionally covered by professional biocurators. The CACAO principle of relying on community members to participate in and shape the future of biocuration in GO is a powerful and scalable model used to promote the scientific enterprise. It also provides undergraduate students with a unique and enriching introduction to critical reading of primary literature and acquisition of marketable skills. Significance StatementThe primary scientific literature catalogs the results from publicly funded scientific research about gene function in human-readable format. Information captured from those studies in a widely adopted, machine-readable standard format comes in the form of Gene Ontology annotations about gene functions from all domains of life. Manual annotations based on inferences directly from the scientific literature, including the evidence used to make such inferences, represents the best return on investment by improving data accessibility across the biological sciences. To supplement professional curation, our CACAO project enabled annotation of the scientific literature by community annotators, in this case undergraduates, which resulted in contribution of thousands of validated entries to public resources. These annotations are now being used by scientists worldwide.

bioinformatics↗

bioRxiv2021

The TPLATE complex mediates membrane bending during plant clathrin-mediated endocytosis

Clathrin-mediated endocytosis in plants is an essential process but the underlying mechanisms are poorly understood, not least because of the extreme intracellular turgor pressure acting against the formation of endocytic vesicles. In contrast to other models, plant endocytosis is independent of actin, indicating a mechanistically distinct solution. Here, by using biochemical and advanced microscopy approaches, we show that the plant-specific TPLATE complex acts outside of endocytic vesicles as a mediator of membrane bending. Cells with disrupted TPLATE fail to generate spherical vesicles, and in vitro biophysical assays identified protein domains with membrane bending capability. These results redefine the role of the TPLATE complex as a key component of the evolutionarily distinct mechanism mediating membrane bending against high turgor pressure to drive endocytosis in plant cells. One Sentence SummaryWhile plant CME is actin independent, we identify that the evolutionarily ancient octameric TPLATE complex mediates membrane bending against high turgor pressure in plant clathrin-mediated endocytosis.

cell biology↗

bioRxiv2020

Disruptions in effort-based decision-making following acute optogenetic stimulation of ventral tegmental area dopamine cells

The dopamine system has been implicated in decision-making particularly when associated with effortful behavior. We examined acute optogenetic stimulation of dopamine cells in the ventral tegmental area (VTA) as mice engaged in an effort-based decision-making task. Tyrosine hydroxylase-Cre mice were injected with Cre-dependent ChR2 or eYFP control in VTA. While eYFP control mice showed effortful discounting, stimulation of dopamine cells in ChR2 mice disrupted effort-based decision-making by reducing choice towards the lever associated with a preferred outcome and greater effort. Surprisingly, disruptions in effortful discounting were observed in subsequent test sessions conducted in the absence of optogenetic stimulation, however during these sessions ChR2 mice displayed enhanced high choice responding across trial blocks. These findings suggest increases in VTA dopamine cell activity can disrupt effort-based decision-making in distinct ways dependent on the timing of optogenetic stimulation.

neuroscience↗

bioRxiv2020

Interactions between axon-like projections extended by Drosophila Follicle Stem Cells dictate cell fate decisions

Stem cells cycle between periods of quiescence and proliferation to promote healthy tissue aging. Once proliferation is initiated, mechanisms that control the balance between self-renewal and differentiation must be engaged to ensure maintenance of stem cell pools until the next quiescent cycle occurs. Here, we demonstrate that dynamic axon-like projections extended by Follicle Stem Cells (FSCs) in the Drosophila ovary control the self-renewal-differentiation balance. Known axon growth regulators still life and sickie are necessary and sufficient for FSC projection growth, mediating organization of germline cyst architecture during follicle formation, controlling targeting of projections to FSCs or germ cells, and regulating expression of the cell fate determinants Eyes Absent (Eya) and Castor (Cas). Our results support a model in which FSC projections function similarly to axons, providing structural organization to a dynamic organ while mediating communication between distinct cell types to effect the key cell fate decision to self-renew or differentiate.

developmental biology↗

bioRxiv2020

Candidate Explorer: a tool for discovery, evaluation, and display of mutations causing significant immune phenotypes

When applied to immunity, forward genetic studies use meiotic mapping to provide strong statistical evidence that a particular mutation is causative of a particular immune phenotype. Notwithstanding this, co-segregation of multiple mutations, occasional unawareness of mutations, and paucity of homozygotes may lead to erroneous declarations of cause and effect. We sought to improve the selection of authentic causative mutations using a machine learning software tool, Candidate Explorer (CE), which integrates 65 data features into a single numeric score, mathematically convertible to the likelihood of verification of any putative mutation-phenotype association. CE has identified most genes within which mutations can be causative of flow cytometric phenovariation in Mus musculus. The majority of these genes were not previously known to support immune function or homeostasis. Mouse geneticists will find CE data informative in identifying causative mutations within quantitative trait loci, while clinical geneticists may use CE to help connect causative variants with rare heritable diseases of immunity, even in the absence of linkage information. CE displays integrated mutation, phenotype, and linkage data, and is freely available for query online.

genetics↗