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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2019

Isolated occurrences of membrane perturbation by mechanosensing from weakly aggregating silver nanoparticles

Silver nanoparticles (AgNPs) have wide-ranging applications, including as additives in consumer products and in medical diagnostics and therapy. Therefore understanding how AgNPs interact with biological systems is important for ascertaining any potential health risks due to the likelihood of high levels of human exposure. Besides any severe, acute effects, it is desirable to understand more subtle interactions that could lead to milder, chronic health impacts. Nanoparticles are small enough to be able to enter biological cells and interfere with their internal biochemistry. The initial contact between nanoparticle and cell is at the plasma membrane. To gain fundamental mechanistic insight into AgNP-membrane interactions, we investigate these phenomena in minimal model systems using a wide-range of biophysical approaches applied to lipid vesicles. We find a strong dependence on the medium composition, where colloidally stable AgNPs in a glucose buffer have negligible effect on the membrane. However, at a physiological salt concentrations, the AgNPs start to weakly aggregate and sporadic but significant membrane perturbation events are observed. Under these latter conditions, transient poration and structural remodelling of some vesicle membranes is observed. We observe that the fluidity of giant vesicle membranes universally decreases by an average of 16% across all vesicles. However, we observe a small population of vesicles display a significant change in mechanical properties with lower bending rigidity and higher membrane tension. Therefore we argue that the isolated occurrences of membrane perturbation by AgNPs are due to low probability mechanosensing events of AgNP aggregation at the membrane.\n\nGRAPHICAL ABSTRACT\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC=\"FIGDIR/small/623678v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (54K):\norg.highwire.dtl.DTLVardef@196aa51org.highwire.dtl.DTLVardef@87c12borg.highwire.dtl.DTLVardef@9b2096org.highwire.dtl.DTLVardef@7877f6_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2019

Frustration and Fidelity in Influenza Genome Assembly

The genome of the influenza virus consists of eight distinct single-stranded RNA segments, each encoding proteins essential for the viral life cycle. When the virus infects a host cell these segments must be replicated and packaged into new budding virions. The viral genome is assembled with remarkably high fidelity: experiments reveal that most virions contain precisely one copy of each of the eight RNA segments. Cell-biological studies suggest that genome assembly is mediated by specific reversible and irreversible interactions between the RNA segments and their associated proteins. However, the precise inter-segment interaction network remains unresolved. Here we computationally predict that tree-like irreversible interaction networks guarantee high-fidelity genome assembly, while cyclic interaction networks lead to futile or frustrated off-pathway products. We test our prediction against multiple experimental datasets. We find that tree-like networks capture the nearest-neighbor statistics of RNA segments in packaged virions, as observed by EM tomography. Just eight tree-like networks (of a possible 262,144) optimally capture both the nearest-neighbor data as well as independently measured RNA-RNA contact propensities. These eight do not include the previously-proposed hub-and-spoke and linear networks. Rather, each predicted network combines hub-like and linear features, consistent with evolutionary models of interaction gain and loss.

biophysics↗

bioRxiv2019

Mitochondrial dynamics quantitatively revealed by STED nanoscopy with an enhanced squaraine variant probe

Mitochondria play a critical role in generating energy to support the entire lifecycle of biological cells, yet it is still unclear how their morphological structures evolve to regulate their functionality. Conventional fluorescence microscopy can only provide [~]300 nm resolution, which is insufficient to visualize mitochondrial cristae. Here, we developed an enhanced squaraine variant dye (MitoESq-635) to study the dynamic structures of mitochondrial cristae in live cells at superresolution. The low saturation intensity and high photostability make it ideal for long-term, high-resolution STED nanoscopy. We demonstrate the time-lapsed imaging of the mitochondrial inner membrane over 50 minutes in living HeLa cells at 35.2 nm resolution for the first time. The forms of the cristae during mitochondrial fusion and fission can be clearly resolved. Our study demonstrates the emerging capability of optical STED nanoscopy to investigate intracellular physiological processes at nanoscale resolution for long periods of time with minimal phototoxicity.

biophysics↗

bioRxiv2019

Fluorescent reporters for functional analysis in rice leaves

Fluorescent reporters have facilitated non-invasive imaging in multiple plant species and thus allowed analysis of processes ranging from gene expression and protein localization through to cellular patterning. However, in rice, a globally important crop and model species, there are relatively few reports of fluorescent proteins being used in leaves. Fluorescence imaging is particularly difficult in the rice leaf blade, likely due to a high degree of light scattering in this tissue. To address this, we investigated approaches to improve deep imaging in mature rice leaf blades. We found that ClearSee treatment, which has previously been used to visualise fluorescent reporters in whole tissues of plants, led to improved imaging in rice. Removing epidermal and subtending mesophyll cell layers was faster than ClearSee, and also reduced light scattering such that imaging of fluorescent proteins in deeper leaf layers was possible. To expand the range of fluorescent proteins suitable for imaging in rice, we screened twelve whose spectral profiles spanned most of the visible spectrum. This identified five proteins, mTurquoise2, mClover3, mNeonGreen, mKO{kappa} and tdTomato that are robustly expressed and visible in mesophyll cells of stably transformed plants. Using microparticle bombardment, we show that mTurquoise2 and mNeonGreen can be used for simultaneous multicolour imaging of different sub-cellular compartments. Overall, we conclude that mTurquoise2, mClover3, mNeonGreen, mKO{kappa} and tdTomato are suitable for high resolution live imaging of rice leaves, both after transient and stable transformation. Along with the rapid microparticle bombardment method, which allows transient transformation of major cell types in the leaf blade, these fluorescent reporters should greatly facilitate the analysis of gene expression and cell biology in rice.\n\nOne sentence summaryWe report five fluorescent reporters suitable for functional analysis in rice leaves.

plant biology↗

bioRxiv2019

The conical shape of DIM lipids promotes Mycobacterium tuberculosis infection of macrophages

Phthiocerol dimycocerosate (DIM) is a major virulence factor of the pathogen Mycobacterium tuberculosis (Mtb). While this lipid promotes the entry of Mtb into macrophages, which occurs via phagocytosis, its molecular mechanism of action is unknown. Here, we combined biophysical, cell biology, and modelling approaches to reveal the molecular mechanism of DIM action on macrophage membranes leading to the first step of Mtb infection. MALDI-TOF mass spectrometry showed that DIM molecules are transferred from the Mtb envelope to macrophage membranes during infection. Multi-scale molecular modeling and 31P-NMR experiments revealed that DIM adopts a conical shape in membranes and aggregate in the stalks formed between two opposing lipid bilayers. Infection of macrophages pre-treated with lipids of various shapes uncovered a general role for conical lipids in promoting phagocytosis. Taken together, these results reveal how the molecular shape of a mycobacterial lipid can modulate the biological function of macrophages.

biophysics↗

bioRxiv2019

Biophysical principles of choanoflagellate self-organization

Inspired by the patterns of multicellularity in choanoflagellates, the closest living relatives of animals, we quantify the biophysical processes underlying the morphogenesis of rosette colonies in the choanoflagellate Salpingoeca rosetta. We find that rosettes reproducibly transition from an early stage of 2D growth to a later stage of 3D growth, despite the underlying stochasticity of the cell lineages. We postulate that the extracellular matrix (ECM) exerts a physical constraint on the packing of proliferating cells, thereby sculpting rosette morphogenesis. Our perturbative experiments coupled with biophysical simulations demonstrates the fundamental importance of a basally-secreted ECM for rosette morphogenesis. In addition, this yields a morphospace for the shapes of these multicellular colonies, consistent with observations of a range of choanoflagellates. Overall, our biophysical perspective on rosette development complements previous genetic perspectives and thus helps illuminate the interplay between cell biology and physics in regulating morphogenesis.\n\nSignificance statementComparisons among animals and their closest living relatives, the choanoflagellates, have begun to shed light on the origin of animal multicellularity and development. Here we complement previous genetic perspectives on this process by focusing on the biophysical principles underlying colony morphology and morphogenesis. Our study reveals the crucial role of the extracellular matrix in shaping the colonies and leads to a phase diagram that delineates the range of morphologies as a function of the biophysical mechanisms at play.

biophysics↗

bioRxiv2019

Engineered illumination devices for optogenetic control of cellular signaling dynamics

Spatially and temporally varying patterns of morphogen signals during development drive cell fate specification at the proper location and time. However, current in vitro methods typically do not allow for precise, dynamic, spatiotemporal control of morphogen signaling and are thus insufficient to readily study how morphogen dynamics impact cell behavior. Here we show that optogenetic Wnt/{beta}-catenin pathway activation can be controlled at user-defined intensities, temporal sequences, and spatial patterns using novel engineered illumination devices for optogenetic photostimulation and light activation at variable amplitudes (LAVA). The optical design of LAVA devices was optimized for uniform illumination of multi-well cell culture plates to enable high-throughput, spatiotemporal optogenetic activation of signaling pathways and protein-protein interactions. Using the LAVA devices, variation in light intensity induced a dose-dependent response in optoWnt activation and downstream Brachyury expression in human embryonic stem cells (hESCs). Furthermore, time-varying and spatially localized patterns of light revealed tissue patterning that models embryonic presentation of Wnt signals in vitro. The engineered LAVA devices thus provide a low-cost, user-friendly method for high-throughput and spatiotemporal optogenetic control of cell signaling for applications in developmental and cell biology.

bioengineering↗

bioRxiv2019

PTMProphet: Fast and Accurate Mass Modification Localization for the Trans-Proteomic Pipeline

Spectral matching sequence database search engines commonly used on mass spectrometry-based proteomics experiments excel at identifying peptide sequence ions, and in addition, possible sequence ions carrying post-translational modifications (PTMs), but most do not provide confidence metrics for the exact localization of those PTMs when several possible sites are available. Localization is absolutely required for downstream molecular cell biology analysis of PTM function in vitro and in vivo. Therefore, we developed PTMProphet, a free and open-source software tool integrated into the Trans-Proteomic Pipeline, which reanalyzes identified spectra from any search engine for which pepXML output is available to provide localization confidence to enable appropriate further characterization of biologic events. Localization of any type of mass modification (e.g., phosphorylation) is supported. PTMProphet applies Bayesian mixture models to compute probabilities for each site/peptide spectrum match where a PTM has been identified. These probabilities can be combined to compute a global false localization rate at any threshold to guide downstream analysis. We describe the PTMProphet tool, its underlying algorithms and demonstrate its performance on ground-truth synthetic peptide reference datasets, one previously published small dataset, one new larger dataset, and also on a previously published phospho-enriched dataset where the correct sites of modification are unknown. Data have been deposited to ProteomeXchange with identifier PXD013210.\n\nO_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

bioinformatics↗

bioRxiv2019

The intracellular symbiont Wolbachia enhances recombination in a dose-dependent manner

Wolbachia pipientis is an intracellular alphaproteobacterium that infects 40-60% of insect species and is well known for host reproductive manipulations. Although Wolbachia are primarily maternally transmitted, evidence of horizontal transmission can be found in incongruent host-symbiont phylogenies and recent acquisitions of the same Wolbachia strain by distantly related species. Parasitoids and predator-prey interactions may indeed facilitate the transfer of Wolbachia between insect lineages but it is likely that Wolbachia are acquired via introgression in many cases. Many hypotheses exist as to explain Wolbachia prevalence and penetrance such as nutritional supplementation, protection from parasites, protection from viruses, or straight up reproductive parasitism. Using classical genetics we show that Wolbachia increase recombination in infected lineages across two genomic intervals. This increase in recombination is titer dependent as the wMelPop variant, which infects at higher load in Drosophila melanogaster, increases recombination 5% more than the wMel variant. In addition, we also show that Spiroplasma poulsonii, the other bacterial intracellular symbiont of Drosophila melanogaster, does not induce an increase in recombination. Our results suggest that Wolbachia infection specifically alters host recombination landscape in a dose dependent manner.\n\nArticle SummaryThe ubiquitous bacterial symbiont Wolbachia is known to alter host reproduction through manipulation of host cell biology, protect from pathogens, and supplement host nutrition. In this work we show that Wolbachia specifically increases host recombination in a dose dependent manner. Flies harboring Wolbachia exhibit elevated rates of recombination across the 2nd and X chromosomes and this increase is proportional to their Wolbachia load. In contrast, another intracellular symbiont, Spiroplasma, does not lead to an increase in recombination across the intervals tested. Our results point to a specific effect of Wolbachia infection that may have a significant effect on infected insect populations.

genetics↗

bioRxiv2019

ORANGE: A CRISPR/Cas9-based genome editing toolbox for epitope tagging of endogenous proteins in neurons

The correct subcellular distribution of protein complexes establishes the complex morphology of neurons and is fundamental to their functioning. Thus, determining the dynamic distribution of proteins is essential to understand neuronal processes. Fluorescence imaging, in particular super-resolution microscopy, has become invaluable to investigate subcellular protein distribution. However, these approaches suffer from the limited ability to efficiently and reliably label endogenous proteins. We developed ORANGE: an Open Resource for the Application of Neuronal Genome Editing, that mediates targeted genomic integration of fluorescent tags in neurons. This toolbox includes a knock-in library for in-depth investigation of endogenous protein distribution, and a detailed protocol explaining how knock-in can be developed for novel targets. In combination with super-resolution microscopy, ORANGE revealed the dynamic nanoscale organization of endogenous neuronal signaling molecules, synaptic scaffolding proteins, and neurotransmitter receptors. Thus, ORANGE enables quantitation of expression and distribution for virtually any protein in neurons at high resolution and will significantly further our understanding of neuronal cell biology.

neuroscience↗

bioRxiv2019

Natural LXRβ agonist stigmasterol confers protection against excitotoxicity after hypoxia-reoxygenation (H/R) injury via regulation of mitophagy in primary hippocampal neurons

Ischemic brain injury represents insufficient oxygen supply to the brain and further damage occurs upon reoxygenation due to elevated intracellular levels excitatory neurotransmitter glutamate and subsequent production of reactive oxygen species (ROS) which has long been related to neuronal cell death of hippocampus brain region. Previously, using cell biological assay and transcriptomics analysis we reported that naturally occurring phytosterol Stigmasterol (ST) promotes brain development and function through the enhancement of neuronal cytoarchitectural complexity and functional maturation in rat hippocampal neurons by induction of immediate early genes (IEGs). In the present study we investigated the STs role in neuroprotection and found that ST also dose-dependently increased the neuronal viability in hypoxia reoxygenation (H/R) induced injury at hippocampal culture. ST, at an optimal concentration of 20 M, significantly reduced the transport of vesicular glutamate (VGULT1), synaptic vesicle pool size, expression of GluN2B, rate of ROS formation (DCFDA) but restore mitochondrial membrane potential (JC1) and DNA fragmentation (H2AX) against H/R induced injury. More interestingly, ST also significantly induces the expression of autophagy marker protein LC3BII and the adaptor protein P62 but not HSC70 which indicates STs capability of induction of chaperon independent autophagy at H/R treated cultures. Furthermore densitometric analysis reveals ST also significantly increases PINK1 (PTEN induced protein kinase 1) expression therefore, indicates its role in mitophagy. In addition, molecular dynamic simulations study indicates that ST bind to LXR{beta} and forms hydrogen bonds with ASN239, GLU281, ARG319, THR316, SER278, ASN239 and SER278 residues at high occupancy with GLU281(20.21%) and ARG319 (21.04%,) residues, which is necessary for sterol binding to the LXR{beta}. Taken together these findings suggest that neuroprotective effect of ST might be associated with anti-excitatory and anti-oxidative actions on CNS neurons and could be a promising drug candidate for the treatment or prevention of ischemic stroke related neurological disorders.

neuroscience↗

bioRxiv2019

Dynamic measurement of cytosolic pH and uncovers the role of the vacuolar transporter AtCLCa in the control of cytosolic pH.

Ion transporters are key players of cellular processes. The mechanistic properties of ion transporters have been well elucidated by biophysical methods. Meanwhile the understanding of their exact functions in the whole cell homeostasis is limited by the difficulty to monitor their activity in vivo. The development of biosensors to track subtle changes in intracellular parameters provides an invaluable key to tackle this challenging issue. Here, we adapted the use of a dual biosensor using guard cells as experimental model to visualize the impact on the cytosol of anion transport from intracellular compartments. To image the activity of AtCLCa, a vacuolar NO3-/H+ exchanger regulating stomata aperture in Arabidopsis thaliana, we expressed a genetically encoded biosensor, ClopHensor allowing monitoring the dynamics of cytosolic anion concentration and pH. We first show that ClopHensor is not only a Cl- but also a NO3- sensor. We were then able to unravel and quantify the variations of NO3- and pH in the cytosol. Our data show that AtCLCa activity modifies cytosolic pH and NO3-, demonstrating that the transport activity of a vacuolar exchanger has a profound impact on cytosolic homeostasis. We propose that a major function of this endomembrane transporter is to adjust cytosolic conditions to cellular needs. This opens a novel perspective on the function of intracellular transporters of the CLC family in eukaryotes: not only controlling the intra organelle lumen but also actively modifying cytosolic conditions.\n\nSignificanceIntracellular transporters are key actors in cell biological processes. Their disruption causes major physiological defects. The role of intracellular ion transporters is usually seen through an \"intra organelle\" lens, meanwhile their potential action on cytosolic ion homeostasis is still a black box. The case of a plant CLC is used as a model to uncover the missing link between the regulation of conditions inside the vacuole and inside the cytosol. The development of an original live imaging workflow to simultaneously measure pH and anion dynamics in the cytosol reveals the role of an Arabidopsis thaliana CLC, AtCLCa, in the modification of cytosolic pH. Our data highlight an unsuspected function of endomembrane transporters in the regulation of cytosolic pH.

plant biology↗

bioRxiv2019

Symmetry breaking during morphogenesis of a mechanosensory organ

Actively regulated symmetry breaking, which is ubiquitous in biological cells, underlies phenomena such as directed cellular movement and morphological polarization. Here we investigate how an organ-level polarity pattern emerges through symmetry breaking at the cellular level during the formation of a mechanosensory organ. Combining theory, genetic perturbations, and in vivo imaging assisted by deep learning, we study the development and regeneration of the fluid-motion sensors in the zebrafishs lateral line. We find that two interacting symmetry-breaking events -- one mediated by biochemical signaling and the other by cellular mechanics -- give rise to a novel form of collective cell migration, which produces a mirror-symmetric polarity pattern in the receptor organ.

biophysics↗

bioRxiv2019

Alpha herpesvirus egress and spread from neurons uses constitutive secretory mechanisms independent of neuronal firing activity

Alpha herpesviruses naturally infect the peripheral nervous system, and can spread to the central nervous system causing severe deadly or debilitating disease. Because alpha herpesviruses spread along synaptic circuits, and infected neurons exhibit altered electrophysiology and increased spontaneous firing, we hypothesized that alpha herpesviruses use activity-dependent synaptic vesicle-like regulated secretory mechanisms for egress and spread from neurons. To address this hypothesis, we used a compartmentalized primary neuron culture system to measure egress and spread of pseudorabies virus (PRV), pharmacological and optogenetics approaches to modulate neuronal firing activity, and a live-cell fluorescence microscopy assay to directly visualize the exocytosis of individual virus particles from infected neurons. Using tetrodotoxin to silence neuronal activity, we observed no inhibition of virus spread, and using potassium chloride or optogenetics to elevate neuronal activity, we also show no increase in virus spread. Using a live-cell fluorescence microscopy method to directly measure virus egress from infected neurons, we observed no association between virus particle exocytosis and intracellular Ca2+ signaling. Finally, we observed virus particle exocytosis occurs in association with constitutive secretory Rab GTPases, Rab6a and Rab8a, not Rab proteins that are associated with the Ca2+-regulated secretory pathway in neurons, Rab3a and Rab11a. Therefore, we conclude that alpha herpesvirus egress and spread is independent of neuronal activity and Ca2+ signaling because virus particle exocytosis uses constitutive secretory mechanisms in neurons.\n\nAuthor SummaryAlpha herpesviruses, including important human pathogens Herpes Simplex Virus 1 and 2, and Varicella-Zoster Virus, are among the very few viruses that naturally infect the nervous system. These viruses cause recurrent herpetic and zosteriform lesions, peripheral neuropathies, and deadly or debilitating central nervous system diseases. Many of the molecular and cellular mechanisms of viral egress and spread remain unknown, particularly in the context of specialized neuronal cell biology. Our results indicate that elevated firing activity of infected neurons is not functionally or mechanistically linked to virus egress and spread; therefore, therapies targeting peripheral neuropathic symptoms, elevated neuronal activity, and synaptic vesicle secretory mechanisms are unlikely to affect virus spread in the nervous system.

microbiology↗

bioRxiv2019

SAPH-ire TFx: A Machine Learning Recommendation Method and Webtool for the Prediction of Functional Post-Translational Modifications

Protein post-translational modifications (PTMs) are a rapidly expanding feature class of significant importance in cell biology. Due to a high burden of experimental proof, the number of functional PTMs in the eukaryotic proteome is currently underestimated. Furthermore, not all PTMs are functionally equivalent. Therefore, computational approaches that can confidently recommend the functional potential of experimental PTMs are essential. To address this challenge, we developed SAPH-ire TFx (https://saphire.biosci.gatech.edu/): a multi-feature neural network model and web resource optimized for recommending experimental PTMs with high potential for biological impact. The model is rigorously benchmarked against independent datasets and alternative models, exhibiting unmatched performance in the recall of known functional PTM sites and the recommendation of PTMs that were later confirmed experimentally. An analysis of feature contributions to model outcome provides further insight on the need for multiple rather than single features to capture the breadth of functional data in the public domain. Contactmtorres35@gatech.edu Supplementary InformationSee Tables S1-S6 & Figures S1-S4. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=189 SRC="FIGDIR/small/731026v2_figS1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@4dbe8dorg.highwire.dtl.DTLVardef@1936d5org.highwire.dtl.DTLVardef@15eef8aorg.highwire.dtl.DTLVardef@1c07cea_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure S1.C_FLOATNO ROC curves at different KFSC thresholds. (unfurled in Figure 1E). C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/731026v2_figS4.gif" ALT="Figure 4"> View larger version (20K): org.highwire.dtl.DTLVardef@48e892org.highwire.dtl.DTLVardef@9cf372org.highwire.dtl.DTLVardef@32da4dorg.highwire.dtl.DTLVardef@99fd89_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure S4.C_FLOATNO Model recall and recommendation comparison for PTMs associated with validated functional SLiMs from the ELM resource. C_FIG

bioinformatics↗

bioRxiv2019

Essential dynamic interdependence of FtsZ and SepF for Z-ring and septum formation in Corynebacterium glutamicum

The mechanisms of Z-ring assembly and regulation in bacteria are poorly understood, particularly in non-model organisms. Actinobacteria, one of the largest bacterial phyla that includes the deadly human pathogen Mycobacterium tuberculosis, lack the canonical FtsZ-membrane anchors as well as all positive and negative Z-ring regulators described for E. coli. Here we investigate the physiological function of Corynebacterium glutamicum SepF, the only cell division-associated protein from Actinobacteria known to directly interact with the conserved C-terminal tail of FtsZ but whose actual mode of action in cytokinesis is yet to be elucidated. We used a mechanistic cell biology approach to unveil the essential interdependence of FtsZ and SepF required for the formation of a functional Z-ring in the actinobacterial model organism C. glutamicum. The crystal structure of the SepF-FtsZ complex reveals a hydrophobic FtsZ-binding pocket, which defines the SepF homodimer as the functional unit, and a reversible oligomerization interface regulated via an alpha helical switch. FtsZ filaments and lipid membranes have opposing effects on SepF polymerization, leading to a complex dynamic role of the protein at the division site, involving FtsZ bundling, Z-ring tethering and membrane reshaping activities that are needed for proper Z-ring assembly and function.

microbiology↗

bioRxiv2019

Single nucleus RNA sequencing maps acinar cell states in a human pancreas cell atlas

Molecular evidence of cellular heterogeneity in the human exocrine pancreas has not been established, due to the local concentration of hydrolytic enzymes that can rapidly degrade cells and RNA upon resection. Here we innovated single-nucleus RNA sequencing protocols, and profiled more than 120,000 cells from adult and neonatal human donors to create the first comprehensive atlas of human pancreas cells, including epithelial and non-epithelial constituents. Adult and neonatal pancreata shared common features, including the presence of previously undetected acinar subtypes, but also showed marked differences in the composition of the endocrine, endothelial, and immune compartments. Spatial cartography, including cell proximity mapping through in situ sequencing, revealed dynamic developmental cell topographies in the endocrine and exocrine pancreas. Our human pancreas cell atlas can be interrogated to understand pancreatic cell biology, and provides a crucial reference set for future comparisons with diseased tissue samples to map the cellular foundations of pancreatic diseases.

genomics↗

bioRxiv2019

Complex+: Aided Decision-Making for the Study of Protein Complexes

Proteins are the chief effectors of cell biology and their functions are typically carried out in the context of multi-protein assemblies; large collections of such interacting protein assemblies are often referred to as interactomes. Knowing the constituents of protein complexes is therefore important for investigating their molecular biology. Many experimental methods are capable of producing data of use for detecting and inferring the existence of physiological protein complexes. Each method has associated pros and cons, affecting the potential quality and utility of the data. Numerous informatic resources exist for the curation, integration, retrieval, and processing of protein interactions data. While each resource may possess different merits, none are definitive and few are wieldy, potentially limiting their effective use by non-experts. In addition, contemporary analyses suggest that we may still be decades away from a comprehensive map of a human protein interactome. Taken together, we are currently unable to maximally impact and improve biomedicine from a protein interactome perspective - motivating the development of experimental and computational techniques that help investigators to address these limitations. Here, we present a resource intended to assist investigators in (i) navigating the cumulative knowledge concerning protein complexes and (ii) forming hypotheses concerning protein interactions that may yet lack conclusive evidence, thus (iii) directing future experiments to address knowledge gaps. To achieve this, we integrated multiple data-types/different properties of protein interactions from multiple sources and after applying various methods of regularization, compared the protein interaction networks computed to those available in the EMBL-EBI Complex Portal, a manually curated, gold-standard catalog of macromolecular complexes. As a result, our resource provides investigators with reliable curation of bona fide and candidate physical interactors of their protein or complex of interest, prompting due scrutiny and further validation when needed. We believe this information will empower a wider range of experimentalists to conduct focused protein interaction studies and to better select research strategies that explicitly target missing information.

bioinformatics↗