Biology subjects

Johnson, M. E.

Publications and source records attributed to Johnson, M. E..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Temporal control by co-factors prevents kinetic trapping in retroviral Gag lattice assembly

For retroviruses like HIV to proliferate, they must form virions shaped by the self-assembly of Gag polyproteins into a rigid lattice. This immature Gag lattice has been structurally characterized and reconstituted in vitro, revealing the sensitivity of lattice assembly to multiple co-factors. Due to this sensitivity, the energetic criterion for forming stable lattices is unknown, as are their corresponding rates. Here, we use a reaction-diffusion model designed from the cryo-ET structure of the immature Gag lattice to map a phase diagram of assembly outcomes controlled by experimentally constrained rates and free energies, over experimentally relevant timescales. We find that productive assembly of complete lattices in bulk solution is extraordinarily difficult due to the large size of this [~]3700 monomer complex. Multiple Gag lattices nucleate before growth can complete, resulting in loss of free monomers and frequent kinetic trapping. We therefore derive a time-dependent protocol to titrate or activate the Gag monomers slowly within the solution volume, mimicking the biological roles of co-factors. This general strategy works remarkably well, yielding productive growth of self-assembled lattices for multiple interaction strengths and binding rates. By comparing to the in vitro assembly kinetics, we can estimate bounds on rates of Gag binding to Gag and the cellular co-factor IP6. Our results show that Gag binding to IP6 can provide the additional time-delay necessary to support smooth growth of the immature lattice with relatively fast assembly kinetics, mostly avoiding kinetic traps. Our work provides a foundation for predicting and disrupting formation of the immature Gag lattice via targeting specific protein- protein binding interactions.

biophysics↗

bioRxiv2022

Defects in the HIV immature lattice support essential lattice remodeling within budded virions

For HIV virions to become infectious, the immature lattice of Gag polyproteins attached to the virion membrane must be cleaved. Cleavage cannot initiate without the protease formed by the homo-dimerization of domains linked to Gag. However, only 5% of the Gag polyproteins, termed Gag-Pol, carry this protease domain, and they are embedded within the structured lattice. The mechanism of Gag-Pol dimerization is unknown. Here, we use reaction-diffusion simulations of the immature Gag lattice as derived from experimental structures, showing that dynamics of the lattice on the membrane is unavoidable due to the missing 1/3 of the spherical protein coat. These dynamics allow for Gag-Pol molecules carrying the protease domains to detach and reattach at new places within the lattice. Surprisingly, dimerization timescales of minutes or less are achievable for realistic binding energies and rates despite retaining most of the large-scale lattice structure. We derive a formula allowing extrapolation of timescales as a function of interaction free energy and binding rate, thus predicting how additional stabilization of the lattice would impact dimerization times. We further show that during assembly, dimerization of Gag-Pol occurs stochastically and therefore must be actively suppressed to prevent early activation. By direct comparison to recent biochemical measurements within budded virions, we find that only moderately stable hexamer contacts (-12kBT<{Delta}G<-8kBT) retain both the dynamics and lattice structures that are consistent with experiment. These dynamics are likely essential for proper maturation, and our models quantify and predict lattice dynamics and protease dimerization timescales that define a key step in understanding formation of infectious viruses. Statement of SignificanceFor retroviruses such as HIV-1, the Gag polyprotein assembles an immature lattice that ensures successful budding from the cell plasma membrane. The first step in the subsequent maturation requires a pair of protease domains embedded within the lattice to form a homodimer. We show here that this homo-dimerization can proceed within minutes despite involving a small subset of Gag monomers, due to the incompleteness of the immature lattice. Using reaction-diffusion simulations, we quantify timescales of first dimerization events between the protease domains and define a formula to extrapolate across a range of energies and rates. Our models illustrate how protein contacts can be weakened to disrupt lattice assembly or stabilized to slow the remodeling essential for viral infectivity.

biophysics↗

bioRxiv2021

Quantitative single-cell transcriptome-based ranking of engineered AAVs in human retinal explants

Gene therapy is a rapidly developing field, and adeno-associated virus (AAV) is a leading viral vector candidate for therapeutic gene delivery. Newly engineered AAVs with improved abilities are now entering the clinic. It has proven challenging, however, to predict the translational potential of gene therapies developed in animal models, due to cross-species differences. Human retinal explants are the only available model of fully developed human retinal tissue, and are thus important for the validation of candidate AAV vectors. In this study, we evaluated 18 wildtype and engineered AAV capsids in human retinal explants using a recently developed single-cell RNA-Seq AAV engineering pipeline (scAAVengr). Human retinal explants retained the same major cell types as fresh retina, with similar expression of cell-specific markers, except for a cone population with altered expression of cone-specific genes. The efficiency and tropism of AAVs in human explants were quantified, with single-cell resolution. The top performing serotypes, K91, K912, and 7m8, were further validated in non-human primate and human retinal explants. Together, this study provides detailed information about the transcriptome profiles of retinal explants, and quantifies the infectivity of leading AAV serotypes in human retina, accelerating the translation of retinal gene therapies to the clinic. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/461256v2_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1e110ddorg.highwire.dtl.DTLVardef@11a2c7eorg.highwire.dtl.DTLVardef@15f1633org.highwire.dtl.DTLVardef@1aa741a_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2021

Mapping the Energy Landscape of PROTAC-Mediated Protein-protein Interactions

One of the principal difficulties in computational modeling of macromolecules is the vast conformational space that arises out of large numbers of atomic degrees of freedom. This problem is a familiar issue in the area of protein-protein docking, where models of protein complexes are generated from the monomeric subunits. Although restriction of molecular flexibility is a commonly used approximation that decreases the dimensionality of the problem, the seemingly endless number of possible ways two binding partners can interact generally necessitates the use of further approximations to explore the search space. Recently, growing interest in using computational tools to build predictive models of PROTAC-mediated complexes has led to the application of state-of-the-art protein-protein docking techniques to tackle this problem. Additionally, the atomic degrees of freedom introduced by flexibility of linkers used in the construction of PROTACs further expands the configurational search space, a problem that can be tackled with conformational sampling tools. However, repurposing existing tools to carry out protein-protein docking and linker conformer generation independently results in extensive sampling of structures incompatible with PROTAC-mediated complex formation. Here we show that it is possible to restrict the search to the space of protein-protein conformations that can be bridged by a PROTAC molecule with a given linker composition by using a cyclic coordinate descent algorithm to position PROTACs into complex-bound configurations. We use this methodology to construct a picture of the energy landscape of PROTAC-mediated interactions in a model test case, and show that the global minimum lies in the space of native-like conformations.

bioinformatics↗

bioRxiv2021

Projecting genetic associations through gene expression patterns highlights disease etiology and drug mechanisms

Genes act in concert with each other in specific contexts to perform their functions. Determining how these genes influence complex traits requires a mechanistic understanding of expression regulation across different conditions. It has been shown that this insight is critical for developing new therapies. In this regard, the role of individual genes in disease-relevant mechanisms can be hypothesized with transcriptome-wide association studies (TWAS), which have represented a significant step forward in testing the mediating role of gene expression in GWAS associations. However, modern models of the architecture of complex traits predict that gene-gene interactions play a crucial role in disease origin and progression. Here we introduce PhenoPLIER, a computational approach that maps gene-trait associations and pharmacological perturbation data into a common latent representation for a joint analysis. This representation is based on modules of genes with similar expression patterns across the same conditions. We observed that diseases were significantly associated with gene modules expressed in relevant cell types, and our approach was accurate in predicting known drug-disease pairs and inferring mechanisms of action. Furthermore, using a CRISPR screen to analyze lipid regulation, we found that functionally important players lacked TWAS associations but were prioritized in trait-associated modules by PhenoPLIER. By incorporating groups of co-expressed genes, PhenoPLIER can contextualize genetic associations and reveal potential targets missed by single-gene strategies.

bioinformatics↗

bioRxiv2021

The bitter end: T2R bitter receptor agonists elevate nuclear calcium and induce apoptosis in non-ciliated airway epithelial cells

Bitter taste receptors (T2Rs) localize to airway motile cilia and initiate innate immune responses in retaliation to bacterial quorum sensing molecules (acyl-homoserine lactones and quinolones). Activation of T2Rs leads to calcium-driven NO production that increases cilia beating and directly kills bacteria. Several airway diseases, including chronic rhinosinusitis, COPD, and cystic fibrosis, are characterized by epithelial remodeling, including loss of motile cilia and/or squamous metaplasia. To understand the function of T2Rs within the altered landscape of airway disease, we studied T2Rs in non-ciliated airway cell lines and primary cells de-differentiated to a squamous phenotype. In differentiated cells, T2Rs localize to cilia, however in de-differentiated, non-ciliated cells they localize to the nucleus. Cilia and nuclear import utilize many shared proteins, thus in the absence of motile cilia some T2Rs may target to the nucleus. T2R agonists selectively elevated both nuclear and mitochondrial calcium through a G-protein-coupled receptor, phospholipase C, and InsP3 receptor-dependent mechanism. Additionally, T2R agonists decreased nuclear cAMP, increased nitric oxide, and increased cGMP, consistent with T2R signaling. Furthermore, exposure to T2R agonists led to nuclear calcium-induced mitochondrial depolarization and caspase activation. T2R agonists induced apoptosis in primary bronchial and nasal cells differentiated at air-liquid interface but then induced to a squamous phenotype by apical submersion. Air-exposed well-differentiated cells did not die. This T2R-induced apoptosis may be a last-resort defense against infection, possibly when bacteria have breached the epithelial barrier and reach non-ciliated cells below. However, it may also increase susceptibility of de-differentiated or remodeled epithelia to damage by bacterial metabolites. Moreover, the T2R-activated apoptosis pathway occurs in airway cancer cells. T2Rs may thus contribute to microbiome-tumor cell crosstalk in airway cancers. T2R agonists may also be useful topical therapeutics (e.g., delivered by nasal rinse or nebulizer) for activating airway cancer cell apoptosis without killing surrounding differentiated tissue.

physiology↗

bioRxiv2021

A continuum membrane model predicts curvature sensing by helix insertion

Protein domains, such as ENTH (Epsin N-terminal homology) and BAR (bin/amphiphysin/rvs), contain amphipathic helices that drive preferential binding to curved membranes. However, predicting how the physical parameters of these domains control this curvature sensing behavior is challenging due to the local membrane deformations generated by the nanoscopic helix on the surface of a large sphere. To overcome this challenge, we here use a deformable continuum model that accounts for the physical properties of the membrane and the helix insertion to predict curvature sensing behavior and is in good agreement with existing experimental data. Specifically, we show that the insertion can be modeled as a local change to the membranes spontaneous curvature,[Formula]. Using physically reasonable ranges of the membrane bending modulus [kcy], and a [Formula] of [~]0.2-0.3 nm-1, this approach provides excellent agreement with the energetics extracted from experiment. For small vesicles with high curvature, the insertion lowers the membrane energy by relieving strain on a membrane that is far from its preferred curvature of zero. For larger vesicles with low curvature, however, the insertion has the inverse effect, de-stabilizing the membrane by introducing more strain. The membrane energy cannot be directly predicted analytically, due to shape changes from surface relaxation around the anisotropic insertion. We formulate here an empirical expression that captures numerically calculated membrane energies as a function of both basic membrane properties (bending modulus [kcy] and radius R) as well as stresses applied by the inserted helix ([Formula] and area Ains). We show that the shape relaxation energy has a similar magnitude to the insertion energy, with a strong nonlinear dependence on [Formula]. We therefore predict how these physical parameters will alter the energetics of helix binding to curved vesicles, which is an essential step in understanding their localization dynamics during membrane remodeling processes.

biophysics↗

bioRxiv2021

Nascent clathrin lattices spontaneously disassemble without sufficient adaptor proteins

Clathrin-coated structures must assemble on cell membranes to perform their primary function of receptor internalization. These structures show marked plasticity and instability, but what conditions are necessary to stabilize against disassembly have not been quantified. Recent in vitro fluorescence experiments have measured kinetics of stable clathrin assembly on membranes as controlled by key adaptor proteins like AP-2. Here, we combine this experimental data with microscopic reaction-diffusion simulations and theory to quantify mechanisms of stable vs unstable clathrin assembly on membranes. Both adaptor binding and dimensional reduction on the 2D surface are necessary to reproduce the cooperative kinetics of assembly. By applying our model to more physiologic-like conditions, where the stoichiometry and volume to area ratio are significantly lower than in vitro, we show that the critical nucleus contains ~25 clathrin, remarkably similar to sizes of abortive structures observed in vivo. Stable nucleation requires a stoichiometry of adaptor to clathrin that exceeds 1:1, meaning that AP-2 on its own has too few copies to nucleate lattices. Increasing adaptor concentration increases lattice sizes and nucleation speeds. For curved clathrin cages, we quantify both the cost of bending the membrane and the stabilization required to nucleate cages in solution. We find the energetics are comparable, suggesting that curving the lattice could offset the bending energy cost. Our model predicts how adaptor density controls stabilization of clathrin-coated structures against spontaneous disassembly, and shows remodeling and disassembly does not require ATPases, which is a critical advance towards predicting control of productive vesicle formation. Significance StatementStochastic self-assembly of clathrin-coated structures on the plasma membrane is essential for transport into cells. We show here that even with abundant clathrin available, robust nucleation and growth into stable structures on membranes is not possible without sufficient adaptor proteins. Our results thus provide quantitative justification for why structures observed to form in vivo can still spontaneously disassemble over many seconds. The ATPases that drive clathrin disassembly after productive vesicle formation are therefore not necessary to control remodeling during growth. With parameterization against in vitro kinetics of assembly on membranes, our reaction-diffusion model provides a powerful and extensible tool for establishing determinants of productive assembly in cells.

biophysics↗

bioRxiv2021

A UVB-responsive common variant at chr7p21.1 confers tanning response and melanoma risk via regulation of the aryl hydrocarbon receptor gene (AHR)

Genome-wide association studies have identified a melanoma-associated locus on chromosome band 7p21.1 with rs117132860 as the lead SNP, and a secondary independent signal marked by rs73069846. rs117132860 is also associated with tanning ability and cutaneous squamous cell carcinoma (cSCC). As ultraviolet radiation (UVR) is a key environmental exposure for all three traits, we investigated the mechanisms by which this locus contributes to melanoma risk, focusing on cellular response to UVR. Fine-mapping of melanoma GWAS identified four independent sets of candidate causal variants. A GWAS region-focused Capture-C study of primary melanocytes identified physical interactions between two causal sets and the promoter of the aryl hydrocarbon receptor gene (AHR). Subsequent chromatin state annotation, eQTL, and luciferase assays identified rs117132860 as a functional variant and reinforced AHR as a likely causal gene. As AHR plays critical roles in cellular response to dioxin and UVR, we explored links between this SNP and AHR expression after both 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and ultraviolet B (UVB) exposure. Allele-specific AHR binding to rs117132860-G was enhanced following both, consistent with predicted weakened AHR binding to the risk/poor-tanning rs117132860-A allele, and allele-preferential AHR expression driven from the protective rs117132860-G allele was observed following UVB exposure. Small deletions surrounding rs117132860 via CRISPR abrogates AHR binding, reduces melanocyte cell growth, and prolongs growth arrest following UVB exposure. These data suggest AHR is a melanoma susceptibility gene at the 7p21.1 risk locus, and rs117132860 is a functional variant within a UVB-responsive element, leading to allelic AHR expression, and altering melanocyte growth phenotypes upon exposure.

genetics↗

bioRxiv2021

Speed limits of protein assembly with reversible membrane localization

Self-assembly is often studied in a three-dimensional (3D) solution, but a significant fraction of binding events involve proteins that can reversibly bind and diffuse along a two-dimensional (2D) surface. In a recent study, we quantified how proteins can exploit the reduced dimension of the membrane to trigger complex formation. Here, we derive a single expression for the characteristic timescale of this multi-step assembly process, where the change in dimensionality renders rates and concentrations effectively time-dependent. We find that proteins can accelerate complex formation due to an increase in relative concentration, driving more frequent collisions which often wins out over slow-downs due to diffusion. Our model contains two protein populations that associate with one another and use a distinct site to bind membrane lipids, creating a complex reaction network. However, by identifying two major rate-limiting pathways to reach an equilibrium steady-state, we derive an accurate approximation for the mean first passage time when lipids are in abundant supply. Our theory highlights how the sticking rate, or effective adsorption coefficient of the membrane is central in controlling timescales. We also derive a corrected localization rate to quantify how the geometry of the system and diffusion can reduce rates of localization. We validate and test our results using kinetic and reaction-diffusion simulations. Our results establish how the speed of key assembly steps can shift by orders-of-magnitude when membrane localization is possible, which is critical to understanding mechanisms used in cells.

biophysics↗

bioRxiv2020

Integrative genomics identifies lncRNA regulatory networks across 1,044 pediatric leukemias and solid tumors

Long non-coding RNAs (lncRNAs) play an important role in gene regulation and contribute to tumorigenesis. While pan-cancer studies of lncRNA expression have been performed for adult malignancies, the lncRNA landscape across pediatric cancers remains largely uncharted. Here, we curate RNA sequencing data for 1,044 pediatric leukemia and solid tumors and integrate paired tumor whole genome sequencing and epigenetic data in relevant cell line models to explore lncRNA expression, regulation, and association with cancer. We report a total of 2,657 robustly expressed lncRNAs across six pediatric cancers, including 1,142 exhibiting histotype-specific expression. DNA copy number alterations contributed to lncRNA dysregulation at a proportion comparable to protein coding genes. Application of a multi-dimensional framework to identify and prioritize lncRNAs impacting gene networks revealed that lncRNAs dysregulated in pediatric cancer are associated with proliferation, metabolism, and DNA damage hallmarks. Analysis of upstream regulation via cell-type specific transcription factors further implicated distinct histotype-specific and developmental lncRNAs. We integrated our analyses to prioritize lncRNAs for experimental validation and showed that silencing of TBX2-AS1, our top-prioritized neuroblastoma-specific lncRNA, resulted in significant growth inhibition of neuroblastoma cells, confirming our computational predictions. Taken together, these data provide a comprehensive characterization of lncRNA regulation and function in pediatric cancers and pave the way for future mechanistic studies.

cancer biology↗

bioRxiv2020

Thermodynamics and free energy landscape of BAR-domain dimerization from molecular simulations

Nearly all proteins interact specifically with other proteins, often forming reversible bound structures whose stability is critical to function. Proteins with BAR domains function to bind to, bend, and remodel biological membranes, where the dimerization of BAR domains is a key step in this function. Here we characterize the binding thermodynamics of homodimerization between the LSP1 BAR domain proteins in solution, using Molecular Dynamics (MD) simulations. By combining the MARTINI coarse-grained protein models with enhanced sampling through metadynamics, we construct a two-dimensional free energy surface quantifying the bound versus unbound ensembles as a function of two distance variables. Our simulations portray a heterogeneous and extraordinarily stable bound ensemble for these modeled LSP1 proteins. The proper crystal structure dimer has a large hydrophobic interface that is part of a stable minima on the free energy surface, which is enthalpically the minima of all bound structures. However, we also find several other stable nonspecific dimers with comparable free energies to the specific dimer. Through structure-based clustering of these bound structures, we find that some of these nonspecific contacts involve extended tail regions that help stabilize the higher-order oligomers formed by BAR-domains, contacts that are separated from the homodimer interface. We find that the known membrane-binding residues of the LSP1 proteins rarely participate in any of the bound interfaces, but that both patches of residues are aligned to interact with the membrane in the specific dimer. Hence, we would expect a strong selection of the specific dimer in binding to the membrane. The effect of a 100mM NaCl buffer reduces the relative stability of nonspecific dimers compared to the specific dimer, indicating that it would help prevent aggregation of the proteins. With these results, we provide the first free energy characterization of interaction pathways in this important class of membrane sculpting domains, revealing a variety of interfacial contacts outside of the specific dimer that may help stabilize its oligomeric assemblies.

biophysics↗

bioRxiv2020

Integrating protein copy numbers with interaction networks to quantify stoichiometry in mammalian endocytosis

Proteins that drive processes like clathrin-mediated endocytosis (CME) are expressed at various copy numbers within a cell, from hundreds (e.g. auxilin) to millions (e.g. clathrin). Between cell types with identical genomes, copy numbers further vary significantly both in absolute and relative abundance. These variations contain essential information about each proteins function, but how significant are these variations and how can they be quantified to infer useful functional behavior? Here, we address this by quantifying the stoichiometry of proteins involved in the CME network. We find robust trends across three cell types in proteins that are sub- vs super-stoichiometric in terms of protein function, network topology (e.g. hubs), and abundance. To perform this analysis, we first constructed the interface resolved network of 82 proteins involved in CME in mammals, plus lipid and cargo binding partners, totaling over 600 specific binding interactions. Our model solves for stoichiometric balance by optimizing each copy of a protein interface to match up to its partner interfaces, keeping the optimized copies as close as possible to observed copies. We find highly expressed, structure-forming proteins such as actin and clathrin do tend to be super-stoichiometric, or in excess of their partners, but they are not the most extreme cases. We test sensitivity of network stoichiometry to protein removal and find that hub proteins tend to be less sensitive to removal of any single partner, thus acting as buffers that compensate dosage changes. As expected, tightly coupled protein pairs (e.g. CAPZA2 and CAPZB) are strongly correlated. Unexpectedly, removal of functionally similar cargo adaptor proteins produces widely variable levels of disruption to the network stoichiometry. Our results predict that knockdown of the adaptor protein DAB2 will globally impact the stoichiometry of most other cargo adaptor proteins in Hela cells, with significantly less impact in fibroblast cells. This inexpensive analysis can be applied to any protein network, synthesizing disparate sources of biological data into a relatively simple and intuitive model of binding stoichiometry that can aid in dynamical modeling and experimental design.

systems biology↗

bioRxiv2020

scAAVengr: Single-cell transcriptome-based quantification of engineered AAVs in non-human primate retina

Adeno-associated virus (AAV)-mediated gene therapies are rapidly advancing to the clinic, and AAV engineering has resulted in vectors with increased ability to deliver therapeutic genes. Although the choice of vector is critical, quantitative comparison of AAVs, especially in large animals, remains challenging. Here, we developed an efficient single-cell AAV engineering pipeline (scAAVengr) to quantify efficiency of AAV-mediated gene expression across all cell types. scAAVengr allows for definitive, head-to-head comparison of vectors in the same animal. To demonstrate proof-of-concept for the scAAVengr workflow, we quantified - with cell-type resolution - the abilities of naturally occurring and newly engineered AAVs to mediate gene expression in primate retina following intravitreal injection. A top performing variant, K912, was used to deliver SaCas9 and edit the rhodopsin gene in macaque retina, resulting in editing efficiency similar to infection rates detected by the scAAVengr workflow. These results validate scAAVengr as a powerful method for development of AAV vectors.

bioengineering↗

bioRxiv2020

Inhibiting the mitochondrial pyruvate carrier does not ameliorate synucleinopathy in the absence of inflammation or metabolic deficits

Epidemiological studies suggest a link between type-2 diabetes and Parkinsons disease (PD) risk. Treatment of type-2 diabetes with insulin sensitizing drugs lowers the risk of PD. We previously showed that the insulin sensitizing drug, MSDC-0160, ameliorates pathogenesis in some animal models of PD. MSDC-0160 reversibly binds the mitochondrial pyruvate carrier (MPC) protein complex, which has an anti-inflammatory effect and restores metabolic deficits. Since PD is characterized by the deposition of -synuclein (Syn), we hypothesized that inhibiting the MPC might directly inhibit Syn aggregation in vivo in mammals. To answer if modulation of MPC can reduce the development of Syn assemblies, and reduce neurodegeneration, we treated two chronic and progressive mouse models; a viral vector-based Syn overexpressing model and a pre-formed fibril (PFF) Syn seeding model with MSDC-0160. These two models present with distinct types of Syn pathology but lack inflammatory or autophagy deficits. Contrary to our hypothesis, we found that a modulation of MPC in these models did not reduce the accumulation Syn aggregates or mitigate neurotoxicity. Instead, MSDC-0160 changed the post-translational modification and aggregation features of the Syn. These results are consistent with the lack of a direct effect on MPC modulation on synuclein clearance in these models.

neuroscience↗

bioRxiv2020

The identity of human tissue-emigrant CD8+ T cells

Lymphocyte migration is essential for human adaptive immune surveillance. However, our current understanding of this process is rudimentary, because most human studies to date have been restricted to immunological analyses of blood and various tissues. To address this issue, we used an integrated approach to characterize tissue-emigrant immune cells in thoracic duct lymph (TDL). In humans and non-human primates, lymphocytes were by far the most abundant immune lineage population in efferent lymph, and a vast majority of these lymphocytes were T cells. Cytolytic CD8+ T cell subsets were clonotypically discrete and selectively confined to the intravascular circulation, persisting for months after inhibition of S1P-dependent tissue egress by FTY-720. In contrast, non-cytolytic CD8+ T cell subsets with stem-like epigenetic and transcriptional signatures predominated in tissues and TDL. Collectively, these data provide an atlas of the migratory immune system and define the nature of tissue-emigrant CD8+ T cells that recirculate via TDL.

immunology↗

bioRxiv2020

The Roles of Space and Stochasticity in Computational Simulations of Cellular Biochemistry: Quantitative Analysis and Qualitative Insights

Most of the fascinating phenomena studied in cell biology emerge from interactions among highly organized multi-molecular structures and rapidly propagating molecular signals embedded into complex and frequently dynamic cellular morphologies. For the exploration of such systems, computational simulation has proved to be an invaluable tool, and many researchers in this field have developed sophisticated computational models for application to specific cell biological questions. However it is often difficult to reconcile conflicting computational results that use different simulation approaches (for example partial differential equations versus particle-based stochastic methods) to describe the same phenomenon. Moreover, the details of the computational implementation of any particular algorithm may give rise to quantitatively or even qualitatively different results for the same set of starting assumptions and parameters. In an effort to address this issue systematically, we have defined a series of computational test cases ranging from very simple (bimolecular binding in solution) to moderately complex (spatial and temporal oscillations generated by proteins binding to membranes) that represent building blocks for comprehensive three-dimensional models of cellular function. Having used two or more distinct computational approaches to solve each of these test cases with consistent parameter sets, we generally find modest but measurable differences in the solutions of the same problem, and a few cases where significant deviations arise. We discuss the strengths and limitations of commonly used computational approaches for exploring cell biological questions and provide a framework for decision-making by researchers wishing to develop new models for cell biology. As computational power and speed continue to increase at a remarkable rate, the dream of a fully comprehensive computational model of a living cell may be drawing closer to reality, but our analysis demonstrates that it will be crucial to evaluate the accuracy of such models critically and systematically.

systems biology↗

bioRxiv2020

Alpha-synuclein expression supports interferon stimulated gene expression in neurons

The protein alpha-synuclein (asyn) is predominantly expressed in neurons and is associated with neurodegenerative diseases like Parkinsons disease (PD); yet, a functional role for asyn in neurons is not clearly established. We have previously shown that asyn expression is up-regulated following viral infection in neurons and is critical for host immune responses to RNA virus infections. Here, we investigate the mechanisms underlying asyn-dependent immune responses to RNA virus infection in the brain. Using asyn knock-out (KO) mice and human neuronal models, we show that asyn is required for expression of the full repertoire of interferon-stimulated genes (ISGs) in neurons following acute RNA virus infection. Furthermore, treatment of asyn KO human neurons with poly I:C or type I interferon also fail to induce expression of the full complement of ISGs suggesting that asyn plays an important role in modulating neuronal innate immune responses. In brain tissue, asyn-dependent ISG expression is independent of microglia activation and supports activation of infiltrating lymphocytes following viral challenge. We also show that virus infections lead to accumulation of phosphorylated S129 asyn in human and non-human primate neuronal tissues. In a model of pS129 asyn pathology, we found that infection with West Nile virus increases microglia activation but does not significantly alter pS129 asyn pathology in the mouse model. Taken together, our results establish asyn as a novel, neuron-specific modulator of innate immunity by a mechanism that promotes interferon-stimulated gene expression and links responses to virus infection with formation of phosphorylated S129-asyn in neuronal tissue.

neuroscience↗