Publications by authors named "Abraham J Koster"

109 Publications

Application of a Highly Selective Cathepsin S Two-step Activity-Based Probe in Multicolor Bio-Orthogonal Correlative Light-Electron Microscopy.

Front Chem 2020 8;8:628433. Epub 2021 Feb 8.

Department of Tumor Immunology and the Institute for Chemical Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, Nijmegen, Netherlands.

Cathepsin S is a lysosomal cysteine protease highly expressed in immune cells such as dendritic cells, B cells and macrophages. Its functions include extracellular matrix breakdown and cleavage of cell adhesion molecules to facilitate immune cell motility, as well as cleavage of the invariant chain during maturation of major histocompatibility complex II. The identification of these diverse specific functions has brought the challenge of delineating cathepsin S activity with great spatial precision, relative to related enzymes and substrates. Here, the development of a potent and highly selective two-step activity-based probe for cathepsin S and the application in multicolor bio-orthogonal correlative light-electron microscopy is presented. LHVS, which has been reported as a selective inhibitor of cathepsin S with nanomolar potency, formed the basis for our probe design. However, in competitive activity-based protein profiling experiments LHVS showed significant cross-reactivity toward Cat L. Introduction of an azide group in the P2 position expanded the selectivity window for cathepsin S, but rendered the probe undetectable, as demonstrated in bio-orthogonal competitive activity-based protein profiling. Incorporation of an additional azide handle for click chemistry on the solvent-exposed P1 position allowed for selective labeling of cathepsin S. This highlights the influence of click handle positioning on probe efficacy. This probe was utilized in multicolor bio-orthogonal confocal and correlative light-electron microscopy to investigate the localization of cathepsin S activity at an ultrastructural level in bone marrow-derived dendritic cells. The tools developed in this study will aid the characterization of the variety of functions of cathepsin S throughout biology.
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http://dx.doi.org/10.3389/fchem.2020.628433DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7903248PMC
February 2021

The adapter protein Myd88 plays an important role in limiting mycobacterial growth in a zebrafish model for tuberculosis.

Virchows Arch 2021 Feb 9. Epub 2021 Feb 9.

Institute of Biology Leiden, Leiden University, Leiden, Netherlands.

Tuberculosis (TB) is the most prevalent bacterial infectious disease in the world, caused by the pathogen Mycobacterium tuberculosis (Mtb). In this study, we have used Mycobacterium marinum (Mm) infection in zebrafish larvae as an animal model for this disease to study the role of the myeloid differentiation factor 88 (Myd88), the key adapter protein of Toll-like receptors. Previously, Myd88 has been shown to enhance innate immune responses against bacterial infections, and in the present study, we have investigated the effect of Myd88 deficiency on the granuloma morphology and the intracellular distribution of bacteria during Mm infection. Our results show that granulomas formed in the tail fin from myd88 mutant larvae have a more compact structure and contain a reduced number of leukocytes compared to the granulomas observed in wild-type larvae. These morphological differences were associated with an increased bacterial burden in the myd88 mutant. Electron microscopy analysis showed that the majority of Mm in the myd88 mutant are located extracellularly, whereas in the wild type, most bacteria were intracellular. In the myd88 mutant, intracellular bacteria were mainly present in compartments that were not electron-dense, suggesting that these compartments had not undergone fusion with a lysosome. In contrast, approximately half of the intracellular bacteria in wild-type larvae were found in electron-dense compartments. These observations in a zebrafish model for tuberculosis suggest a role for Myd88-dependent signalling in two important phenomena that limit mycobacterial growth in the infected tissue. It reduces the number of leukocytes at the site of infection and the acidification of bacteria-containing compartments inside these cells.
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http://dx.doi.org/10.1007/s00428-021-03043-3DOI Listing
February 2021

Bioorthogonal Correlative Light-Electron Microscopy of in Macrophages Reveals the Effect of Antituberculosis Drugs on Subcellular Bacterial Distribution.

ACS Cent Sci 2020 Nov 16;6(11):1997-2007. Epub 2020 Oct 16.

Leiden Institute of Chemistry and The Institute of Chemical Immunology, Leiden University, Einsteinweg 55, Leiden 2300 RA, The Netherlands.

Bioorthogonal correlative light-electron microscopy (B-CLEM) can give a detailed overview of multicomponent biological systems. It can provide information on the ultrastructural context of bioorthogonal handles and other fluorescent signals, as well as information about subcellular organization. We have here applied B-CLEM to the study of the intracellular pathogen () by generating a triply labeled through combined metabolic labeling of the cell wall and the proteome of a DsRed-expressing strain. Study of this pathogen in a B-CLEM setting was used to provide information about the intracellular distribution of the pathogen, as well as its response to various clinical antibiotics, supported by flow cytometric analysis of the bacteria, after recovery from the host cell (). The RNA polymerase-targeting drug rifampicin displayed the most prominent effect on subcellular distribution, suggesting the most direct effect on pathogenicity and/or viability, while the cell wall synthesis-targeting drugs isoniazid and ethambutol effectively rescued bacterial division-induced loss of metabolic labels. The three drugs combined did not give a more pronounced effect but rather an intermediate response, whereas gentamicin displayed a surprisingly strong additive effect on subcellular distribution.
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http://dx.doi.org/10.1021/acscentsci.0c00539DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7706097PMC
November 2020

A molecular pore spans the double membrane of the coronavirus replication organelle.

Science 2020 09 6;369(6509):1395-1398. Epub 2020 Aug 6.

Section Electron Microscopy, Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden 2333 ZC, Netherlands.

Coronavirus genome replication is associated with virus-induced cytosolic double-membrane vesicles, which may provide a tailored microenvironment for viral RNA synthesis in the infected cell. However, it is unclear how newly synthesized genomes and messenger RNAs can travel from these sealed replication compartments to the cytosol to ensure their translation and the assembly of progeny virions. In this study, we used cellular cryo-electron microscopy to visualize a molecular pore complex that spans both membranes of the double-membrane vesicle and would allow export of RNA to the cytosol. A hexameric assembly of a large viral transmembrane protein was found to form the core of the crown-shaped complex. This coronavirus-specific structure likely plays a key role in coronavirus replication and thus constitutes a potential drug target.
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http://dx.doi.org/10.1126/science.abd3629DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7665310PMC
September 2020

A unifying structural and functional model of the coronavirus replication organelle: Tracking down RNA synthesis.

PLoS Biol 2020 06 8;18(6):e3000715. Epub 2020 Jun 8.

Section Electron Microscopy, Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, the Netherlands.

Zoonotic coronavirus (CoV) infections, such as those responsible for the current severe acute respiratory syndrome-CoV 2 (SARS-CoV-2) pandemic, cause grave international public health concern. In infected cells, the CoV RNA-synthesizing machinery associates with modified endoplasmic reticulum membranes that are transformed into the viral replication organelle (RO). Although double-membrane vesicles (DMVs) appear to be a pan-CoV RO element, studies to date describe an assortment of additional CoV-induced membrane structures. Despite much speculation, it remains unclear which RO element(s) accommodate viral RNA synthesis. Here we provide detailed 2D and 3D analyses of CoV ROs and show that diverse CoVs essentially induce the same membrane modifications, including the small open double-membrane spherules (DMSs) previously thought to be restricted to gamma- and delta-CoV infections and proposed as sites of replication. Metabolic labeling of newly synthesized viral RNA followed by quantitative electron microscopy (EM) autoradiography revealed abundant viral RNA synthesis associated with DMVs in cells infected with the beta-CoVs Middle East respiratory syndrome-CoV (MERS-CoV) and SARS-CoV and the gamma-CoV infectious bronchitis virus. RNA synthesis could not be linked to DMSs or any other cellular or virus-induced structure. Our results provide a unifying model of the CoV RO and clearly establish DMVs as the central hub for viral RNA synthesis and a potential drug target in CoV infection.
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http://dx.doi.org/10.1371/journal.pbio.3000715DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7302735PMC
June 2020

Human-iPSC-Derived Cardiac Stromal Cells Enhance Maturation in 3D Cardiac Microtissues and Reveal Non-cardiomyocyte Contributions to Heart Disease.

Cell Stem Cell 2020 06 26;26(6):862-879.e11. Epub 2020 May 26.

Department of Anatomy and Embryology, Leiden University Medical Center, 2333 Leiden, the Netherlands; Department of Applied Stem Cell Technologies, University of Twente, 7500 Enschede, the Netherlands. Electronic address:

Cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) are functionally immature, but this is improved by incorporation into engineered tissues or forced contraction. Here, we showed that tri-cellular combinations of hiPSC-derived CMs, cardiac fibroblasts (CFs), and cardiac endothelial cells also enhance maturation in easily constructed, scaffold-free, three-dimensional microtissues (MTs). hiPSC-CMs in MTs with CFs showed improved sarcomeric structures with T-tubules, enhanced contractility, and mitochondrial respiration and were electrophysiologically more mature than MTs without CFs. Interactions mediating maturation included coupling between hiPSC-CMs and CFs through connexin 43 (CX43) gap junctions and increased intracellular cyclic AMP (cAMP). Scaled production of thousands of hiPSC-MTs was highly reproducible across lines and differentiated cell batches. MTs containing healthy-control hiPSC-CMs but hiPSC-CFs from patients with arrhythmogenic cardiomyopathy strikingly recapitulated features of the disease. Our MT model is thus a simple and versatile platform for modeling multicellular cardiac diseases that will facilitate industry and academic engagement in high-throughput molecular screening.
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http://dx.doi.org/10.1016/j.stem.2020.05.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7284308PMC
June 2020

Progression and Classification of Granular Osmiophilic Material (GOM) Deposits in Functionally Characterized Human NOTCH3 Transgenic Mice.

Transl Stroke Res 2020 06 30;11(3):517-527. Epub 2019 Oct 30.

Department of Clinical Genetics, Leiden University Medical Center, Albinusdreef 2, 2300 RC, Leiden, The Netherlands.

CADASIL is a NOTCH3-associated cerebral small vessel disease. A pathological ultrastructural disease hallmark is the presence of NOTCH3-protein containing deposits called granular osmiophilic material (GOM), in small arteries. How these GOM deposits develop over time and what their role is in disease progression is largely unknown. Here, we studied the progression of GOM deposits in humanized transgenic NOTCH3 mice, compared them to GOM deposits in patient material, and determined whether GOM deposits in mice are associated with a functional CADASIL phenotype. We found that GOM deposits are not static, but rather progress in ageing mice, both in terms of size and aspect. We devised a GOM classification system, reflecting size, morphology and electron density. Six-month-old mice showed mostly early stage GOM, whereas older mice and patient vessels showed predominantly advanced stage GOM, but also early stage GOM. Mutant mice did not develop the most severe GOM stage seen in patient material. This absence of end-stage GOM in mice was associated with an overall lack of histological vascular pathology, which may explain why the mice did not reveal functional deficits in cerebral blood flow, cognition and motor function. Taken together, our data indicate that GOM progress over time, and that new GOM deposits are continuously being formed. The GOM staging system we introduce here allows for uniform GOM deposit classification in future mouse and human studies, which may lead to more insight into a potential association between GOM stage and CADASIL disease severity, and the role of GOM in disease progression.
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http://dx.doi.org/10.1007/s12975-019-00742-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7235067PMC
June 2020

Mind the gap: Micro-expansion joints drastically decrease the bending of FIB-milled cryo-lamellae.

J Struct Biol 2019 12 16;208(3):107389. Epub 2019 Sep 16.

Department of Cell and Chemical Biology, Section Electron Microscopy, Leiden University Medical Center, Leiden 2333 ZC, The Netherlands. Electronic address:

Cryo-focused ion beam (FIB)-milling of biological samples can be used to generate thin electron-transparent slices from cells grown or deposited on EM grids. These so called cryo-lamellae allow high-resolution structural studies of the natural cellular environment by in situ cryo-electron tomography. However, the cryo-lamella workflow is a low-throughput technique and can easily be hindered by technical issues like the bending of the lamellae during the final cryo-FIB-milling steps. The severity of lamella bending seems to correlate with crinkling of the EM grid support film at cryogenic temperatures, which could generate tensions that may be transferred onto the thin lamella, leading to its bending and breakage. To protect the lamellae from such forces, we milled "micro-expansion joints" alongside the lamellae, creating gaps in the support that can act as physical buffers to safely absorb material motion. We demonstrate that the presence of micro-expansion joints drastically decreases bending of lamellae milled from eukaryotic cells grown and frozen on EM grids. Furthermore, we show that this adaptation does not create additional instabilities that could impede subsequent parts of the cryo-lamella workflow, as we obtained high-quality Volta phase plate tomograms revealing macromolecules in their natural structural context. The minimal additional effort required to implement micro-expansion joints in the cryo-FIB-milling workflow makes them a straightforward solution against cryo-lamella bending to increase the throughput of in situ structural biology studies.
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http://dx.doi.org/10.1016/j.jsb.2019.09.006DOI Listing
December 2019

Glomerular permeability is not affected by heparan sulfate glycosaminoglycan deficiency in zebrafish embryos.

Am J Physiol Renal Physiol 2019 11 28;317(5):F1211-F1216. Epub 2019 Aug 28.

Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.

Proteinuria develops when specific components in the glomerular filtration barrier have impaired function. Although the precise components involved in maintaining this barrier have not been fully identified, heparan sulfate proteoglycans are believed to play an essential role in maintaining glomerular filtration. Although in situ studies have shown that a loss of heparan sulfate glycosaminoglycans increases the permeability of the glomerular filtration barrier, recent studies using experimental models have shown that podocyte-specific deletion of heparan sulfate glycosaminoglycan assembly does not lead to proteinuria. However, tubular reabsorption of leaked proteins might have masked an increase in glomerular permeability in these models. Furthermore, not only podocytes but also glomerular endothelial cells are involved in heparan sulfate synthesis in the glomerular filtration barrier. Therefore, we investigated the effect of a global heparan sulfate glycosaminoglycan deficiency on glomerular permeability. We used a zebrafish embryo model carrying a homozygous germline mutation in the gene. Glomerular permeability was assessed with a quantitative dextran tracer injection method. In this model, we accounted for tubular reabsorption. Loss of anionic sites in the glomerular basement membrane was measured using polyethyleneimine staining. Although mutant animals had significantly fewer negatively charged areas in the glomerular basement membrane, glomerular permeability was unaffected. Moreover, heparan sulfate glycosaminoglycan-deficient embryos had morphologically intact podocyte foot processes. Glomerular filtration remains fully functional despite a global reduction of heparan sulfate.
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http://dx.doi.org/10.1152/ajprenal.00126.2019DOI Listing
November 2019

CRB2 Loss in Rod Photoreceptors Is Associated with Progressive Loss of Retinal Contrast Sensitivity.

Int J Mol Sci 2019 Aug 21;20(17). Epub 2019 Aug 21.

Department of Ophthalmology, Leiden University Medical Center (LUMC), Albinusdreef 2, 2333 ZA Leiden, The Netherlands.

Variations in the Crumbs homolog-1 () gene are associated with a wide variety of autosomal recessive retinal dystrophies, including early onset retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA). CRB1 belongs to the Crumbs family, which in mammals includes CRB2 and CRB3. Here, we studied the specific roles of CRB2 in rod photoreceptor cells and whether ablation of CRB2 in rods exacerbates the -disease. Therefore, we assessed the morphological, retinal, and visual functional consequences of specific ablation of CRB2 from rods with or without concomitant loss of CRB1. Our data demonstrated that loss of CRB2 in mature rods resulted in RP. The retina showed gliosis and disruption of the subapical region and adherens junctions at the outer limiting membrane. Rods were lost at the peripheral and central superior retina, while gross retinal lamination was preserved. Rod function as measured by electroretinography was impaired in adult mice. Additional loss of CRB1 exacerbated the retinal phenotype leading to an early reduction of the dark-adapted rod photoreceptor a-wave and reduced contrast sensitivity from 3-months-of-age, as measured by optokinetic tracking reflex (OKT) behavior testing. The data suggest that CRB2 present in rods is required to prevent photoreceptor degeneration and vision loss.
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http://dx.doi.org/10.3390/ijms20174069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6747345PMC
August 2019

Origins of Enterovirus Replication Organelles Established by Whole-Cell Electron Microscopy.

mBio 2019 06 11;10(3). Epub 2019 Jun 11.

Section Electron Microscopy, Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, The Netherlands

Enterovirus genome replication occurs at virus-induced structures derived from cellular membranes and lipids. However, the origin of these replication organelles (ROs) remains uncertain. Ultrastructural evidence of the membrane donor is lacking, suggesting that the sites of its transition into ROs are rare or fleeting. To overcome this challenge, we combined live-cell imaging and serial block-face scanning electron microscopy of whole cells to capture emerging enterovirus ROs. The first foci of fluorescently labeled viral protein correlated with ROs connected to the endoplasmic reticulum (ER) and preceded the appearance of ROs stemming from the -Golgi network. Whole-cell data sets further revealed striking contact regions between ROs and lipid droplets that may represent a route for lipid shuttling to facilitate RO proliferation and genome replication. Our data provide direct evidence that enteroviruses use ER and then Golgi membranes to initiate RO formation, demonstrating the remarkable flexibility with which enteroviruses usurp cellular organelles. Enteroviruses are causative agents of a range of human diseases. The replication of these viruses within cells relies on specialized membranous structures termed replication organelles (ROs) that form during infection but whose origin remains elusive. To capture the emergence of enterovirus ROs, we use correlative light and serial block-face scanning electron microscopy, a powerful method to pinpoint rare events in their whole-cell ultrastructural context. RO biogenesis was found to occur first at ER and then at Golgi membranes. Extensive contacts were found between early ROs and lipid droplets (LDs), which likely serve to provide LD-derived lipids required for replication. Together, these data establish the dual origin of enterovirus ROs and the chronology of their biogenesis at different supporting cellular membranes.
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http://dx.doi.org/10.1128/mBio.00951-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6561026PMC
June 2019

Insights into IgM-mediated complement activation based on in situ structures of IgM-C1-C4b.

Proc Natl Acad Sci U S A 2019 06 30;116(24):11900-11905. Epub 2019 May 30.

Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Faculty of Science, Utrecht University, 3584 CH Utrecht, The Netherlands.

Antigen binding by serum Ig-M (IgM) protects against microbial infections and helps to prevent autoimmunity, but causes life-threatening diseases when mistargeted. How antigen-bound IgM activates complement-immune responses remains unclear. We present cryoelectron tomography structures of IgM, C1, and C4b complexes formed on antigen-bearing lipid membranes by normal human serum at 4 °C. The IgM-C1-C4b complexes revealed C4b product release as the temperature-limiting step in complement activation. Both IgM hexamers and pentamers adopted hexagonal, dome-shaped structures with Fab pairs, dimerized by hinge domains, bound to surface antigens that support a platform of Fc regions. C1 binds IgM through widely spread C1q-collagen helices, with C1r proteases pointing outward and C1s bending downward and interacting with surface-attached C4b, which further interacts with the adjacent IgM-Fab and globular C1q-recognition unit. Based on these data, we present mechanistic models for antibody-mediated, C1q-transmitted activation of C1 and for C4b deposition, while further conformational rearrangements are required to form C3 convertases.
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http://dx.doi.org/10.1073/pnas.1901841116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6575175PMC
June 2019

Human iPSC-Derived Retinas Recapitulate the Fetal CRB1 CRB2 Complex Formation and Demonstrate that Photoreceptors and Müller Glia Are Targets of AAV5.

Stem Cell Reports 2019 05 4;12(5):906-919. Epub 2019 Apr 4.

Department of Ophthalmology, Leiden University Medical Center (LUMC), 2333 ZA Leiden, The Netherlands; Netherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and Sciences (KNAW), 1105 BA Amsterdam, The Netherlands. Electronic address:

Human retinal organoids from induced pluripotent stem cells (hiPSCs) can be used to confirm the localization of proteins in retinal cell types and to test transduction and expression patterns of gene therapy vectors. Here, we compared the onset of CRB protein expression in human fetal retina with human iPSC-derived retinal organoids. We show that CRB2 protein precedes the expression of CRB1 in the developing human retina. Our data suggest the presence of CRB1 and CRB2 in human photoreceptors and Müller glial cells. Thus the fetal CRB complex formation is replicated in hiPSC-derived retina. CRB1 patient iPSC retinal organoids showed disruptions at the outer limiting membrane as found in Crb1 mutant mice. Furthermore, AAV serotype 5 (AAV5) is potent in infecting human Müller glial cells and photoreceptors in hiPSC-derived retinas and retinal explants. Our data suggest that human photoreceptors can be efficiently transduced by AAVs in the presence of photoreceptor segments.
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http://dx.doi.org/10.1016/j.stemcr.2019.03.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6522954PMC
May 2019

Localization of active endogenous and exogenous β-glucocerebrosidase by correlative light-electron microscopy in human fibroblasts.

Traffic 2019 05;20(5):346-356

Department of Medical Biochemistry, Leiden Institute of Chemistry, Leiden University, Leiden, the Netherlands.

β-Glucocerebrosidase (GBA) is the enzyme that degrades glucosylceramide in lysosomes. Defects in GBA that result in overall loss of enzymatic activity give rise to the lysosomal storage disorder Gaucher disease, which is characterized by the accumulation of glucosylceramide in tissue macrophages. Gaucher disease is currently treated by infusion of mannose receptor-targeted recombinant GBA. The recombinant GBA is thought to reach the lysosomes of macrophages, based on the impressive clinical response that is observed in Gaucher patients (type 1) receiving this enzyme replacement therapy. In this study, we used cyclophellitol-derived activity-based probes (ABPs) with a fluorescent reporter that irreversibly bind to the catalytic pocket of GBA, to visualize the active enzymes in a correlative microscopy approach. The uptake of pre-labeled recombinant enzyme was monitored by fluorescence and electron microscopy in human fibroblasts that stably expressed the mannose receptor. The endogenous active enzyme was simultaneously visualized by in situ labeling with the ABP containing an orthogonal fluorophore. This method revealed the efficient delivery of recombinant GBA to lysosomal target compartments that contained endogenous active enzyme.
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http://dx.doi.org/10.1111/tra.12641DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6519279PMC
May 2019

Correlative cryo super-resolution light and electron microscopy on mammalian cells using fluorescent proteins.

Sci Rep 2019 02 4;9(1):1369. Epub 2019 Feb 4.

Section Electron Microscopy, Dept. of Cell and Chemical Biology, Leiden University Medical Center, 2300 RC, Leiden, The Netherlands.

Sample fixation by vitrification is critical for the optimal structural preservation of biomolecules and subsequent high-resolution imaging by cryo-correlative light and electron microscopy (cryoCLEM). There is a large resolution gap between cryo fluorescence microscopy (cryoFLM), ~400-nm, and the sub-nanometre resolution achievable with cryo-electron microscopy (cryoEM), which hinders interpretation of cryoCLEM data. Here, we present a general approach to increase the resolution of cryoFLM using cryo-super-resolution (cryoSR) microscopy that is compatible with successive cryoEM investigation in the same region. We determined imaging parameters to avoid devitrification of the cryosamples without the necessity for cryoprotectants. Next, we examined the applicability of various fluorescent proteins (FPs) for single-molecule localisation cryoSR microscopy and found that all investigated FPs display reversible photoswitchable behaviour, and demonstrated cryoSR on lipid nanotubes labelled with rsEGFP2 and rsFastLime. Finally, we performed SR-cryoCLEM on mammalian cells expressing microtubule-associated protein-2 fused to rsEGFP2 and performed 3D cryo-electron tomography on the localised areas. The method we describe exclusively uses commercially available equipment to achieve a localisation precision of 30-nm. Furthermore, all investigated FPs displayed behaviour compatible with cryoSR microscopy, making this technique broadly available without requiring specialised equipment and will improve the applicability of this emerging technique for cellular and structural biology.
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http://dx.doi.org/10.1038/s41598-018-37728-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6362030PMC
February 2019

Loss of CRB2 in Müller glial cells modifies a CRB1-associated retinitis pigmentosa phenotype into a Leber congenital amaurosis phenotype.

Hum Mol Genet 2019 01;28(1):105-123

Department of Ophthalmology, Leiden University Medical Center, RC Leiden, The Netherlands.

Variations in the human Crumbs homolog-1 (CRB1) gene lead to an array of retinal dystrophies including early onset of retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) in children. To investigate the physiological roles of CRB1 and CRB2 in retinal Müller glial cells (MGCs), we analysed mouse retinas lacking both proteins in MGC. The peripheral retina showed a faster progression of dystrophy than the central retina. The central retina showed retinal folds, disruptions at the outer limiting membrane, protrusion of photoreceptor nuclei into the inner and outer segment layers and ingression of photoreceptor nuclei into the photoreceptor synaptic layer. The peripheral retina showed a complete loss of the photoreceptor synapse layer, intermingling of photoreceptor nuclei within the inner nuclear layer and ectopic photoreceptor cells in the ganglion cell layer. Electroretinography showed severe attenuation of the scotopic a-wave at 1 month of age with responses below detection levels at 3 months of age. The double knockout mouse retinas mimicked a phenotype equivalent to a clinical LCA phenotype due to loss of CRB1. Localization of CRB1 and CRB2 in non-human primate (NHP) retinas was analyzed at the ultrastructural level. We found that NHP CRB1 and CRB2 proteins localized to the subapical region adjacent to adherens junctions at the outer limiting membrane in MGC and photoreceptors. Our data suggest that loss of CRB2 in MGC aggravates the CRB1-associated RP-like phenotype towards an LCA-like phenotype.
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http://dx.doi.org/10.1093/hmg/ddy337DOI Listing
January 2019

Weibel-Palade Body Localized Syntaxin-3 Modulates Von Willebrand Factor Secretion From Endothelial Cells.

Arterioscler Thromb Vasc Biol 2018 07 7;38(7):1549-1561. Epub 2018 Jun 7.

From the Plasma Proteins, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, The Netherlands (M.S., E.K., B.L.v.d.E., A.G., M.H., D.v.B., H.M., M.v.d.B., J.V., R.B.)

Objective: Endothelial cells store VWF (von Willebrand factor) in rod-shaped secretory organelles, called Weibel-Palade bodies (WPBs). WPB exocytosis is coordinated by a complex network of Rab GTPases, Rab effectors, and SNARE (soluble NSF attachment protein receptor) proteins. We have previously identified STXBP1 as the link between the Rab27A-Slp4-a complex on WPBs and the SNARE proteins syntaxin-2 and -3. In this study, we investigate the function of syntaxin-3 in VWF secretion.

Approach And Results: In human umbilical vein endothelial cells and in blood outgrowth endothelial cells (BOECs) from healthy controls, endogenous syntaxin-3 immunolocalized to WPBs. A detailed analysis of BOECs isolated from a patient with variant microvillus inclusion disease, carrying a homozygous mutation in (STX3), showed a loss of syntaxin-3 protein and absence of WPB-associated syntaxin-3 immunoreactivity. Ultrastructural analysis revealed no detectable differences in morphology or prevalence of immature or mature WPBs in control versus STX3 BOECs. VWF multimer analysis showed normal patterns in plasma of the microvillus inclusion disease patient, and media from STX3 BOECs, together indicating WPB formation and maturation are unaffected by absence of syntaxin-3. However, a defect in basal as well as Ca- and cAMP-mediated VWF secretion was found in the STX3 BOECs. We also show that syntaxin-3 interacts with the WPB-associated SNARE protein VAMP8 (vesicle-associated membrane protein-8).

Conclusions: Our data reveal syntaxin-3 as a novel WPB-associated SNARE protein that controls WPB exocytosis.
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http://dx.doi.org/10.1161/ATVBAHA.117.310701DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6039413PMC
July 2018

Ultrastructural Imaging of Salmonella-Host Interactions Using Super-resolution Correlative Light-Electron Microscopy of Bioorthogonal Pathogens.

Chembiochem 2018 Jun 5. Epub 2018 Jun 5.

Leiden Institute of Chemistry and, The Institute for Chemical Immunology, Leiden University, Einsteinweg 55, 2333, CC, Leiden, The Netherlands.

The imaging of intracellular pathogens inside host cells is complicated by the low resolution and sensitivity of fluorescence microscopy and by the lack of ultrastructural information to visualize the pathogens. Herein, we present a new method to visualize these pathogens during infection that circumvents these problems: by using a metabolic hijacking approach to bioorthogonally label the intracellular pathogen Salmonella Typhimurium and by using these bioorthogonal groups to introduce fluorophores compatible with stochastic optical reconstruction microscopy (STORM) and placing this in a correlative light electron microscopy (CLEM) workflow, the pathogen can be imaged within its host cell context Typhimurium with a resolution of 20 nm. This STORM-CLEM approach thus presents a new approach to understand these pathogens during infection.
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http://dx.doi.org/10.1002/cbic.201800230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6120560PMC
June 2018

Advances in cryo-electron tomography for biology and medicine.

Ann Anat 2018 May 8;217:82-96. Epub 2018 Mar 8.

Department of Cell and Chemical Biology, Leiden University Medical Center, 2300 RC Leiden, The Netherlands. Electronic address:

Cryo-electron tomography (CET) utilizes a combination of specimen cryo-fixation and multi-angle electron microscopy imaging to produce three-dimensional (3D) volume reconstructions of native-state macromolecular and subcellular biological structures with nanometer-scale resolution. In recent years, cryo-electron microscopy (cryoEM) has experienced a dramatic increase in the attainable resolution of 3D reconstructions, resulting from technical improvements of electron microscopes, improved detector sensitivity, the implementation of phase plates, automated data acquisition schemes, and improved image reconstruction software and hardware. These developments also greatly increased the usability and applicability of CET as a diagnostic and research tool, which is now enabling structural biologists to determine the structure of proteins in their native cellular environment to sub-nanometer resolution. These recent technical developments have stimulated us to update on our previous review (Koning, R.I., Koster, A.J., 2009. Cryo-electron tomography in biology and medicine. Ann Anat 191, 427-445) in which we described the fundamentals of CET. In this follow-up, we extend this basic description in order to explain the aforementioned recent advances, and describe related 3D techniques that can be applied to the anatomy of biological systems that are relevant for medicine.
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http://dx.doi.org/10.1016/j.aanat.2018.02.004DOI Listing
May 2018

Renal Subcapsular Transplantation of PSC-Derived Kidney Organoids Induces Neo-vasculogenesis and Significant Glomerular and Tubular Maturation In Vivo.

Stem Cell Reports 2018 03 1;10(3):751-765. Epub 2018 Mar 1.

Department of Internal Medicine - Nephrology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, the Netherlands; Einthoven Laboratory of Vascular and Regenerative Medicine, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, the Netherlands.

Human pluripotent stem cell (hPSC)-derived kidney organoids may facilitate disease modeling and the generation of tissue for renal replacement. Long-term application, however, will require transferability between hPSC lines and significant improvements in organ maturation. A key question is whether time or a patent vasculature is required for ongoing morphogenesis. Here, we show that hPSC-derived kidney organoids, derived in fully defined medium conditions and in the absence of any exogenous vascular endothelial growth factor, develop host-derived vascularization. In vivo imaging of organoids under the kidney capsule confirms functional glomerular perfusion as well as connection to pre-existing vascular networks in the organoids. Wide-field electron microscopy demonstrates that transplantation results in formation of a glomerular basement membrane, fenestrated endothelial cells, and podocyte foot processes. Furthermore, compared with non-transplanted organoids, polarization and segmental specialization of tubular epithelium are observed. These data demonstrate that functional vascularization is required for progressive morphogenesis of human kidney organoids.
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http://dx.doi.org/10.1016/j.stemcr.2018.01.041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5918682PMC
March 2018

Structures of C1-IgG1 provide insights into how danger pattern recognition activates complement.

Science 2018 02;359(6377):794-797

Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, Netherlands.

Danger patterns on microbes or damaged host cells bind and activate C1, inducing innate immune responses and clearance through the complement cascade. How these patterns trigger complement initiation remains elusive. Here, we present cryo-electron microscopy analyses of C1 bound to monoclonal antibodies in which we observed heterogeneous structures of single and clustered C1-immunoglobulin G1 (IgG1) hexamer complexes. Distinct C1q binding sites are observed on the two Fc-CH2 domains of each IgG molecule. These are consistent with known interactions and also reveal additional interactions, which are supported by functional IgG1-mutant analysis. Upon antibody binding, the C1q arms condense, inducing rearrangements of the C1rs proteases and tilting C1q's cone-shaped stalk. The data suggest that C1r may activate C1s within single, strained C1 complexes or between neighboring C1 complexes on surfaces.
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http://dx.doi.org/10.1126/science.aao4988DOI Listing
February 2018

Correlative microscopy for structural microbiology.

Curr Opin Microbiol 2018 06 4;43:132-138. Epub 2018 Feb 4.

Leiden University Medical Centre, Department of Molecular Cell Biology, PO Box 9600, 2300 RC Leiden, The Netherlands; Netherlands Centre for Electron Nanoscopy, Institute of Biology, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands. Electronic address:

Understanding how microbes utilize their environment is aided by visualizing them in their natural context at high resolution. Correlative imaging enables efficient targeting and identification of labelled viral and bacterial components by light microscopy combined with high resolution imaging by electron microscopy. Advances in genetic and bioorthogonal labelling, improved workflows for targeting and image correlation, and large-scale data collection are increasing the applicability of correlative imaging methods. Furthermore, developments in mass spectroscopy and soft X-ray imaging are expanding the correlative imaging modalities available. Investigating the structure and organization of microbes within their host by combined imaging methods provides important insights into mechanisms of infection and disease which cannot be obtained by other techniques.
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http://dx.doi.org/10.1016/j.mib.2018.01.009DOI Listing
June 2018

Postmortem MRI and histology demonstrate differential iron accumulation and cortical myelin organization in early- and late-onset Alzheimer's disease.

Neurobiol Aging 2018 02 28;62:231-242. Epub 2017 Oct 28.

Department of Radiology, Leiden University Medical Center, Leiden, the Netherlands; Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands.

Previous MRI studies reported cortical iron accumulation in early-onset (EOAD) compared to late-onset (LOAD) Alzheimer disease patients. However, the pattern and origin of iron accumulation is poorly understood. This study investigated the histopathological correlates of MRI contrast in both EOAD and LOAD. T2*-weighted MRI was performed on postmortem frontal cortex of controls, EOAD, and LOAD. Images were ordinally scored using predefined criteria followed by histology. Nonlinear histology-MRI registration was used to calculate pixel-wise spatial correlations based on the signal intensity. EOAD and LOAD were distinguishable based on 7T MRI from controls and from each other. Histology-MRI correlation analysis of the pixel intensities showed that the MRI contrast is best explained by increased iron accumulation and changes in cortical myelin, whereas amyloid and tau showed less spatial correspondence with T2*-weighted MRI. Neuropathologically, subtypes of Alzheimer's disease showed different patterns of iron accumulation and cortical myelin changes independent of amyloid and tau that may be detected by high-field susceptibility-based MRI.
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http://dx.doi.org/10.1016/j.neurobiolaging.2017.10.017DOI Listing
February 2018

Expression and Cleavage of Middle East Respiratory Syndrome Coronavirus nsp3-4 Polyprotein Induce the Formation of Double-Membrane Vesicles That Mimic Those Associated with Coronaviral RNA Replication.

mBio 2017 11 21;8(6). Epub 2017 Nov 21.

Section Electron Microscopy, Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands

Betacoronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV), are important pathogens causing potentially lethal infections in humans and animals. Coronavirus RNA synthesis is thought to be associated with replication organelles (ROs) consisting of modified endoplasmic reticulum (ER) membranes. These are transformed into double-membrane vesicles (DMVs) containing viral double-stranded RNA and into other membranous elements such as convoluted membranes, together forming a reticulovesicular network. Previous evidence suggested that the nonstructural proteins (nsp's) 3, 4, and 6 of the severe acute respiratory syndrome coronavirus (SARS-CoV), which contain transmembrane domains, would all be required for DMV formation. We have now expressed MERS-CoV replicase self-cleaving polyprotein fragments encompassing nsp3-4 or nsp3-6, as well as coexpressed nsp3 and nsp4 of either MERS-CoV or SARS-CoV, to characterize the membrane structures induced. Using electron tomography, we demonstrate that for both MERS-CoV and SARS-CoV coexpression of nsp3 and nsp4 is required and sufficient to induce DMVs. Coexpression of MERS-CoV nsp3 and nsp4 either as individual proteins or as a self-cleaving nsp3-4 precursor resulted in very similar DMVs, and in both setups we observed proliferation of zippered ER that appeared to wrap into nascent DMVs. Moreover, when inactivating nsp3-4 polyprotein cleavage by mutagenesis, we established that cleavage of the nsp3/nsp4 junction is essential for MERS-CoV DMV formation. Addition of the third MERS-CoV transmembrane protein, nsp6, did not noticeably affect DMV formation. These findings provide important insight into the biogenesis of coronavirus DMVs, establish strong similarities with other nidoviruses (specifically, the arteriviruses), and highlight possible general principles in viral DMV formation. The RNA replication of positive stranded RNA viruses of eukaryotes is thought to take place at cytoplasmic membranous replication organelles (ROs). Double-membrane vesicles are a prominent type of viral ROs. They are induced by coronaviruses, such as SARS-CoV and MERS-CoV, as well as by a number of other important pathogens, yet little is known about their biogenesis. In this study, we explored the viral protein requirements for the formation of MERS-CoV- and SARS-CoV-induced DMVs and established that coexpression of two of the three transmembrane subunits of the coronavirus replicase polyprotein, nonstructural proteins (nsp's) 3 and 4, is required and sufficient to induce DMV formation. Moreover, release of nsp3 and nsp4 from the polyprotein by proteolytic maturation is essential for this process. These findings provide a strong basis for further research on the biogenesis and functionality of coronavirus ROs and may point to more general principles of viral DMV formation.
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http://dx.doi.org/10.1128/mBio.01658-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5698553PMC
November 2017

Escaping Host Factor PI4KB Inhibition: Enterovirus Genomic RNA Replication in the Absence of Replication Organelles.

Cell Rep 2017 Oct;21(3):587-599

Department of Infectious Diseases & Immunology, Utrecht University, Utrecht 3584 CL, the Netherlands. Electronic address:

Enteroviruses reorganize cellular endomembranes into replication organelles (ROs) for genome replication. Although enterovirus replication depends on phosphatidylinositol 4-kinase type IIIβ (PI4KB), its role, and that of its product, phosphatidylinositol 4-phosphate (PI4P), is only partially understood. Exploiting a mutant coxsackievirus resistant to PI4KB inhibition, we show that PI4KB activity has distinct functions both in proteolytic processing of the viral polyprotein and in RO biogenesis. The escape mutation rectifies a proteolytic processing defect imposed by PI4KB inhibition, pointing to a possible escape mechanism. Remarkably, under PI4KB inhibition, the mutant virus could replicate its genome in the absence of ROs, using instead the Golgi apparatus. This impaired RO biogenesis provided an opportunity to investigate the proposed role of ROs in shielding enteroviral RNA from cellular sensors. Neither accelerated sensing of viral RNA nor enhanced innate immune responses was observed. Together, our findings challenge the notion that ROs are indispensable for enterovirus genome replication and immune evasion.
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http://dx.doi.org/10.1016/j.celrep.2017.09.068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5656745PMC
October 2017

Intradermal vaccination with hollow microneedles: A comparative study of various protein antigen and adjuvant encapsulated nanoparticles.

J Control Release 2017 Nov 21;266:109-118. Epub 2017 Sep 21.

Division of Drug Delivery Technology, Cluster BioTherapeutics, Leiden Academic Centre for Drug Research, Leiden University, Leiden, The Netherlands.

In this study, we investigated the potential of intradermal delivery of nanoparticulate vaccines to modulate the immune response of protein antigen using hollow microneedles. Four types of nanoparticles covering a broad range of physiochemical parameters, namely poly (lactic-co-glycolic) (PLGA) nanoparticles, liposomes, mesoporous silica nanoparticles (MSNs) and gelatin nanoparticles (GNPs) were compared. The developed nanoparticles were loaded with a model antigen (ovalbumin (OVA)) with and without an adjuvant (poly(I:C)), followed by the characterization of size, zeta potential, morphology, and loading and release of antigen and adjuvant. An in-house developed hollow-microneedle applicator was used to inject nanoparticle suspensions precisely into murine skin at a depth of about 120μm. OVA/poly(I:C)-loaded nanoparticles and OVA/poly(I:C) solution elicited similarly strong total IgG and IgG1 responses. However, the co-encapsulation of OVA and poly(I:C) in nanoparticles significantly increased the IgG2a response compared to OVA/poly(I:C) solution. PLGA nanoparticles and liposomes induced stronger IgG2a responses than MSNs and GNPs, correlating with sustained release of the antigen and adjuvant and a smaller nanoparticle size. When examining cellular responses, the highest CD8 and CD4 T cell responses were induced by OVA/poly(I:C)-loaded liposomes. In conclusion, the applicator controlled hollow microneedle delivery is an excellent method for intradermal injection of nanoparticle vaccines, allowing selection of optimal nanoparticle formulations for humoral and cellular immune responses.
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http://dx.doi.org/10.1016/j.jconrel.2017.09.021DOI Listing
November 2017

Inducing fluorescence of uranyl acetate as a dual-purpose contrast agent for correlative light-electron microscopy with nanometre precision.

Sci Rep 2017 09 5;7(1):10442. Epub 2017 Sep 5.

Section Electron Microscopy, Department of Molecular Cell Biology, Leiden University Medical Center, 2300 RC, Leiden, The Netherlands.

Correlative light-electron microscopy (CLEM) combines the high spatial resolution of transmission electron microscopy (TEM) with the capability of fluorescence light microscopy (FLM) to locate rare or transient cellular events within a large field of view. CLEM is therefore a powerful technique to study cellular processes. Aligning images derived from both imaging modalities is a prerequisite to correlate the two microscopy data sets, and poor alignment can limit interpretability of the data. Here, we describe how uranyl acetate, a commonly-used contrast agent for TEM, can be induced to fluoresce brightly at cryogenic temperatures (-195 °C) and imaged by cryoFLM using standard filter sets. This dual-purpose contrast agent can be used as a general tool for CLEM, whereby the equivalent staining allows direct correlation between fluorescence and TEM images. We demonstrate the potential of this approach by performing multi-colour CLEM of cells containing equine arteritis virus proteins tagged with either green- or red-fluorescent protein, and achieve high-precision localization of virus-induced intracellular membrane modifications. Using uranyl acetate as a dual-purpose contrast agent, we achieve an image alignment precision of ~30 nm, twice as accurate as when using fiducial beads, which will be essential for combining TEM with the evolving field of super-resolution light microscopy.
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http://dx.doi.org/10.1038/s41598-017-10905-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5585351PMC
September 2017

Best practices for managing large CryoEM facilities.

J Struct Biol 2017 09 4;199(3):225-236. Epub 2017 Aug 4.

New York Structural Biology Center, United States. Electronic address:

This paper provides an overview of the discussion and presentations from the Workshop on the Management of Large CryoEM Facilities held at the New York Structural Biology Center, New York, NY on February 6-7, 2017. A major objective of the workshop was to discuss best practices for managing cryoEM facilities. The discussions were largely focused on supporting single-particle methods for cryoEM and topics included: user access, assessing projects, workflow, sample handling, microscopy, data management and processing, and user training.
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http://dx.doi.org/10.1016/j.jsb.2017.07.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5605453PMC
September 2017

Building bridges between cellular and molecular structural biology.

Elife 2017 07 6;6. Epub 2017 Jul 6.

Molecular and Cellular Structure Cluster, European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, United Kingdom.

The integration of cellular and molecular structural data is key to understanding the function of macromolecular assemblies and complexes in their in vivo context. Here we report on the outcomes of a workshop that discussed how to integrate structural data from a range of public archives. The workshop identified two main priorities: the development of tools and file formats to support segmentation (that is, the decomposition of a three-dimensional volume into regions that can be associated with defined objects), and the development of tools to support the annotation of biological structures.
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http://dx.doi.org/10.7554/eLife.25835DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5524535PMC
July 2017

Human CD8 T Cells Damage Noninfected Epithelial Cells during Influenza Virus Infection In Vitro.

Am J Respir Cell Mol Biol 2017 11;57(5):536-546

1 Department of Viroscience, Erasmus Medical Center, Rotterdam, the Netherlands.

During severe influenza A virus (IAV) infections, a large amount of damage to the pulmonary epithelium is the result of the antiviral immune response. Specifically, whilst CD8 T cells are important for killing IAV-infected cells, during a severe IAV infection, they can damage uninfected epithelial cells. At present, the mechanisms by which this occurs are unclear. Here, we used a novel in vitro coculture model of human NCl-H441 cells and CD8 T cells to provide a new insight into how CD8 T cells may affect uninfected epithelial cells during severe IAV infections. Using this model, we show that human IAV-specific CD8 T cells produce soluble factors that reduce the barrier integrity of noninfected epithelial cells (referred to as "bystander damage"). We show that this bystander damage is the result of a combination of TNF-α and IFN-γ. This bystander damage occurred in the absence of widespread epithelial cell death and was instead associated with decreased expression of epithelial cell ion channels and pumps. Together, these data suggest that ameliorating the function of epithelial cell ion channels and pumps may help reduce immunopathology during severe IAV infections.
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http://dx.doi.org/10.1165/rcmb.2016-0377OCDOI Listing
November 2017