Publications by authors named "Coralie Spiegelhalter"

19 Publications

  • Page 1 of 1

Ral GTPases promote breast cancer metastasis by controlling biogenesis and organ targeting of exosomes.

Elife 2021 Jan 6;10. Epub 2021 Jan 6.

INSERM UMR_S1109, Tumor Biomechanics, Strasbourg, France.

Cancer extracellular vesicles (EVs) shuttle at distance and fertilize pre-metastatic niches facilitating subsequent seeding by tumor cells. However, the link between EV secretion mechanisms and their capacity to form pre-metastatic niches remains obscure. Using mouse models, we show that GTPases of the Ral family control, through the phospholipase D1, multi-vesicular bodies homeostasis and tune the biogenesis and secretion of pro-metastatic EVs. Importantly, EVs from RalA or RalB depleted cells have limited organotropic capacities and are less efficient in promoting metastasis. RalA and RalB reduce the EV levels of the adhesion molecule MCAM/CD146, which favors EV-mediated metastasis by allowing EVs targeting to the lungs. Finally, RalA, RalB, and MCAM/CD146, are factors of poor prognosis in breast cancer patients. Altogether, our study identifies RalGTPases as central molecules linking the mechanisms of EVs secretion and cargo loading to their capacity to disseminate and induce pre-metastatic niches in a CD146-dependent manner.
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http://dx.doi.org/10.7554/eLife.61539DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822591PMC
January 2021

Functional analyses of STIM1 mutations reveal a common pathomechanism for tubular aggregate myopathy and Stormorken syndrome.

Neuropathology 2020 Dec 19;40(6):559-569. Epub 2020 Oct 19.

Department of Translational Medicine and Neurogenetics, Institute of Genetics and Molecular and Cellular Biology (IGBMC), Illkirch, France.

Tubular aggregate myopathy (TAM) is a progressive disorder characterized by muscle weakness, cramps, and myalgia. TAM clinically overlaps with Stormorken syndrome (STRMK), combining TAM with miosis, thrombocytopenia, hyposplenism, ichthyosis, short stature, and dyslexia. TAM and STRMK arise from gain-of-function mutations in STIM1 (stromal interaction molecule 1) or ORAI1, both encoding key regulators of Ca homeostasis, and mutations in either gene result in excessive extracellular Ca entry. The pathomechanistic similarities and differences between TAM and STRMK are only partially understood. Here we provide functional in vitro experiments demonstrating that STIM1 harboring the TAM D84G or the STRMK R304W mutation similarly cluster and exert a dominant effect on the wild-type protein. Both mutants recruit ORAI1 to the clusters, increase cytosolic Ca levels, promote major nuclear import of the Ca -dependent transcription factor NFAT (nuclear factor of activated T cells), and trigger the formation of circular membrane stacks. In conclusion, the analyzed TAM and STRMK mutations have a comparable impact on STIM1 protein function and downstream effects of excessive Ca entry, highlighting that TAM and STRMK involve a common pathomechanism.
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http://dx.doi.org/10.1111/neup.12692DOI Listing
December 2020

Aggregation and Amyloidogenicity of the Nuclear Coactivator Binding Domain of CREB-Binding Protein.

Chemistry 2020 Aug 14;26(44):9889-9899. Epub 2020 Jul 14.

ISIS (Institut de Science et d'Ingénierie Supramoléculaires) and, icFRC (International Center for Frontier Research in Chemistry), University of Strasbourg, CNRS-UMR 7006, 8 allée Gaspard Monge, 67083, Strasbourg, France.

The nuclear coactivator binding domain (NCBD) of transcriptional co-regulator CREB-binding protein (CBP) is an example of conformationally malleable proteins that can bind to structurally unrelated protein targets and adopt distinct folds in the respective protein complexes. Here, we show that the folding landscape of NCBD contains an alternative pathway that results in protein aggregation and self-assembly into amyloid fibers. The initial steps of such protein misfolding are driven by intermolecular interactions of its N-terminal α-helix bringing multiple NCBD molecules into contact. These oligomers then undergo slow but progressive interconversion into β-sheet-containing aggregates. To reveal the concealed aggregation potential of NCBD we used a chemically synthesized mirror-image d-NCBD form. The addition of d-NCBD promoted self-assembly into amyloid precipitates presumably due to formation of thermodynamically more stable racemic β-sheet structures. The unexpected aggregation of NCBD needs to be taken into consideration given the multitude of protein-protein interactions and resulting biological functions mediated by CBP.
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http://dx.doi.org/10.1002/chem.202001847DOI Listing
August 2020

Lysosomal degradation of newly formed insulin granules contributes to β cell failure in diabetes.

Nat Commun 2019 07 25;10(1):3312. Epub 2019 Jul 25.

Institut de Génétique et de Biologie Moléculaire et Cellulaire, 67404, Illkirch, France.

Compromised function of insulin-secreting pancreatic β cells is central to the development and progression of Type 2 Diabetes (T2D). However, the mechanisms underlying β cell failure remain incompletely understood. Here, we report that metabolic stress markedly enhances macroautophagy-independent lysosomal degradation of nascent insulin granules. In different model systems of diabetes including of human origin, stress-induced nascent granule degradation (SINGD) contributes to loss of insulin along with mammalian/mechanistic Target of Rapamycin (mTOR)-dependent suppression of macroautophagy. Expression of Protein Kinase D (PKD), a negative regulator of SINGD, is reduced in diabetic β cells. Pharmacological activation of PKD counters SINGD and delays the onset of T2D. Conversely, inhibition of PKD exacerbates SINGD, mitigates insulin secretion and accelerates diabetes. Finally, reduced levels of lysosomal tetraspanin CD63 prevent SINGD, leading to increased insulin secretion. Overall, our findings implicate aberrant SINGD in the pathogenesis of diabetes and suggest new therapeutic strategies to prevent β cell failure.
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http://dx.doi.org/10.1038/s41467-019-11170-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6658524PMC
July 2019

Macrophage functionality and homeostasis in response to oligoethyleneglycol-coated IONPs: Impact of a dendritic architecture.

Int J Pharm 2019 Feb 14;556:287-300. Epub 2018 Dec 14.

Université de Strasbourg, CNRS, CAMB UMR 7199, F-67000 Strasbourg, France.

The engineering of iron oxide nanoparticles (IONPs) for biomedical use has received great interest over the past decade. In the present study we investigated the biocompatibility of IONPs grafted with linear (2P) or generation 1 (2PG1) or 2 (2PG2) dendronized oligoethyleneglycol units in THP-1-derived macrophages. To evaluate IONP effects on cell functionality and homeostasis, mitochondrial function (MTT assay), membrane permeability (LDH release), inflammation (IL-8), oxidative stress (reduced glutathione, GSH), NLRP3 inflammasome activation (IL-1β) and nanoparticle cellular uptake (intracellular iron content) were quantified after a 4-h or 24-h cell exposure to increasing IONP concentrations (0-300 µg Fe/mL). IONPs coated with a linear molecule, [email protected], were highly taken up by cells and induced significant dose-dependent IL-8 release, oxidative stress and NLRP3 inflammasome activation. In comparison, IONPs coated with dendrons of generation 1 ([email protected]) and 2 ([email protected]) exhibited better biocompatibility. Effect of the dendritic architecture of the surface coating was investigated in a kinetic experiment involving cell short-term exposure (30 min or 1 h 30) to the two dendronized IONPs. [email protected] disrupted cellular homeostasis (LDH release, IL-1β and IL-8 secretion) to a greater extend than [email protected], which makes this last IONP the best candidate as MRI contrast or theranostic agent.
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http://dx.doi.org/10.1016/j.ijpharm.2018.12.024DOI Listing
February 2019

ReAsH/tetracystein-based correlative light-electron microscopy for HIV-1 imaging during the early stages of infection.

Methods Appl Fluoresc 2018 Jul 9;6(4):045001. Epub 2018 Jul 9.

Laboratoire de Bioimagerie et Pathologies, CNRS UMR 7021, Strasbourg University, Faculty of Pharmacy, 74 route du Rhin, Illkirch, France.

Visualization of viruses in the host cell during the course of infection by correlative light-electron microscopy (CLEM) requires a specific labelling of the viral structures in order to recognize the nanometric viral cores in the intracellular environment. For Human immunodeficiency virus type 1 (HIV-1), the labelling approaches developed for fluorescence microscopy are generally not suited for transmission electron microscopy (TEM), so that imaging of HIV-1 particles in infected cells by CLEM is not straightforward. Herein, we adapt the labeling approach with a tetracystein tag (TC) and a biarsenical resorufin-based label (ReAsH) for monitoring the HIV-1 particles during the early stages of HIV-1 infection by CLEM. In this approach, the ReAsH fluorophore triggers the photo-conversion of 3,3-diaminobenzidine tetrahydrochloride (DAB), generating a precipitate sensitive to osmium tetroxide staining that can be visualized by transmission electron microscopy. The TC tag is fused to the nucleocapsid protein NCp7, a nucleic acid chaperone that binds to the viral genome. HeLa cells, infected by ReAsH-labeled pseudoviruses containg NCp7-TC proteins exhibit strong fluorescent cytoplasmic spots that overlap with dark precipitates in the TEM sections. The DAB precipitates corresponding to single viral cores are observed all over the cytoplasm, and notably near microtubules and nuclear pores. This work describes for the first time a specific contrast given by HIV-1 viral proteins in TEM images and opens new perspectives for the use of CLEM to monitor the intracellular traffic of viral complexes.
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http://dx.doi.org/10.1088/2050-6120/aacec1DOI Listing
July 2018

Modulation of large dense core vesicle insulin content mediates rhythmic hormone release from pancreatic beta cells over the 24h cycle.

PLoS One 2018 15;13(3):e0193882. Epub 2018 Mar 15.

CSGA, AgroSup Dijon, Centre National de la Recherche Scientifique, Institut National de la Recherche Agronomique, Université de Bourgogne Franche-Comté, 9E Boulevard Jeanne d'Arc, Dijon, France.

The rhythmic nature of insulin secretion over the 24h cycle in pancreatic islets has been mostly investigated using transcriptomics studies showing that modulation of insulin secretion over this cycle is achieved via distal stages of insulin secretion. We set out to measure β-cell exocytosis using in depth cell physiology techniques at several time points. In agreement with the activity and feeding pattern of nocturnal rodents, we find that C57/Bl6J islets in culture for 24h exhibit higher insulin secretion during the corresponding dark phase than in the light phase (Zeitgeber Time ZT20 and ZT8, respectively, in vivo). Glucose-induced insulin secretion is increased by 21% despite normal intracellular Ca2+ transients and depolarization-evoked exocytosis, as measured by whole-cell capacitance measurements. This paradox is explained by a 1.37-fold increase in beta cell insulin content. Ultramorphological analyses show that vesicle size and density are unaltered, demonstrating that intravesicular insulin content per granule is modulated over the 24h cycle. Proinsulin levels did not change between ZT8 and ZT20. Islet glucagon content was inversely proportional to insulin content indicating that this unique feature is likely to support a physiological role. Microarray data identified the differential expression of 301 transcripts, of which 26 are miRNAs and 54 are known genes (including C2cd4b, a gene previously involved in insulin processing, and clock genes such as Bmal1 and Rev-erbα). Mouse β-cell secretion over the full course of the 24h cycle may rely on several distinct cellular functions but late night increase in insulin secretion depends solely on granule insulin content.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0193882PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5854349PMC
June 2018

RAL-1 controls multivesicular body biogenesis and exosome secretion.

J Cell Biol 2015 Oct;211(1):27-37

Institut de Génétique et de Biologie Moléculaire et Cellulaire, Development and Stem Cells Program, Centre National de la Recherche Scientifique (UMR7104), Institut National de la Santé et de la Recherche Médicale (U964), Université de Strasbourg, 67400 Illkirch, France Institut de Biologie Paris (UMR7622), UPMC, 75005 Paris, France

Exosomes are secreted vesicles arising from the fusion of multivesicular bodies (MVBs) with the plasma membrane. Despite their importance in various processes, the molecular mechanisms controlling their formation and release remain unclear. Using nematodes and mammary tumor cells, we show that Ral GTPases are involved in exosome biogenesis. In Caenorhabditis elegans, RAL-1 localizes at the surface of secretory MVBs. A quantitative electron microscopy analysis of RAL-1-deficient animals revealed that RAL-1 is involved in both MVB formation and their fusion with the plasma membrane. These functions do not involve the exocyst complex, a common Ral guanosine triphosphatase (GTPase) effector. Furthermore, we show that the target membrane SNARE protein SYX-5 colocalizes with a constitutively active form of RAL-1 at the plasma membrane, and MVBs accumulate under the plasma membrane when SYX-5 is absent. In mammals, RalA and RalB are both required for the secretion of exosome-like vesicles in cultured cells. Therefore, Ral GTPases represent new regulators of MVB formation and exosome release.
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http://dx.doi.org/10.1083/jcb.201504136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4602040PMC
October 2015

Developmental Alterations in Heart Biomechanics and Skeletal Muscle Function in Desmin Mutants Suggest an Early Pathological Root for Desminopathies.

Cell Rep 2015 Jun 4;11(10):1564-76. Epub 2015 Jun 4.

Institut de Génétique et de Biologie Moléculaire et Cellulaire, 67404 Illkirch, France; Centre National de la Recherche Scientifique, UMR7104, 67404 Illkirch, France; Institut National de la Santé et de la Recherche Médicale, U964, 67404 Illkirch, France; Université de Strasbourg, 67404 Illkirch, France. Electronic address:

Desminopathies belong to a family of muscle disorders called myofibrillar myopathies that are caused by Desmin mutations and lead to protein aggregates in muscle fibers. To date, the initial pathological steps of desminopathies and the impact of desmin aggregates in the genesis of the disease are unclear. Using live, high-resolution microscopy, we show that Desmin loss of function and Desmin aggregates promote skeletal muscle defects and alter heart biomechanics. In addition, we show that the calcium dynamics associated with heart contraction are impaired and are associated with sarcoplasmic reticulum dilatation as well as abnormal subcellular distribution of Ryanodine receptors. Our results demonstrate that desminopathies are associated with perturbed excitation-contraction coupling machinery and that aggregates are more detrimental than Desmin loss of function. Additionally, we show that pharmacological inhibition of aggregate formation and Desmin knockdown revert these phenotypes. Our data suggest alternative therapeutic approaches and further our understanding of the molecular determinants modulating Desmin aggregate formation.
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http://dx.doi.org/10.1016/j.celrep.2015.05.010DOI Listing
June 2015

Insulin granules. Insulin secretory granules control autophagy in pancreatic β cells.

Science 2015 Feb;347(6224):878-82

Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), INSERM, CNRS, Université de Strasbourg, 67404 Illkirch, France. Nouvel Hôpital Civil, Laboratoire de Biochimie et de Biologie Moléculaire, Université de Strasbourg, 67091 Strasbourg, France.

Pancreatic β cells lower insulin release in response to nutrient depletion. The question of whether starved β cells induce macroautophagy, a predominant mechanism maintaining energy homeostasis, remains poorly explored. We found that, in contrast to many mammalian cells, macroautophagy in pancreatic β cells was suppressed upon starvation. Instead, starved β cells induced lysosomal degradation of nascent secretory insulin granules, which was controlled by protein kinase D (PKD), a key player in secretory granule biogenesis. Starvation-induced nascent granule degradation triggered lysosomal recruitment and activation of mechanistic target of rapamycin that suppressed macroautophagy. Switching from macroautophagy to insulin granule degradation was important to keep insulin secretion low upon fasting. Thus, β cells use a PKD-dependent mechanism to adapt to nutrient availability and couple autophagy flux to secretory function.
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http://dx.doi.org/10.1126/science.aaa2628DOI Listing
February 2015

Higher chromatin mobility supports totipotency and precedes pluripotency in vivo.

Genes Dev 2014 May;28(10):1042-7

CNRS/INSERM U964, Université de Strasbourg, Institut de Génétique et de Biologie Moléculaire et Cellulaire, F-67404 Illkirch, France;

The fusion of the gametes upon fertilization results in the formation of a totipotent cell. Embryonic chromatin is expected to be able to support a large degree of plasticity. However, whether this plasticity relies on a particular conformation of the embryonic chromatin is unknown. Moreover, whether chromatin plasticity is functionally linked to cellular potency has not been addressed. Here, we adapted fluorescence recovery after photobleaching (FRAP) in the developing mouse embryo and show that mobility of the core histones H2A, H3.1, and H3.2 is unusually high in two-cell stage embryos and decreases as development proceeds. The transition toward pluripotency is accompanied by a decrease in histone mobility, and, upon lineage allocation, pluripotent cells retain higher mobility than the differentiated trophectoderm. Importantly, totipotent two-cell-like embryonic stem cells also display high core histone mobility, implying that reprogramming toward totipotency entails changes in chromatin mobility. Our data suggest that changes in chromatin dynamics underlie the transitions in cellular plasticity and that higher chromatin mobility is at the nuclear foundations of totipotency.
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http://dx.doi.org/10.1101/gad.238881.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4035533PMC
May 2014

Autistic-like syndrome in mu opioid receptor null mice is relieved by facilitated mGluR4 activity.

Neuropsychopharmacology 2014 Aug 12;39(9):2049-60. Epub 2014 Mar 12.

Département de Médecine Translationelle et Neurogénétique, Institut de Génétique et de Biologie Moléculaire et Cellulaire, INSERM U-964, CNRS UMR-7104, Université de Strasbourg, Illkirch, France.

The etiology of Autism Spectrum Disorders (ASDs) remains largely unknown. Identifying vulnerability genes for autism represents a major challenge in the field and allows the development of animal models for translational research. Mice lacking the mu opioid receptor gene (Oprm1(-/-)) were recently proposed as a monogenic mouse model of autism, based on severe deficits in social behavior and communication skills. We confirm this hypothesis by showing that adult Oprm1(-/-) animals recapitulate core and multiple comorbid behavioral symptoms of autism and also display anatomical, neurochemical, and genetic landmarks of the disease. Chronic facilitation of mGluR4 signaling, which we identified as a novel pharmacological target in ASDs in these mice, was more efficient in alleviating behavioral deficits than the reference molecule risperidone. Altogether, our data provide first evidence that disrupted mu opioid receptor signaling is sufficient to trigger a comprehensive autistic syndrome, maybe through blunted social reward processes, and this mouse model opens promising avenues for therapeutic innovation.
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http://dx.doi.org/10.1038/npp.2014.59DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4104328PMC
August 2014

Correlative light and electron microscopy: from live cell dynamic to 3D ultrastructure.

Methods Mol Biol 2014 ;1117:485-501

Imaging Center, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France.

Correlative light and electron microscopy (CLEM) aims at combining data acquired from the same sample through both imaging modalities. Many combinations can be found in the literature where almost any kind of light microscopy (LM) has been associated to different processing in electron microscopy (EM) and applied to a wide variety of specimen, from cultured cells to multicellular organisms. In this chapter, we focus on a technique that intends to combine LM acquisition on living cells with transmission EM (TEM) analysis. A specific attention is given to the description of a method to bring precise coordinates to the object of interest, to allow a straightforward correlation between LM and EM. Moreover, we describe how, by using high-pressure freezing as a fixation technique, dynamic events observed at the LM are captured and studied at the ultrastructural level.
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http://dx.doi.org/10.1007/978-1-62703-776-1_21DOI Listing
August 2014

STARD3 or STARD3NL and VAP form a novel molecular tether between late endosomes and the ER.

J Cell Sci 2013 Dec 8;126(Pt 23):5500-12. Epub 2013 Oct 8.

Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Functional Genomics and Cancer Department, 1 rue Laurent Fries, Illkirch, 67404, France.

Inter-organelle membrane contacts sites (MCSs) are specific subcellular regions favoring the exchange of metabolites and information. We investigated the potential role of the late-endosomal membrane-anchored proteins StAR related lipid transfer domain-3 (STARD3) and STARD3 N-terminal like (STARD3NL) in the formation of MCSs involving late-endosomes (LEs). We demonstrate that both STARD3 and STARD3NL create MCSs between LEs and the endoplasmic reticulum (ER). STARD3 and STARD3NL use a conserved two phenylalanines in an acidic tract (FFAT)-motif to interact with ER-anchored VAP proteins. Together, they form an LE-ER tethering complex allowing heterologous membrane apposition. This LE-ER tethering complex affects organelle dynamics by altering the formation of endosomal tubules. An in situ proximity ligation assay between STARD3, STARD3NL and VAP proteins identified endogenous LE-ER MCS. Thus, we report here the identification of proteins involved in inter-organellar interaction.
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http://dx.doi.org/10.1242/jcs.139295DOI Listing
December 2013

Birbeck granule-like "organized smooth endoplasmic reticulum" resulting from the expression of a cytoplasmic YFP-tagged langerin.

PLoS One 2013 5;8(4):e60813. Epub 2013 Apr 5.

Unité Mixte de Recherche Santé 725, Institut National de la Santé et de la Recherche Médicale, Strasbourg, France.

Langerin is required for the biogenesis of Birbeck granules (BGs), the characteristic organelles of Langerhans cells. We previously used a Langerin-YFP fusion protein having a C-terminal luminal YFP tag to dynamically decipher the molecular and cellular processes which accompany the traffic of Langerin. In order to elucidate the interactions of Langerin with its trafficking effectors and their structural impact on the biogenesis of BGs, we generated a YFP-Langerin chimera with an N-terminal, cytosolic YFP tag. This latter fusion protein induced the formation of YFP-positive large puncta. Live cell imaging coupled to a fluorescence recovery after photobleaching approach showed that this coalescence of proteins in newly formed compartments was static. In contrast, the YFP-positive structures present in the pericentriolar region of cells expressing Langerin-YFP chimera, displayed fluorescent recovery characteristics compatible with active membrane exchanges. Using correlative light-electron microscopy we showed that the coalescent structures represented highly organized stacks of membranes with a pentalaminar architecture typical of BGs. Continuities between these organelles and the rough endoplasmic reticulum allowed us to identify the stacks of membranes as a form of "Organized Smooth Endoplasmic Reticulum" (OSER), with distinct molecular and physiological properties. The involvement of homotypic interactions between cytoplasmic YFP molecules was demonstrated using an A206K variant of YFP, which restored most of the Langerin traffic and BG characteristics observed in Langerhans cells. Mutation of the carbohydrate recognition domain also blocked the formation of OSER. Hence, a "double-lock" mechanism governs the behavior of YFP-Langerin, where asymmetric homodimerization of the YFP tag and homotypic interactions between the lectin domains of Langerin molecules participate in its retention and the subsequent formation of BG-like OSER. These observations confirm that BG-like structures appear wherever Langerin accumulates and confirm that membrane trafficking effectors dictate their physiology and, illustrate the importance of molecular interactions in the architecture of intracellular membranes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0060813PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3618057PMC
October 2013

Immunomodulatory properties of multi-walled carbon nanotubes in peripheral blood mononuclear cells from healthy subjects and allergic patients.

Toxicol Lett 2013 Feb 20;217(2):91-101. Epub 2012 Dec 20.

Laboratoire de Conception et Application de Molécules Bioactives, CNRS-Université de Strasbourg, Faculté de Pharmacie, Illkirch, France.

In the present study, we investigated the immunomodulatory activity of multi-walled carbon nanotubes (MWCNTs) in peripheral blood mononuclear cells (PBMCs) from healthy donors and mite-allergic subjects. Freshly prepared PBMCs, stimulated or not with Toll-like receptor (TLR)1-9 agonists, a T cell mitogen (phytohemagglutinin A) or mite allergen extract were cultured in the presence or absence of MWCNTs. Secretion of TNF-α, IL-2, IL-5, IL-6, IL-12/23p40 or IFN-γ was quantified in the culture supernatants by ELISA. Basal secretion of all the cytokines was not altered by MWCNTs in PBMCs from both healthy donors and allergic subjects. In PBMCs from healthy donors, TNF-α, IL-6 and IL-12/23p40 secretion in response to the TLR4 agonist, lipopolysaccharide was however increased in a dose-dependent manner by MWCNTs. Significant increases in the release of these cytokines were also observed in PBMCs stimulated with a TLR2 or TLR3 agonist. MWCNTs also increased the release of IL-2 and IFN-γ by PBMCs stimulated with a T cell mitogen. In contrast, MWCNTs inhibited allergen-induced IL-5 secretion by PBMCs from mite-allergic subjects. As well, MWCNTs altered the capacity of PBMC-derived monocytes to differentiate into functional dendritic cells. All together, our data suggest that according to its immune cell target, MWCNTs may either promote or suppress immune responses in humans. Further investigations are necessary to fully understand the complexity behind interactions of engineered nanoparticles with the immune system.
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http://dx.doi.org/10.1016/j.toxlet.2012.12.008DOI Listing
February 2013

Perispeckles are major assembly sites for the exon junction core complex.

Mol Biol Cell 2012 May 14;23(9):1765-82. Epub 2012 Mar 14.

Institut de Génétique et de Biologie Moléculaire et Cellulaire, Unité Mixte de Recherche 7104, Centre National de la Recherche Scientifique/U964 Institut National de Santé et de Recherche Médicale/Université de Strasbourg, 67404 Illkirch, France.

The exon junction complex (EJC) is loaded onto mRNAs as a consequence of splicing and regulates multiple posttranscriptional events. MLN51, Magoh, Y14, and eIF4A3 form a highly stable EJC core, but where this tetrameric complex is assembled in the cell remains unclear. Here we show that EJC factors are enriched in domains that we term perispeckles and are visible as doughnuts around nuclear speckles. Fluorescence resonance energy transfer analyses and EJC assembly mutants show that perispeckles do not store free subunits, but instead are enriched for assembled cores. At the ultrastructural level, perispeckles are distinct from interchromatin granule clusters that may function as storage sites for splicing factors and intermingle with perichromatin fibrils, where nascent RNAs and active RNA Pol II are present. These results support a model in which perispeckles are major assembly sites for the tetrameric EJC core. This subnuclear territory thus represents an intermediate region important for mRNA maturation, between transcription sites and splicing factor reservoirs and assembly sites.
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http://dx.doi.org/10.1091/mbc.E12-01-0040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3338441PMC
May 2012

Lung deposition and toxicological responses evoked by multi-walled carbon nanotubes dispersed in a synthetic lung surfactant in the mouse.

Arch Toxicol 2012 Jan 30;86(1):137-49. Epub 2011 Jul 30.

Faculté de Pharmacie, Laboratoire de Conception et Application de Molécules Bioactives, CNRS, Université de Strasbourg, Illkirch, France.

In the present work, we elaborated a synthetic lung surfactant composed of dipalmitoyl phosphatidylcholine (DPPC), phosphatidylglycerol, cholesterol and bovine serum albumin (BSA), as a vehicle to study the lung toxicity of pristine multi-walled carbon nanotubes (MWCNT). MWCNT were dispersed in surfactant, saline or saline containing DPPC, BSA, Pluronic(®) F68 or sodium dodecyl sulfate, for comparison. Dispersions were characterized visually, and by light microscopy, dynamic light scattering and transmission electronic microscopy (TEM). Deposition of surfactant-dispersed MWCNT in the lung of BALB/c mice upon single or repeated administrations was analyzed by histology and TEM. Inflammation and airway remodeling were assessed in bronchoalveolar lavage fluid (BALF) or lung tissue of mice by counting cells and quantifying cytokines, tumor growth factor (TGF)-β1 and collagen, and by histology. We found that the elaborated surfactant is more effective in dispersing MWCNT when compared to the other agents, while being biocompatible. Surfactant-dispersed MWCNT distributed all throughout the mouse airways upon single and repeated administrations and were observed in alveolar macrophages and epithelial cells, and in infiltrated neutrophils. Mice that received a single administration of MWCNT showed neutrophil infiltrate and greater concentrations of tumor necrosis factor (TNF)-α, keratinocyte-derived chemokine (KC) and interleukin (IL)-17 in BALF when compared to controls. After repeated MWCNT administrations, increases in macrophage number, KC and TGF-β1 levels in BALF, and collagen deposition and mucus hyperplasia in lung tissue were observed. Altogether, the elaborated lung surfactant could be a valuable tool to further study the toxicological impact of pristine MWCNT in laboratory animals.
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http://dx.doi.org/10.1007/s00204-011-0741-yDOI Listing
January 2012

From dynamic live cell imaging to 3D ultrastructure: novel integrated methods for high pressure freezing and correlative light-electron microscopy.

PLoS One 2010 Feb 3;5(2):e9014. Epub 2010 Feb 3.

Imaging Centre, IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), Illkirch, France.

Background: In cell biology, the study of proteins and organelles requires the combination of different imaging approaches, from live recordings with light microscopy (LM) to electron microscopy (EM).

Methodology: To correlate dynamic events in adherent cells with both ultrastructural and 3D information, we developed a method for cultured cells that combines confocal time-lapse images of GFP-tagged proteins with electron microscopy. With laser micro-patterned culture substrate, we created coordinates that were conserved at every step of the sample preparation and visualization processes. Specifically designed for cryo-fixation, this method allowed a fast freezing of dynamic events within seconds and their ultrastructural characterization. We provide examples of the dynamic oligomerization of GFP-tagged myotubularin (MTM1) phosphoinositides phosphatase induced by osmotic stress, and of the ultrastructure of membrane tubules dependent on amphiphysin 2 (BIN1) expression.

Conclusion: Accessible and versatile, we show that this approach is efficient to routinely correlate functional and dynamic LM with high resolution morphology by EM, with immuno-EM labeling, with 3D reconstruction using serial immuno-EM or tomography, and with scanning-EM.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0009014PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2815783PMC
February 2010
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