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1.  Localization and Relative Quantification of Carbon Nanotubes in Cells with Multispectral Imaging Flow Cytometry 
Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich cell structures and de facto there is still no quantitative method to assess their distribution at cell and tissue levels. What we propose here is an innovative method allowing the detection and quantification of CNTs in cells using a multispectral imaging flow cytometer (ImageStream, Amnis). This newly developed device integrates both a high-throughput of cells and high resolution imaging, providing thus images for each cell directly in flow and therefore statistically relevant image analysis. Each cell image is acquired on bright-field (BF), dark-field (DF), and fluorescent channels, giving access respectively to the level and the distribution of light absorption, light scattered and fluorescence for each cell. The analysis consists then in a pixel-by-pixel comparison of each image, of the 7,000-10,000 cells acquired for each condition of the experiment. Localization and quantification of CNTs is made possible thanks to some particular intrinsic properties of CNTs: strong light absorbance and scattering; indeed CNTs appear as strongly absorbed dark spots on BF and bright spots on DF with a precise colocalization.
This methodology could have a considerable impact on studies about interactions between nanomaterials and cells given that this protocol is applicable for a large range of nanomaterials, insofar as they are capable of absorbing (and/or scattering) strongly enough the light.
doi:10.3791/50566
PMCID: PMC4048057  PMID: 24378540
Bioengineering; Issue 82; bioengineering; imaging flow cytometry; Carbon Nanotubes; bio-nano-interactions; cellular uptake; cell trafficking
2.  Homotypic and Heterotypic Adhesion Induced by Odorant Receptors and the β2-Adrenergic Receptor 
PLoS ONE  2013;8(12):e80100.
In the mouse olfactory system regulated expression of a large family of G Protein-Coupled Receptors (GPCRs), the Odorant Receptors (ORs), provides each sensory neuron with a single OR identity. In the wiring of the olfactory sensory neuron projections, a complex axon sorting process ensures the segregation of >1,000 subpopulations of axons of the same OR identity into homogeneously innervated glomeruli. ORs are critical determinants in axon sorting, and their presence on olfactory axons raises the intriguing possibility that they may participate in axonal wiring through direct or indirect trans-interactions mediating adhesion or repulsion between axons. In the present work, we used a biophysical assay to test the capacity of ORs to induce adhesion of cell doublets overexpressing these receptors. We also tested the β2 Adrenergic Receptor, a non-OR GPCR known to recapitulate the functions of ORs in olfactory axon sorting. We report here the first evidence for homo- and heterotypic adhesion between cells overexpressing the ORs MOR256-17 or M71, supporting the hypothesis that ORs may contribute to olfactory axon sorting by mediating differential adhesion between axons.
doi:10.1371/journal.pone.0080100
PMCID: PMC3846556  PMID: 24312457
3.  Deciphering the mechanisms of cellular uptake of engineered nanoparticles by accurate evaluation of internalization using imaging flow cytometry 
Background
The uptake of nanoparticles (NPs) by cells remains to be better characterized in order to understand the mechanisms of potential NP toxicity as well as for a reliable risk assessment. Real NP uptake is still difficult to evaluate because of the adsorption of NPs on the cellular surface.
Results
Here we used two approaches to distinguish adsorbed fluorescently labeled NPs from the internalized ones. The extracellular fluorescence was either quenched by Trypan Blue or the uptake was analyzed using imaging flow cytometry. We used this novel technique to define the inside of the cell to accurately study the uptake of fluorescently labeled (SiO2) and even non fluorescent but light diffracting NPs (TiO2). Time course, dose-dependence as well as the influence of surface charges on the uptake were shown in the pulmonary epithelial cell line NCI-H292. By setting up an integrative approach combining these flow cytometric analyses with confocal microscopy we deciphered the endocytic pathway involved in SiO2 NP uptake. Functional studies using energy depletion, pharmacological inhibitors, siRNA-clathrin heavy chain induced gene silencing and colocalization of NPs with proteins specific for different endocytic vesicles allowed us to determine macropinocytosis as the internalization pathway for SiO2 NPs in NCI-H292 cells.
Conclusion
The integrative approach we propose here using the innovative imaging flow cytometry combined with confocal microscopy could be used to identify the physico-chemical characteristics of NPs involved in their uptake in view to redesign safe NPs.
doi:10.1186/1743-8977-10-2
PMCID: PMC3599262  PMID: 23388071
Lung epithelial cells; ImageStreamX; Endocytosis; Clathrin; Macropinocytosis; TiO2; SiO2
4.  LFA-1 and ICAM-1 expression induced during melanoma-endothelial cell co-culture favors the transendothelial migration of melanoma cell lines in vitro 
BMC Cancer  2012;12:455.
Background
Patients with metastatic melanoma have a poor median rate of survival. It is therefore necessary to increase our knowledge about melanoma cell dissemination which includes extravasation, where cancer cells cross the endothelial barrier. Extravasation is well understood during travelling of white blood cells, and involves integrins such as LFA-1 (composed of two chains, CD11a and CD18) expressed by T cells, while ICAM-1 is induced during inflammation by endothelial cells. Although melanoma cell lines cross endothelial cell barriers, they do not express LFA-1. We therefore hypothesized that melanoma-endothelial cell co-culture might induce the LFA-1/ICAM ligand/receptor couple during melanoma transmigration.
Methods
A transwell approach has been used as well as blocking antibodies against CD11a, CD18 and ICAM-1. Data were analyzed with an epifluorescence microscope. Fluorescence intensity was quantified with the ImageJ software.
Results
We show here that HUVEC-conditioned medium induce cell-surface expression of LFA-1 on melanoma cell lines. Similarly melanoma-conditioned medium activates ICAM-1 expression in endothelial cells. Accordingly blocking antibodies of ICAM-1, CD11a or CD18 strongly decrease melanoma transmigration. We therefore demonstrate that melanoma cells can cross endothelial monolayers in vitro due to the induction of ICAM-1 and LFA-1 occurring during the co-culture of melanoma and endothelial cells. Our data further suggest a role of LFA-1 and ICAM-1 in the formation of melanoma cell clumps enhancing tumor cell transmigration.
Conclusion
Melanoma-endothelial cell co-culture induces LFA-1 and ICAM-1 expression, thereby favoring in vitro melanoma trans-migration.
doi:10.1186/1471-2407-12-455
PMCID: PMC3495854  PMID: 23039186
Melanoma; Transendothelial migration; Metastasis; LFA-1; ICAM-1; HUVEC
5.  Apn1 AP-endonuclease is essential for the repair of oxidatively damaged DNA bases in yeast frataxin-deficient cells 
Human Molecular Genetics  2012;21(18):4060-4072.
Frataxin deficiency results in mitochondrial dysfunction and oxidative stress and it is the cause of the hereditary neurodegenerative disease Friedreich ataxia (FA). Here, we present evidence that one of the pleiotropic effects of oxidative stress in frataxin-deficient yeast cells (Δyfh1 mutant) is damage to nuclear DNA and that repair requires the Apn1 AP-endonuclease of the base excision repair pathway. Major phenotypes of Δyfh1 cells are respiratory deficit, disturbed iron homeostasis and sensitivity to oxidants. These phenotypes are weak or absent under anaerobiosis. We show here that exposure of anaerobically grown Δyfh1 cells to oxygen leads to down-regulation of antioxidant defenses, increase in reactive oxygen species, delay in G1- and S-phases of the cell cycle and damage to mitochondrial and nuclear DNA. Nuclear DNA lesions in Δyfh1 cells are primarily caused by oxidized bases and single-strand breaks that can be detected 15–30 min after oxygen exposition. The Apn1 enzyme is essential for the repair of the DNA lesions in Δyfh1 cells. Compared with Δyfh1, the double Δyfh1Δapn1 mutant shows growth impairment, increased mutagenesis and extreme sensitivity to H2O2. On the contrary, overexpression of the APN1 gene in Δyfh1 cells decreases spontaneous and induced mutagenesis. Our results show that frataxin deficiency in yeast cells leads to increased DNA base oxidation and requirement of Apn1 for repair, suggesting that DNA damage and repair could be important features in FA disease progression.
doi:10.1093/hmg/dds230
PMCID: PMC3428155  PMID: 22706278
6.  USF Binding Sequences from the HS4 Insulator Element Impose Early Replication Timing on a Vertebrate Replicator 
PLoS Biology  2012;10(3):e1001277.
A combination of cis-regulatory elements can impose the formation of an early replicating domain in a naturally late replicating region and might constitute the basic unit of early replicating domains.
The nuclear genomes of vertebrates show a highly organized program of DNA replication where GC-rich isochores are replicated early in S-phase, while AT-rich isochores are late replicating. GC-rich regions are gene dense and are enriched for active transcription, suggesting a connection between gene regulation and replication timing. Insulator elements can organize independent domains of gene transcription and are suitable candidates for being key regulators of replication timing. We have tested the impact of inserting a strong replication origin flanked by the β-globin HS4 insulator on the replication timing of naturally late replicating regions in two different avian cell types, DT40 (lymphoid) and 6C2 (erythroid). We find that the HS4 insulator has the capacity to impose a shift to earlier replication. This shift requires the presence of HS4 on both sides of the replication origin and results in an advance of replication timing of the target locus from the second half of S-phase to the first half when a transcribed gene is positioned nearby. Moreover, we find that the USF transcription factor binding site is the key cis-element inside the HS4 insulator that controls replication timing. Taken together, our data identify a combination of cis-elements that might constitute the basic unit of multi-replicon megabase-sized early domains of DNA replication.
Author Summary
All eukaryotic organisms must duplicate their genome precisely once before cell division. This occurs according to an established temporal program during S-phase (when DNA synthesis takes place) of the cell cycle. In vertebrates, this program is regulated at the level of large chromosomal domains ranging from 200 kb to 2 Mb, but the molecular mechanisms that control the temporal firing order of animal replication origins are not clearly understood. Using the genetically tractable chicken DT40 cell system, we identified a minimal combination of cis-regulatory DNA elements that is able to shift the timing of a naturally “mid-late” replicated region to “mid-early.” This critical group of elements is composed of one strong replication origin flanked by binding sequences for the upstream stimulatory factor (USF) protein. The additional presence of a strongly transcribed gene shifted the region towards an even earlier replication time, suggesting cooperation between cis-elements when establishing temporal programs of replication. We speculate that USF binding sequences cooperate with sites of replication initiation and transcribed genes to promote the establishment of early replicating domains along vertebrate genomes.
doi:10.1371/journal.pbio.1001277
PMCID: PMC3295818  PMID: 22412349
7.  A Novel Role for Dbx1-Derived Cajal-Retzius Cells in Early Regionalization of the Cerebral Cortical Neuroepithelium 
PLoS Biology  2010;8(7):e1000440.
Patterning of the cerebral cortex during embryogenesis depends not only on passive diffusion of morphogens but also on signal delivery by Cajal-Retzius neurons that migrate over long distances.
Patterning of the cortical neuroepithelium occurs at early stages of embryonic development in response to secreted molecules from signaling centers. These signals have been shown to establish the graded expression of transcription factors in progenitors within the ventricular zone and to control the size and positioning of cortical areas. Cajal-Retzius (CR) cells are among the earliest generated cortical neurons and migrate from the borders of the developing pallium to cover the cortical primordium by E11.5. We show that molecularly distinct CR subtypes distribute in specific combinations in pallial territories at the time of cortical regionalization. By means of genetic ablation experiments in mice, we report that loss of septum Dbx1-derived CR cells in the rostromedial pallium between E10.5 and E11.5 results in the redistribution of CR subtypes. This leads to changes in the expression of transcription factors within the neuroepithelium and in the proliferation properties of medial and dorsal cortical progenitors. Early regionalization defects correlate with shifts in the positioning of cortical areas at postnatal stages in the absence of alterations of gene expression at signaling centers. We show that septum-derived CR neurons express a highly specific repertoire of signaling factors. Our results strongly suggest that these cells, migrating over long distances and positioned in the postmitotic compartment, signal to ventricular zone progenitors and, thus, function as modulators of early cortical patterning.
Author Summary
Patterning of the cerebral cortex occurs early during embryonic development in response to secreted molecules or morphogens produced at signaling centers. These morphogens establish the graded expression of transcription factors (TFs) in progenitor cells and control the size and positioning of cortical areas in the postnatal animal. CR cells are among the earliest born cortical neurons and play a crucial role in cortical lamination. They are generated at signaling centers and migrate over long distances to cover its entire surface. We show that three different CR subtypes distribute in specific proportions in cortical territories. Genetic ablation of one subpopulation leads to a highly dynamic redistribution of the two others. This results in defects in expression of transcription factors and in progenitor cell proliferation, which correlate with the resulting changes in the size and positioning of cortical areas. Given our additional evidence that CR subtypes express specific repertoires of signaling factors, the ablation phenotypes point to a novel early role for CR cells as mediators of cortical patterning and suggest that CR cells are able to signal to progenitor cells. Our data thus add to the conventional model that morphogens act by passive diffusion and point to a strategy of morphogen delivery over long distance by migrating cells.
doi:10.1371/journal.pbio.1000440
PMCID: PMC2910656  PMID: 20668538
8.  Polycyclic aromatic hydrocarbon components contribute to the mitochondria-antiapoptotic effect of fine particulate matter on human bronchial epithelial cells via the aryl hydrocarbon receptor 
Background
Nowadays, effects of fine particulate matter (PM2.5) are well-documented and related to oxidative stress and pro-inflammatory response. Nevertheless, epidemiological studies show that PM2.5 exposure is correlated with an increase of pulmonary cancers and the remodeling of the airway epithelium involving the regulation of cell death processes. Here, we investigated the components of Parisian PM2.5 involved in either the induction or the inhibition of cell death quantified by different parameters of apoptosis and delineated the mechanism underlying this effect.
Results
In this study, we showed that low levels of Parisian PM2.5 are not cytotoxic for three different cell lines and primary cultures of human bronchial epithelial cells. Conversely, a 4 hour-pretreatment with PM2.5 prevent mitochondria-driven apoptosis triggered by broad spectrum inducers (A23187, staurosporine and oligomycin) by reducing the mitochondrial transmembrane potential loss, the subsequent ROS production, phosphatidylserine externalization, plasma membrane permeabilization and typical morphological outcomes (cell size decrease, massive chromatin and nuclear condensation, formation of apoptotic bodies). The use of recombinant EGF and specific inhibitor led us to rule out the involvement of the classical EGFR signaling pathway as well as the proinflammatory cytokines secretion. Experiments performed with different compounds of PM2.5 suggest that endotoxins as well as carbon black do not participate to the antiapoptotic effect of PM2.5. Instead, the water-soluble fraction, washed particles and organic compounds such as polycyclic aromatic hydrocarbons (PAH) could mimic this antiapoptotic activity. Finally, the activation or silencing of the aryl hydrocarbon receptor (AhR) showed that it is involved into the molecular mechanism of the antiapoptotic effect of PM2.5 at the mitochondrial checkpoint of apoptosis.
Conclusions
The PM2.5-antiapoptotic effect in addition to the well-documented inflammatory response might explain the maintenance of a prolonged inflammation state induced after pollution exposure and might delay repair processes of injured tissues.
doi:10.1186/1743-8977-7-18
PMCID: PMC2914693  PMID: 20663163

Results 1-8 (8)