Publications by authors named "Spencer Shorte"

57 Publications

LocalZProjector and DeProj: a toolbox for local 2D projection and accurate morphometrics of large 3D microscopy images.

BMC Biol 2021 Jul 2;19(1):136. Epub 2021 Jul 2.

Image Analysis Hub, C2RT / DTPS, Institut Pasteur, Paris, France.

Background: Quantitative imaging of epithelial tissues requires bioimage analysis tools that are widely applicable and accurate. In the case of imaging 3D tissues, a common preprocessing step consists of projecting the acquired 3D volume on a 2D plane mapping the tissue surface. While segmenting the tissue cells is amenable on 2D projections, it is still very difficult and cumbersome in 3D. However, for many specimen and models used in developmental and cell biology, the complex content of the image volume surrounding the epithelium in a tissue often reduces the visibility of the biological object in the projection, compromising its subsequent analysis. In addition, the projection may distort the geometry of the tissue and can lead to strong artifacts in the morphology measurement.

Results: Here we introduce a user-friendly toolbox built to robustly project epithelia on their 2D surface from 3D volumes and to produce accurate morphology measurement corrected for the projection distortion, even for very curved tissues. Our toolbox is built upon two components. LocalZProjector is a configurable Fiji plugin that generates 2D projections and height-maps from potentially large 3D stacks (larger than 40 GB per time-point) by only incorporating signal of the planes with local highest variance/mean intensity, despite a possibly complex image content. DeProj is a MATLAB tool that generates correct morphology measurements by combining the height-map output (such as the one offered by LocalZProjector) and the results of a cell segmentation on the 2D projection, hence effectively deprojecting the 2D segmentation in 3D. In this paper, we demonstrate their effectiveness over a wide range of different biological samples. We then compare its performance and accuracy against similar existing tools.

Conclusions: We find that LocalZProjector performs well even in situations where the volume to project also contains unwanted signal in other layers. We show that it can process large images without a pre-processing step. We study the impact of geometrical distortions on morphological measurements induced by the projection. We measured very large distortions which are then corrected by DeProj, providing accurate outputs.
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http://dx.doi.org/10.1186/s12915-021-01037-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8254216PMC
July 2021

Unveiling Interindividual Variability of Human Fibroblast Innate Immune Response Using Robust Cell-Based Protocols.

Front Immunol 2020 11;11:569331. Epub 2021 Jan 11.

UTechS Photonic BioImaging, C2RT, Institut Pasteur, Paris, France.

The () project is a clinical study centered on the detailed characterization of the baseline and induced immune responses in blood samples from 1,000 healthy donors. Analyses of these samples has lay ground for seminal studies on the genetic and environmental determinants of immunologic variance in a healthy cohort population. In the current study we developed methods enabling standardized quantification of MI-cohort-derived primary fibroblasts responses. Our results show that human donor cohort fibroblast responses to stimulation by different MAMPs analogs allows to characterize individual donor immune-phenotype variability. The results provide proof-of-concept foundation to a new experimental framework for such studies. A bio-bank of primary fibroblast lines was generated from 323 out of 1,000 healthy individuals selected from the MI-study cohort. To study inter-donor variability of innate immune response in primary human dermal fibroblasts we chose to measure the TLR3 and TLR4 response pathways, both receptors being expressed and previously studied in fibroblasts. We established high-throughput automation compatible methods for standardized primary fibroblast cell activation, using purified MAMPS analogs, poly I:C and LPS that stimulate TLR3 and TLR4 pathways respectively. These results were in turn compared with a stimulation method using infection by HSV-1 virus. Our "Add-only" protocol minimizes high-throughput automation system variability facilitating whole process automation from cell plating through stimulation to recovery of cell supernatants, and fluorescent labeling. Images were acquired automatically by high-throughput acquisition on an automated high-content imaging microscope. Under these methodological conditions standardized image acquisition provided for quantification of cellular responses allowing biological variability to be measured with low system noise and high biological signal fidelity. Optimal for automated analysis of immuno-phenotype of primary human cell responses our method and experimental framework as reported here is highly compatible to high-throughput screening protocols like those necessary for chemo-genomic screening. In context of primary fibroblasts derived from donors enrolled to the MI-clinical-study our results open the way to assert the utility of studying immune-phenotype characteristics relevant to a human clinical cohort.
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http://dx.doi.org/10.3389/fimmu.2020.569331DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7829859PMC
May 2021

Shigella-mediated oxygen depletion is essential for intestinal mucosa colonization.

Nat Microbiol 2019 11 5;4(11):2001-2009. Epub 2019 Aug 5.

Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur, Paris, France.

Pathogenic enterobacteria face various oxygen (O) levels during intestinal colonization from the O-deprived lumen to oxygenated tissues. Using Shigella flexneri as a model, we have previously demonstrated that epithelium invasion is promoted by O in a type III secretion system-dependent manner. However, subsequent pathogen adaptation to tissue oxygenation modulation remained unknown. Assessing single-cell distribution, together with tissue oxygenation, we demonstrate here that the colonic mucosa O is actively depleted by S. flexneri aerobic respiration-and not host neutrophils-during infection, leading to the formation of hypoxic foci of infection. This process is promoted by type III secretion system inactivation in infected tissues, favouring colonizers over explorers. We identify the molecular mechanisms supporting infectious hypoxia induction, and demonstrate here how enteropathogens optimize their colonization capacity in relation to their ability to manipulate tissue oxygenation during infection.
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http://dx.doi.org/10.1038/s41564-019-0525-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6817363PMC
November 2019

Next-Generation Phenotypic Screening in Early Drug Discovery for Infectious Diseases.

Trends Parasitol 2019 07 5;35(7):559-570. Epub 2019 Jun 5.

Institut Pasteur Paris, UTechS-PBI/Imagopole, 25-28 rue du Docteur Roux, 75015, France; Institut Pasteur Korea, 16 Daewangpangyo-ro 712 beon-gil, Bundang-gu, Seongnam-si, Gyeonggi-do, 13488, Republic of Korea. Electronic address:

Cell-based phenotypic screening has proven to be valuable, notably in recapitulating relevant biological conditions, for example, the host cell/pathogen niche. However, the corresponding methodological complexity is not readily compatible with high-throughput pipelines, and fails to inform either molecular target or mechanism of action, which frustrates conventional drug-discovery roadmaps. We review the state-of-the-art and emerging technologies that suggest new strategies for harnessing value from the complexity of phenotypic screening and augmenting powerful utility for translational drug discovery. Advances in cellular, molecular, and bioinformatics technologies are converging at a cutting edge where the complexity of phenotypic screening may no longer be considered a hinderance but rather a catalyst to chemotherapeutic discovery for infectious diseases.
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http://dx.doi.org/10.1016/j.pt.2019.05.004DOI Listing
July 2019

Imaging of Red-Shifted Light From Bioluminescent Tumors Using Fluorescence by Unbound Excitation From Luminescence.

Front Bioeng Biotechnol 2019 5;7:73. Epub 2019 Apr 5.

UTechS Photonic BioImaging, C2RT, Unité Pathogénie Microbienne Moléculaire, Institut Pasteur, INSERM U1202, Paris, France.

Early detection of tumors is today a major challenge and requires sensitive imaging methodologies coupled with new efficient probes. optical bioluminescence imaging has been widely used in the field of preclinical oncology to visualize tumors and several cancer cell lines have been genetically modified to provide bioluminescence signals. However, the light emitted by the majority of commonly used luciferases is usually in the blue part of the visible spectrum, where tissue absorption is still very high, making deep tissue imaging non-optimal, and calling for optimized optical imaging methodologies. We have previously shown that red-shifting of bioluminescence signal by Fluorescence Unbound Excitation from Luminescence (FUEL) is a mean to increase bioluminescence signal sensitivity detection . Here, we applied FUEL to tumor detection in two different subcutaneous tumor models: the auto-luminescent human embryonic kidney (HEK293) cell line and the murine B16-F10 melanoma cell line previously transfected with a plasmid encoding the Luc2 firefly luciferase. Tumor size and bioluminescence were measured over time and tumor vascularization characterized. We then locally injected near infrared emitting Quantum Dots (NIR QDs) in the tumor site and observed a red-shifting of bioluminescence signal by (FUEL) indicating that FUEL could be used to allow deeper tumor detection in mice.
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http://dx.doi.org/10.3389/fbioe.2019.00073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6460942PMC
April 2019

Adhesion to nanofibers drives cell membrane remodeling through one-dimensional wetting.

Nat Commun 2018 10 25;9(1):4450. Epub 2018 Oct 25.

Pathogenesis of Vascular Infections Unit, INSERM, Institut Pasteur, Paris, 75015, France.

The shape of cellular membranes is highly regulated by a set of conserved mechanisms that can be manipulated by bacterial pathogens to infect cells. Remodeling of the plasma membrane of endothelial cells by the bacterium Neisseria meningitidis is thought to be essential during the blood phase of meningococcal infection, but the underlying mechanisms are unclear. Here we show that plasma membrane remodeling occurs independently of F-actin, along meningococcal type IV pili fibers, by a physical mechanism that we term 'one-dimensional' membrane wetting. We provide a theoretical model that describes the physical basis of one-dimensional wetting and show that this mechanism occurs in model membranes interacting with nanofibers, and in human cells interacting with extracellular matrix meshworks. We propose one-dimensional wetting as a new general principle driving the interaction of cells with their environment at the nanoscale that is diverted by meningococci during infection.
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http://dx.doi.org/10.1038/s41467-018-06948-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6202395PMC
October 2018

MuscleJ: a high-content analysis method to study skeletal muscle with a new Fiji tool.

Skelet Muscle 2018 08 6;8(1):25. Epub 2018 Aug 6.

Experimental Neuropathology Unit, Infection and Epidemiology Department, Institut Pasteur, 25, rue du Docteur Roux, 75015, Paris, France.

Background: Skeletal muscle has the capacity to adapt to environmental changes and regenerate upon injury. To study these processes, most experimental methods use quantification of parameters obtained from images of immunostained skeletal muscle. Muscle cross-sectional area, fiber typing, localization of nuclei within the muscle fiber, the number of vessels, and fiber-associated stem cells are used to assess muscle physiology. Manual quantification of these parameters is time consuming and only poorly reproducible. While current state-of-the-art software tools are unable to analyze all these parameters simultaneously, we have developed MuscleJ, a new bioinformatics tool to do so.

Methods: Running on the popular open source Fiji software platform, MuscleJ simultaneously analyzes parameters from immunofluorescent staining, imaged by different acquisition systems in a completely automated manner.

Results: After segmentation of muscle fibers, up to three other channels can be analyzed simultaneously. Dialog boxes make MuscleJ easy-to-use for biologists. In addition, we have implemented color in situ cartographies of results, allowing the user to directly visualize results on reconstituted muscle sections.

Conclusion: We report here that MuscleJ results were comparable to manual observations made by five experts. MuscleJ markedly enhances statistical analysis by allowing reliable comparison of skeletal muscle physiology-pathology results obtained from different laboratories using different acquisition systems. Providing fast robust multi-parameter analyses of skeletal muscle physiology-pathology, MuscleJ is available as a free tool for the skeletal muscle community.
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http://dx.doi.org/10.1186/s13395-018-0171-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6091189PMC
August 2018

Multi-view light-sheet imaging and tracking with the MaMuT software reveals the cell lineage of a direct developing arthropod limb.

Elife 2018 03 29;7. Epub 2018 Mar 29.

Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States.

During development, coordinated cell behaviors orchestrate tissue and organ morphogenesis. Detailed descriptions of cell lineages and behaviors provide a powerful framework to elucidate the mechanisms of morphogenesis. To study the cellular basis of limb development, we imaged transgenic fluorescently-labeled embryos from the crustacean with multi-view light-sheet microscopy at high spatiotemporal resolution over several days of embryogenesis. The cell lineage of outgrowing thoracic limbs was reconstructed at single-cell resolution with new software called Massive Multi-view Tracker (MaMuT). In silico clonal analyses suggested that the early limb primordium becomes subdivided into anterior-posterior and dorsal-ventral compartments whose boundaries intersect at the distal tip of the growing limb. Limb-bud formation is associated with spatial modulation of cell proliferation, while limb elongation is also driven by preferential orientation of cell divisions along the proximal-distal growth axis. Cellular reconstructions were predictive of the expression patterns of limb development genes including the BMP morphogen Decapentaplegic.
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http://dx.doi.org/10.7554/eLife.34410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5929908PMC
March 2018

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy.

J Vis Exp 2018 03 6(133). Epub 2018 Mar 6.

Cell Polarity, Migration and Cancer Unit, UMR 3691, CNRS, Institut Pasteur.

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, polarity, and motility. The organization of the actin and microtubule networks has been extensively studied but that of IFs is not yet fully characterized. IFs have an average diameter of 10 nm and form a network extending throughout the cell cytoplasm. They are physically associated with actin and microtubules through molecular motors and cytoskeletal linkers. This tight association is at the heart of the regulatory mechanisms that ensure the coordinated regulation of the three cytoskeletal networks required for most cell functions. It is therefore crucial to visualize IFs alone and also together with each of the other cytoskeletal networks. However, IF networks are extremely dense in most cell types, especially in glial cells, which makes its resolution very difficult to achieve with standard fluorescence microscopy (lateral resolution of ~250 nm). Direct STochastic Optical Reconstruction Microscopy (dSTORM) is a technique allowing a gain in lateral resolution of one order of magnitude. Here, we show that lateral dSTORM resolution is sufficient to resolve the dense organization of the IF networks and, in particular, of IF bundles surrounding microtubules. Such tight association is likely to participate in the coordinated regulation of these two networks and may, explain how vimentin IFs template and stabilize microtubule organization as well as could influence microtubule dependent vesicular trafficking. More generally, we show how the observation of two cytoskeletal components with dual-color dSTORM technique brings new insight into their mutual interaction.
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http://dx.doi.org/10.3791/57087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5931441PMC
March 2018

Fluorescence imaging host pathogen interactions: fifteen years benefit of hindsight….

Curr Opin Microbiol 2018 06 19;43:193-198. Epub 2018 Mar 19.

Institut Pasteur, Citech, Imagopole-UTechS-PBI Photonic BioImaging, 25-28 rue du Dr Roux, 75015 Paris, France.

We consider in review current state-of-the-art fluorescence microscopy for investigating the host-pathogen interface. Our perspective is honed from years with literally thousands of microbiologists using the variety of imaging technologies available within our dedicated BSL2/BSL3 optical imaging research service facilities at the Institut Pasteur Paris founded from scratch in 2001. During fifteen years learning from the success and failures of introducing different fluorescence imaging technologies, methods, and technical development strategies we provide here a synopsis review of our experience to date and a synthesis of how we see the future in perspective for fluorescence imaging at the host-pathogen interface.
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http://dx.doi.org/10.1016/j.mib.2018.03.001DOI Listing
June 2018

Assessing Vacuolar Escape of Listeria Monocytogenes.

Methods Mol Biol 2017 ;1535:173-195

Institut Pasteur, Unité des Interactions Bactéries-Cellules, 25, rue du Docteur Roux, Paris, 75015, France.

Listeria monocytogenes is a bacterial pathogen which invades and multiplies within non-professional phagocytes. Signaling cascades involved in cellular entry have been extensively analyzed, but the events leading to vacuolar escape remain less clear. In this chapter, we detail a microscopy FRET-based assay which allows quantitatively measuring L. monocytogenes infection and escape from its internalization vacuole, as well as a correlative light/electron microscopy method to investigate the morphological features of the vacuolar compartments containing L. monocytogenes.
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http://dx.doi.org/10.1007/978-1-4939-6673-8_11DOI Listing
January 2018

Screening out irrelevant cell-based models of disease.

Nat Rev Drug Discov 2016 11 12;15(11):751-769. Epub 2016 Sep 12.

Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XR, UK.

The common and persistent failures to translate promising preclinical drug candidates into clinical success highlight the limited effectiveness of disease models currently used in drug discovery. An apparent reluctance to explore and adopt alternative cell- and tissue-based model systems, coupled with a detachment from clinical practice during assay validation, contributes to ineffective translational research. To help address these issues and stimulate debate, here we propose a set of principles to facilitate the definition and development of disease-relevant assays, and we discuss new opportunities for exploiting the latest advances in cell-based assay technologies in drug discovery, including induced pluripotent stem cells, three-dimensional (3D) co-culture and organ-on-a-chip systems, complemented by advances in single-cell imaging and gene editing technologies. Funding to support precompetitive, multidisciplinary collaborations to develop novel preclinical models and cell-based screening technologies could have a key role in improving their clinical relevance, and ultimately increase clinical success rates.
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http://dx.doi.org/10.1038/nrd.2016.175DOI Listing
November 2016

TrackMate: An open and extensible platform for single-particle tracking.

Methods 2017 02 3;115:80-90. Epub 2016 Oct 3.

Laboratory for Optical and Computational Instrumentation, University of Wisconsin-Madison, Madison, WI 53706, USA; Morgridge Institute for Research, Madison, WI 53719, USA.

We present TrackMate, an open source Fiji plugin for the automated, semi-automated, and manual tracking of single-particles. It offers a versatile and modular solution that works out of the box for end users, through a simple and intuitive user interface. It is also easily scriptable and adaptable, operating equally well on 1D over time, 2D over time, 3D over time, or other single and multi-channel image variants. TrackMate provides several visualization and analysis tools that aid in assessing the relevance of results. The utility of TrackMate is further enhanced through its ability to be readily customized to meet specific tracking problems. TrackMate is an extensible platform where developers can easily write their own detection, particle linking, visualization or analysis algorithms within the TrackMate environment. This evolving framework provides researchers with the opportunity to quickly develop and optimize new algorithms based on existing TrackMate modules without the need of having to write de novo user interfaces, including visualization, analysis and exporting tools. The current capabilities of TrackMate are presented in the context of three different biological problems. First, we perform Caenorhabditis-elegans lineage analysis to assess how light-induced damage during imaging impairs its early development. Our TrackMate-based lineage analysis indicates the lack of a cell-specific light-sensitive mechanism. Second, we investigate the recruitment of NEMO (NF-κB essential modulator) clusters in fibroblasts after stimulation by the cytokine IL-1 and show that photodamage can generate artifacts in the shape of TrackMate characterized movements that confuse motility analysis. Finally, we validate the use of TrackMate for quantitative lifetime analysis of clathrin-mediated endocytosis in plant cells.
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http://dx.doi.org/10.1016/j.ymeth.2016.09.016DOI Listing
February 2017

Phenotypic clustering: a novel method for microglial morphology analysis.

J Neuroinflammation 2016 06 17;13(1):153. Epub 2016 Jun 17.

Imagopole - CITech, Institut Pasteur, Paris, France.

Background: Microglial cells are tissue-resident macrophages of the central nervous system. They are extremely dynamic, sensitive to their microenvironment and present a characteristic complex and heterogeneous morphology and distribution within the brain tissue. Many experimental clues highlight a strong link between their morphology and their function in response to aggression. However, due to their complex "dendritic-like" aspect that constitutes the major pool of murine microglial cells and their dense network, precise and powerful morphological studies are not easy to realize and complicate correlation with molecular or clinical parameters.

Methods: Using the knock-in mouse model CX3CR1(GFP/+), we developed a 3D automated confocal tissue imaging system coupled with morphological modelling of many thousands of microglial cells revealing precise and quantitative assessment of major cell features: cell density, cell body area, cytoplasm area and number of primary, secondary and tertiary processes. We determined two morphological criteria that are the complexity index (CI) and the covered environment area (CEA) allowing an innovative approach lying in (i) an accurate and objective study of morphological changes in healthy or pathological condition, (ii) an in situ mapping of the microglial distribution in different neuroanatomical regions and (iii) a study of the clustering of numerous cells, allowing us to discriminate different sub-populations.

Results: Our results on more than 20,000 cells by condition confirm at baseline a regional heterogeneity of the microglial distribution and phenotype that persists after induction of neuroinflammation by systemic injection of lipopolysaccharide (LPS). Using clustering analysis, we highlight that, at resting state, microglial cells are distributed in four microglial sub-populations defined by their CI and CEA with a regional pattern and a specific behaviour after challenge.

Conclusions: Our results counteract the classical view of a homogenous regional resting state of the microglial cells within the brain. Microglial cells are distributed in different defined sub-populations that present specific behaviour after pathological challenge, allowing postulating for a cellular and functional specialization. Moreover, this new experimental approach will provide a support not only to neuropathological diagnosis but also to study microglial function in various disease models while reducing the number of animals needed to approach the international ethical statements.
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http://dx.doi.org/10.1186/s12974-016-0614-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4912769PMC
June 2016

A Dual Microscopy-Based Assay To Assess Listeria monocytogenes Cellular Entry and Vacuolar Escape.

Appl Environ Microbiol 2016 01 23;82(1):211-7. Epub 2015 Oct 23.

Institut Pasteur, Unité des Interactions Bactéries-Cellules, Paris, France INSERM, U604, Paris, France INRA, USC2020, Paris, France.

Listeria monocytogenes is a Gram-positive bacterium and a facultative intracellular pathogen that invades mammalian cells, disrupts its internalization vacuole, and proliferates in the host cell cytoplasm. Here, we describe a novel image-based microscopy assay that allows discrimination between cellular entry and vacuolar escape, enabling high-content screening to identify factors specifically involved in these two steps. We first generated L. monocytogenes and Listeria innocua strains expressing a β-lactamase covalently attached to the bacterial cell wall. These strains were then incubated with HeLa cells containing the Förster resonance energy transfer (FRET) probe CCF4 in their cytoplasm. The CCF4 probe was cleaved by the bacterial surface β-lactamase only in cells inoculated with L. monocytogenes but not those inoculated with L. innocua, thereby demonstrating bacterial access to the host cytoplasm. Subsequently, we performed differential immunofluorescence staining to distinguish extracellular versus total bacterial populations in samples that were also analyzed by the FRET-based assay. With this two-step analysis, bacterial entry can be distinguished from vacuolar rupture in a single experiment. Our novel approach represents a powerful tool for identifying factors that determine the intracellular niche of L. monocytogenes.
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http://dx.doi.org/10.1128/AEM.02302-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4702615PMC
January 2016

Bioimage analysis of Shigella infection reveals targeting of colonic crypts.

Proc Natl Acad Sci U S A 2015 Jun 8;112(25):E3282-90. Epub 2015 Jun 8.

Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur, 75724 Paris, France; INSERM, U1202, 75015 Paris, France; Collège de France, 75005 Paris, France

Few studies within the pathogenic field have used advanced imaging and analytical tools to quantitatively measure pathogenicity in vivo. In this work, we present a novel approach for the investigation of host-pathogen processes based on medium-throughput 3D fluorescence imaging. The guinea pig model for Shigella flexneri invasion of the colonic mucosa was used to monitor the infectious process over time with GFP-expressing S. flexneri. A precise quantitative imaging protocol was devised to follow individual S. flexneri in a large tissue volume. An extensive dataset of confocal images was obtained and processed to extract specific quantitative information regarding the progression of S. flexneri infection in an unbiased and exhaustive manner. Specific parameters included the analysis of S. flexneri positions relative to the epithelial surface, S. flexneri density within the tissue, and volume of tissue destruction. In particular, at early time points, there was a clear association of S. flexneri with crypts, key morphological features of the colonic mucosa. Numerical simulations based on random bacterial entry confirmed the bias of experimentally measured S. flexneri for early crypt targeting. The application of a correlative light and electron microscopy technique adapted for thick tissue samples further confirmed the location of S. flexneri within colonocytes at the mouth of crypts. This quantitative imaging approach is a novel means to examine host-pathogen systems in a tailored and robust manner, inclusive of the infectious agent.
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http://dx.doi.org/10.1073/pnas.1509091112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4485126PMC
June 2015

Microtubule-associated proteins 1 (MAP1) promote human immunodeficiency virus type I (HIV-1) intracytoplasmic routing to the nucleus.

J Biol Chem 2015 Feb 11;290(8):4631-4646. Epub 2014 Dec 11.

From INSERM U941, Institut Universitaire d'Hématologie de l'Hôpital Saint-Louis, 75010 Paris, France,. Electronic address:

After cell entry, HIV undergoes rapid transport toward the nucleus using microtubules and microfilaments. Neither the cellular cytoplasmic components nor the viral proteins that interact to mediate transport have yet been identified. Using a yeast two-hybrid screen, we identified four cytoskeletal components as putative interaction partners for HIV-1 p24 capsid protein: MAP1A, MAP1S, CKAP1, and WIRE. Depletion of MAP1A/MAP1S in indicator cell lines and primary human macrophages led to a profound reduction in HIV-1 infectivity as a result of impaired retrograde trafficking, demonstrated by a characteristic accumulation of capsids away from the nuclear membrane, and an overall defect in nuclear import. MAP1A/MAP1S did not impact microtubule network integrity or cell morphology but contributed to microtubule stabilization, which was shown previously to facilitate infection. In addition, we found that MAP1 proteins interact with HIV-1 cores both in vitro and in infected cells and that interaction involves MAP1 light chain LC2. Depletion of MAP1 proteins reduced the association of HIV-1 capsids with both dynamic and stable microtubules, suggesting that MAP1 proteins help tether incoming viral capsids to the microtubular network, thus promoting cytoplasmic trafficking. This work shows for the first time that following entry into target cells, HIV-1 interacts with the cytoskeleton via its p24 capsid protein. Moreover, our results support a role for MAP1 proteins in promoting efficient retrograde trafficking of HIV-1 by stimulating the formation of stable microtubules and mediating the association of HIV-1 cores with microtubules.
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http://dx.doi.org/10.1074/jbc.M114.613133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4335204PMC
February 2015

Conical diffraction illumination opens the way for low phototoxicity super-resolution imaging.

Cell Adh Migr 2014 ;8(5):430-9

a Bioaxial SAS ; Paris , France.

We present a new technology for super-resolution fluorescence imaging, based on conical diffraction. Conical diffraction is a linear, singular phenomenon, taking place when a laser beam is diffracted through a biaxial crystal. We use conical diffraction in a thin biaxial crystal to generate illumination patterns that are more compact than the classical Gaussian beam, and use them to generate a super-resolution imaging modality. While there already exist several super-resolution modalities, our technology (biaxial super-resolution: BSR) is distinguished by the unique combination of several performance features. Using BSR super-resolution data are achieved using low light illumination significantly less than required for classical confocal imaging, which makes BSR ideal for live-cell, long-term time-lapse super-resolution imaging. Furthermore, no specific sample preparation is required, and any fluorophore can be used. Perhaps most exciting, improved resolution BSR-imaging resolution enhancement can be achieved with any type of objective no matter the magnification, numerical aperture, working distance, or the absence or presence of immersion medium. In this article, we present the first implementation of BSR modality on a commercial confocal microscope. We acquire and analyze validation data, showing high quality super-resolved images of biological objects, and demonstrate the wide applicability of the technology. We report live-cell super-resolution imaging over a long period, and show that the light dose required for super-resolution imaging is far below the threshold likely to generate phototoxicity.
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http://dx.doi.org/10.4161/cam.29358DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4594584PMC
October 2015

Shigella subverts the host recycling compartment to rupture its vacuole.

Cell Host Microbe 2014 Oct;16(4):517-30

Institut Pasteur, Dynamics of Host-Pathogen interactions Unit, 25 Rue du Dr. Roux, 75724 Paris, France. Electronic address:

Shigella enters epithlial cells via internalization into a vacuole. Subsequent vacuolar membrane rupture allows bacterial escape into the cytosol for replication and cell-to-cell spread. Bacterial effectors such as IpgD, a PI(4,5)P2 phosphatase that generates PI(5)P and alters host actin, facilitate this internalization. Here, we identify host proteins involved in Shigella uptake and vacuolar membrane rupture by high-content siRNA screening and subsequently focus on Rab11, a constituent of the recycling compartment. Rab11-positive vesicles are recruited to the invasion site before vacuolar rupture, and Rab11 knockdown dramatically decreases vacuolar membrane rupture. Additionally, Rab11 recruitment is absent and vacuolar rupture is delayed in the ipgD mutant that does not dephosphorylate PI(4,5)P₂ into PI(5)P. Ultrastructural analyses of Rab11-positive vesicles further reveal that ipgD mutant-containing vacuoles become confined in actin structures that likely contribute to delayed vacular rupture. These findings provide insight into the underlying molecular mechanism of vacuole progression and rupture during Shigella invasion.
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http://dx.doi.org/10.1016/j.chom.2014.09.005DOI Listing
October 2014

A step beyond BRET: Fluorescence by Unbound Excitation from Luminescence (FUEL).

J Vis Exp 2014 May 23(87). Epub 2014 May 23.

Plate-Forme d'Imagerie Dynamique, Imagopole, Institut Pasteur;

Fluorescence by Unbound Excitation from Luminescence (FUEL) is a radiative excitation-emission process that produces increased signal and contrast enhancement in vitro and in vivo. FUEL shares many of the same underlying principles as Bioluminescence Resonance Energy Transfer (BRET), yet greatly differs in the acceptable working distances between the luminescent source and the fluorescent entity. While BRET is effectively limited to a maximum of 2 times the Förster radius, commonly less than 14 nm, FUEL can occur at distances up to µm or even cm in the absence of an optical absorber. Here we expand upon the foundation and applicability of FUEL by reviewing the relevant principles behind the phenomenon and demonstrate its compatibility with a wide variety of fluorophores and fluorescent nanoparticles. Further, the utility of antibody-targeted FUEL is explored. The examples shown here provide evidence that FUEL can be utilized for applications where BRET is not possible, filling the spatial void that exists between BRET and traditional whole animal imaging.
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http://dx.doi.org/10.3791/51549DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4207116PMC
May 2014

Silencing the radicals improves Click Chemistry.

Biotechnol J 2014 May 16;9(5):595-6. Epub 2014 Apr 16.

Institut Pasteur, Imagopole, Plateforme d'imagerie dynamique, Paris, France; INSERM U786, Paris, France.

Modern fluorescence imaging microscopy in living and fixed material makes use of fluorescent probes to label targeted entities. Common labelling approaches include classical immunocytochemistry, expression of chimerically tagged fluorescent protein domains, and chemical affinity-binding or covalent labelling. Of these methods, the so-called "Click Chemistry", is emerging as one of the most influential labelling chemistries introduced in recent times, offering enormous utility for bio-orthoganol attachment of fluorescent probes to biological target entities. In this issue of Biotechnology Journal, Löschberger, Niehörster and Sauer report "ClickOx", a Click Chemistry protocol that uses an enzymatic oxygen scavenger system to reduce concurrent ROS-associated damage during Click labeling.
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http://dx.doi.org/10.1002/biot.201400103DOI Listing
May 2014

Real-time label-free detection of dividing cells by means of lensfree video-microscopy.

J Biomed Opt 2014 Mar;19(3):36004

Commissariat à l'énergie atomique et aux énergies alternatives (CEA), LETI, MINATEC, 17 rue des martyrs, Grenoble cedex 9, 38054 France.

Quantification of cell proliferation and monitoring its kinetics are essential in fields of research such as developmental biology, oncology, etc. Although several proliferation assays exist, monitoring cell proliferation kinetics remains challenging. We present a novel cell proliferation assay based on real-time monitoring of cell culture inside a standard incubator using a lensfree video-microscope, combined with automated detection of single cell divisions over a population of several thousand cells. Since the method is based on direct visualization of dividing cells, it is label-free, continuous, and not sample destructive. Kinetics of cell proliferation can be monitored from a few hours to several days. We compare our method to a standard assay, the EdU proliferation assay, and as proof of principle, we demonstrate concentration-dependent and time-dependent effect of actinomycin D-a cell proliferation inhibitor.
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http://dx.doi.org/10.1117/1.JBO.19.3.036004DOI Listing
March 2014

Objective comparison of particle tracking methods.

Nat Methods 2014 Mar 19;11(3):281-9. Epub 2014 Jan 19.

Molecular Biotechnology Group, Institute of Biology, Leiden University, Leiden, The Netherlands.

Particle tracking is of key importance for quantitative analysis of intracellular dynamic processes from time-lapse microscopy image data. Because manually detecting and following large numbers of individual particles is not feasible, automated computational methods have been developed for these tasks by many groups. Aiming to perform an objective comparison of methods, we gathered the community and organized an open competition in which participating teams applied their own methods independently to a commonly defined data set including diverse scenarios. Performance was assessed using commonly defined measures. Although no single method performed best across all scenarios, the results revealed clear differences between the various approaches, leading to notable practical conclusions for users and developers.
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http://dx.doi.org/10.1038/nmeth.2808DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4131736PMC
March 2014

In vitro and in vivo demonstrations of Fluorescence by Unbound Excitation from Luminescence (FUEL).

Methods Mol Biol 2014 ;1098:259-70

Plate-Forme d'Imagerie Dynamique, Imagopole, Institut Pasteur, Paris, France.

Bioluminescence imaging is a powerful technique that allows for deep-tissue analysis in living, intact organisms. However, in vivo optical imaging is compounded by difficulties due to light scattering and absorption. While light scattering is relatively difficult to overcome and compensate, light absorption by biological tissue is strongly dependent upon wavelength. For example, light absorption by mammalian tissue is highest in the blue-yellow part of the visible energy spectrum. Many natural bioluminescent molecules emit photonic energy in this range, thus in vivo optical detection of these molecules is primarily limited by absorption. This has driven efforts for probe development aimed to enhance photonic emission of red light that is absorbed much less by mammalian tissue using either direct genetic manipulation, and/or resonance energy transfer methods. Here we describe a recently identified alternative approach termed Fluorescence by Unbound Excitation from Luminescence (FUEL), where bioluminescent molecules are able to induce a fluorescent response from fluorescent nanoparticles through an epifluorescence mechanism, thereby significantly increasing both the total number of detectable photons as well as the number of red photons produced.
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http://dx.doi.org/10.1007/978-1-62703-718-1_20DOI Listing
May 2014

In vitro characterization of Fluorescence by Unbound Excitation from Luminescence: broadening the scope of energy transfer.

Methods 2014 Mar 14;66(2):353-61. Epub 2013 Sep 14.

Plate-Forme d'Imagerie Dynamique, Imagopole, Institut Pasteur, 25-28 Rue du Dr. Roux, 75724 Paris cedex 15, France. Electronic address:

Energy transfer mechanisms represent the basis for an array of valuable tools to infer interactions in vitro and in vivo, enhance detection or resolve interspecies distances such as with resonance. Based upon our own previously published studies and new results shown here we present a novel framework describing for the first time a model giving a view of the biophysical relationship between Fluorescence by Unbound Excitation from Luminescence (FUEL), a conventional radiative excitation-emission process, and bioluminescence resonance energy transfer. We show here that in homogeneous solutions and in fluorophore-targeted bacteria, FUEL is the dominant mechanism responsible for the production of red-shifted photons. The minor resonance contribution was ascertained by comparing the intensity of the experimental signal to its theoretical resonance counterpart. Distinctive features of the in vitro FUEL signal include a macroscopic depth dependency, a lack of enhancement upon targeting at a constant fluorophore concentration cf and a non-square dependency on cf. Significantly, FUEL is an important, so far overlooked, component of all resonance phenomena which should guide the design of appropriate controls when elucidating interactions. Last, our results highlight the potential for FUEL as a means to enhance in vivo and in vitro detection through complex media while alleviating the need for targeting.
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http://dx.doi.org/10.1016/j.ymeth.2013.09.005DOI Listing
March 2014

The giant spectrin βV couples the molecular motors to phototransduction and Usher syndrome type I proteins along their trafficking route.

Hum Mol Genet 2013 Sep 23;22(18):3773-88. Epub 2013 May 23.

Institut Pasteur, Unité de génétique et physiologie de l'audition, Paris, France.

Mutations in the myosin VIIa gene cause Usher syndrome type IB (USH1B), characterized by deaf-blindness. A delay of opsin trafficking has been observed in the retinal photoreceptor cells of myosin VIIa-deficient mice. We identified spectrin βV, the mammalian β-heavy spectrin, as a myosin VIIa- and rhodopsin-interacting partner in photoreceptor cells. Spectrin βV displays a polarized distribution from the Golgi apparatus to the base of the outer segment, which, unlike that of other β spectrins, matches the trafficking route of opsin and other phototransduction proteins. Formation of spectrin βV-rhodopsin complex could be detected in the differentiating photoreceptors as soon as their outer segment emerges. A failure of the spectrin βV-mediated coupling between myosin VIIa and opsin molecules thus probably accounts for the opsin transport delay in myosin VIIa-deficient mice. We showed that spectrin βV also associates with two USH1 proteins, sans (USH1G) and harmonin (USH1C). Spectrins are supposed to function as heteromers of α and β subunits, but fluorescence resonance energy transfer and in vitro binding experiments indicated that spectrin βV can also form homodimers, which likely supports its αII-independent βV functions. Finally, consistent with its distribution along the connecting cilia axonemes, spectrin βV binds to several subunits of the microtubule-based motor proteins, kinesin II and the dynein complex. We therefore suggest that spectrin βV homomers couple some USH1 proteins, opsin and other phototransduction proteins to both actin- and microtubule-based motors, thereby contributing to their transport towards the photoreceptor outer disks.
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http://dx.doi.org/10.1093/hmg/ddt228DOI Listing
September 2013

High content analysis of primary macrophages hosting proliferating Leishmania amastigotes: application to anti-leishmanial drug discovery.

PLoS Negl Trop Dis 2013 4;7(4):e2154. Epub 2013 Apr 4.

Institut Pasteur, Imagopole, Paris, France.

Background/objectives: Human leishmaniases are parasitic diseases causing severe morbidity and mortality. No vaccine is available and numerous factors limit the use of current therapies. There is thus an urgent need for innovative initiatives to identify new chemotypes displaying selective activity against intracellular Leishmania amastigotes that develop and proliferate inside macrophages, thereby causing the pathology of leishmaniasis.

Methodology/principal Findings: We have developed a biologically sound High Content Analysis assay, based on the use of homogeneous populations of primary mouse macrophages hosting Leishmania amazonensis amastigotes. In contrast to classical promastigote-based screens, our assay more closely mimics the environment where intracellular amastigotes are growing within acidic parasitophorous vacuoles of their host cells. This multi-parametric assay provides quantitative data that accurately monitors the parasitic load of amastigotes-hosting macrophage cultures for the discovery of leishmanicidal compounds, but also their potential toxic effect on host macrophages. We validated our approach by using a small set of compounds of leishmanicidal drugs and recently published chemical entities. Based on their intramacrophagic leishmanicidal activity and their toxicity against host cells, compounds were classified as irrelevant or relevant for entering the next step in the drug discovery pipeline.

Conclusions/significance: Our assay represents a new screening platform that overcomes several limitations in anti-leishmanial drug discovery. First, the ability to detect toxicity on primary macrophages allows for discovery of compounds able to cross the membranes of macrophage, vacuole and amastigote, thereby accelerating the hit to lead development process for compounds selectively targeting intracellular parasites. Second, our assay allows discovery of anti-leishmanials that interfere with biological functions of the macrophage required for parasite development and growth, such as organelle trafficking/acidification or production of microbicidal effectors. These data thus validate a novel phenotypic screening assay using virulent Leishmania amastigotes growing inside primary macrophage to identify new chemical entities with bona fide drug potential.
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http://dx.doi.org/10.1371/journal.pntd.0002154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3617141PMC
November 2013

Human nucleoporins promote HIV-1 docking at the nuclear pore, nuclear import and integration.

PLoS One 2012 25;7(9):e46037. Epub 2012 Sep 25.

Molecular Virology and Vaccinology Unit, CNRS URA 3015, Department of Virology, Institut Pasteur, Paris, France.

The nuclear pore complex (NPC) mediates nucleo-cytoplasmic transport of macromolecules and is an obligatory point of passage and functional bottleneck in the replication of some viruses. The Human Immunodeficiency Virus (HIV) has evolved the required mechanisms for active nuclear import of its genome through the NPC. However the mechanisms by which the NPC allows or even assists HIV translocation are still unknown. We investigated the involvement of four key nucleoporins in HIV-1 docking, translocation, and integration: Nup358/RanBP2, Nup214/CAN, Nup98 and Nup153. Although all induce defects in infectivity when depleted, only Nup153 actually showed any evidence of participating in HIV-1 translocation through the nuclear pore. We show that Nup358/RanBP2 mediates docking of HIV-1 cores on NPC cytoplasmic filaments by interacting with the cores and that the C-terminus of Nup358/RanBP2 comprising a cyclophilin-homology domain contributes to binding. We also show that Nup214/CAN and Nup98 play no role in HIV-1 nuclear import per se: Nup214/CAN plays an indirect role in infectivity read-outs through its effect on mRNA export, while the reduction of expression of Nup98 shows a slight reduction in proviral integration. Our work shows the involvement of nucleoporins in diverse and functionally separable steps of HIV infection and nuclear import.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0046037PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3457934PMC
April 2013

In vivo excitation of nanoparticles using luminescent bacteria.

Proc Natl Acad Sci U S A 2012 Jun 21;109(23):8890-5. Epub 2012 May 21.

Plate-Forme d'Imagerie Dynamique, Imagopole, Institut Pasteur, 25-28 Rue du Docteur Roux, 75724 Paris cedex 15, France.

The lux operon derived from Photorhabdus luminescens incorporated into bacterial genomes, elicits the production of biological chemiluminescence typically centered on 490 nm. The light-producing bacteria are widely used for in vivo bioluminescence imaging. However, in living samples, a common difficulty is the presence of blue-green absorbers such as hemoglobin. Here we report a characterization of fluorescence by unbound excitation from luminescence, a phenomenon that exploits radiating luminescence to excite nearby fluorophores by epifluorescence. We show that photons from bioluminescent bacteria radiate over mesoscopic distances and induce a red-shifted fluorescent emission from appropriate fluorophores in a manner distinct from bioluminescence resonance energy transfer. Our results characterizing fluorescence by unbound excitation from luminescence, both in vitro and in vivo, demonstrate how the resulting blue-to-red wavelength shift is both necessary and sufficient to yield contrast enhancement revealing mesoscopic proximity of luminescent and fluorescent probes in the context of living biological tissues.
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http://dx.doi.org/10.1073/pnas.1204516109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3384150PMC
June 2012
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