Publications by authors named "Jean-Yves Tinevez"

35 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

Highlights from the 2016-2020 NEUBIAS training schools for Bioimage Analysts: a success story and key asset for analysts and life scientists.

F1000Res 2021 30;10:334. Epub 2021 Apr 30.

Nikon Imaging Center, University of Heidelberg, Heidelberg, Germany.

NEUBIAS, the European Network of Bioimage Analysts, was created in 2016 with the goal of improving the communication and the knowledge transfer among the various stakeholders involved in the acquisition, processing and analysis of biological image data, and to promote the establishment and recognition of the profession of Bioimage Analyst. One of the most successful initiatives of the NEUBIAS programme was its series of 15 training schools, which trained over 400 new Bioimage Analysts, coming from over 40 countries. Here we outline the rationale behind the innovative three-level program of the schools, the curriculum, the trainer recruitment and turnover strategy, the outcomes for the community and the career path of analysts, including some success stories. We discuss the future of the materials created during this programme and some of the new initiatives emanating from the community of NEUBIAS-trained analysts, such as the NEUBIAS Academy. Overall, we elaborate on how this training programme played a key role in collectively leveraging Bioimaging and Life Science research by bringing the latest innovations into structured, frequent and intensive training activities, and on why we believe this should become a model to further develop in Life Sciences.
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http://dx.doi.org/10.12688/f1000research.25485.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8215561PMC
April 2021

Dynamic spatiotemporal coordination of neural stem cell fate decisions occurs through local feedback in the adult vertebrate brain.

Cell Stem Cell 2021 Apr 2. Epub 2021 Apr 2.

Zebrafish Neurogenetics Unit, Institut Pasteur, UMR3738, CNRS, team supported by La Ligue Nationale Contre le Cancer, 75015 Paris, France. Electronic address:

Neural stem cell (NSC) populations persist in the adult vertebrate brain over a lifetime, and their homeostasis is controlled at the population level through unknown mechanisms. Here, we combine dynamic imaging of entire NSC populations in their in vivo niche over several weeks with pharmacological manipulations, mathematical modeling, and spatial statistics and demonstrate that NSCs use spatiotemporally resolved local feedback signals to coordinate their decision to divide in adult zebrafish brains. These involve Notch-mediated short-range inhibition from transient neural progenitors and a dispersion effect from the dividing NSCs themselves exerted with a delay of 9-12 days. Simulations from a stochastic NSC lattice model capturing these interactions demonstrate that these signals are linked by lineage progression and control the spatiotemporal distribution of output neurons. These results highlight how local and temporally delayed interactions occurring between brain germinal cells generate self-propagating dynamics that maintain NSC population homeostasis and coordinate specific spatiotemporal correlations.
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http://dx.doi.org/10.1016/j.stem.2021.03.014DOI Listing
April 2021

Automated cell tracking using StarDist and TrackMate.

F1000Res 2020 28;9:1279. Epub 2020 Oct 28.

Turku Bioscience Centre, University of Turku and Åbo Akademi University, Turku, Finland.

The ability of cells to migrate is a fundamental physiological process involved in embryonic development, tissue homeostasis, immune surveillance, and wound healing. Therefore, the mechanisms governing cellular locomotion have been under intense scrutiny over the last 50 years. One of the main tools of this scrutiny is live-cell quantitative imaging, where researchers image cells over time to study their migration and quantitatively analyze their dynamics by tracking them using the recorded images. Despite the availability of computational tools, manual tracking remains widely used among researchers due to the difficulty setting up robust automated cell tracking and large-scale analysis. Here we provide a detailed analysis pipeline illustrating how the deep learning network StarDist can be combined with the popular tracking software TrackMate to perform 2D automated cell tracking and provide fully quantitative readouts. Our proposed protocol is compatible with both fluorescent and widefield images. It only requires freely available and open-source software (ZeroCostDL4Mic and Fiji), and does not require any coding knowledge from the users, making it a versatile and powerful tool for the field. We demonstrate this pipeline's usability by automatically tracking cancer cells and T cells using fluorescent and brightfield images. Importantly, we provide, as supplementary information, a detailed step-by-step protocol to allow researchers to implement it with their images.
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http://dx.doi.org/10.12688/f1000research.27019.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7670479PMC
April 2021

Shigella impairs human T lymphocyte responsiveness by hijacking actin cytoskeleton dynamics and T cell receptor vesicular trafficking.

Cell Microbiol 2020 05 1;22(5):e13166. Epub 2020 Mar 1.

Molecular Microbial Pathogenesis Unit, Institut Pasteur, INSERM U1202, Paris, France.

Strategies employed by pathogenic enteric bacteria, such as Shigella, to subvert the host adaptive immunity are not well defined. Impairment of T lymphocyte chemotaxis by blockage of polarised edge formation has been reported upon Shigella infection. However, the functional impact of Shigella on T lymphocytes remains to be determined. Here, we show that Shigella modulates CD4+ T cell F-actin dynamics and increases cell cortical stiffness. The scanning ability of T lymphocytes when encountering antigen-presenting cells (APC) is subsequently impaired resulting in decreased cell-cell contacts (or conjugates) between the two cell types, as compared with non-infected T cells. In addition, the few conjugates established between the invaded T cells and APCs display no polarised delivery and accumulation of the T cell receptor to the contact zone characterising canonical immunological synapses. This is most likely due to the targeting of intracellular vesicular trafficking by the bacterial type III secretion system (T3SS) effectors IpaJ and VirA. The collective impact of these cellular reshapings by Shigella eventually results in T cell activation dampening. Altogether, these results highlight the combined action of T3SS effectors leading to T cell defects upon Shigella infection.
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http://dx.doi.org/10.1111/cmi.13166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7187243PMC
May 2020

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

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

Altered spinogenesis in iPSC-derived cortical neurons from patients with autism carrying de novo SHANK3 mutations.

Sci Rep 2019 01 14;9(1):94. Epub 2019 Jan 14.

Human Genetics and Cognitive Functions, Institut Pasteur, Paris, France.

The synaptic protein SHANK3 encodes a multidomain scaffold protein expressed at the postsynaptic density of neuronal excitatory synapses. We previously identified de novo SHANK3 mutations in patients with autism spectrum disorders (ASD) and showed that SHANK3 represents one of the major genes for ASD. Here, we analyzed the pyramidal cortical neurons derived from induced pluripotent stem cells from four patients with ASD carrying SHANK3 de novo truncating mutations. At 40-45 days after the differentiation of neural stem cells, dendritic spines from pyramidal neurons presented variable morphologies: filopodia, thin, stubby and muschroom, as measured in 3D using GFP labeling and immunofluorescence. As compared to three controls, we observed a significant decrease in SHANK3 mRNA levels (less than 50% of controls) in correlation with a significant reduction in dendritic spine densities and whole spine and spine head volumes. These results, obtained through the analysis of de novo SHANK3 mutations in the patients' genomic background, provide further support for the presence of synaptic abnormalities in a subset of patients with ASD.
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http://dx.doi.org/10.1038/s41598-018-36993-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6331634PMC
January 2019

Bidirectional intraflagellar transport is restricted to two sets of microtubule doublets in the trypanosome flagellum.

J Cell Biol 2018 12 1;217(12):4284-4297. Epub 2018 Oct 1.

Trypanosome Cell Biology Unit, INSERM U1201, Institut Pasteur, Paris, France

Intraflagellar transport (IFT) is the rapid bidirectional movement of large protein complexes driven by kinesin and dynein motors along microtubule doublets of cilia and flagella. In this study, we used a combination of high-resolution electron and light microscopy to investigate how and where these IFT trains move within the flagellum of the protist Focused ion beam scanning electron microscopy (FIB-SEM) analysis of trypanosomes showed that trains are found almost exclusively along two sets of doublets (3-4 and 7-8) and distribute in two categories according to their length. High-resolution live imaging of cells expressing mNeonGreen::IFT81 or GFP::IFT52 revealed for the first time IFT trafficking on two parallel lines within the flagellum. Anterograde and retrograde IFT occurs on each of these lines. At the distal end, a large individual anterograde IFT train is converted in several smaller retrograde trains in the space of 3-4 s while remaining on the same side of the axoneme.
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http://dx.doi.org/10.1083/jcb.201805030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6279389PMC
December 2018

Anisotropic organization of circumferential actomyosin characterizes hematopoietic stem cells emergence in the zebrafish.

Elife 2018 08 22;7. Epub 2018 Aug 22.

Department of Developmental and Stem Cell Biology, Institut Pasteur, Paris, France.

Hematopoiesis leads to the formation of blood and immune cells. Hematopoietic stem cells emerge during development, from vascular components, via a process called the endothelial-to-hematopoietic transition (EHT). Here, we reveal essential biomechanical features of the EHT, using the zebrafish embryo imaged at unprecedented spatio-temporal resolution and an algorithm to unwrap the aorta into 2D-cartography. We show that the transition involves anisotropic contraction along the antero-posterior axis, with heterogenous organization of contractile circumferential actomyosin. The biomechanics of the contraction is oscillatory, with unusually long periods in comparison to other apical constriction mechanisms described so far in morphogenesis, and is supported by the anisotropic reinforcement of junctional contacts. Finally, we show that abrogation of blood flow impairs the actin cytoskeleton, the morphodynamics of EHT cells, and the orientation of the emergence. Overall, our results underline the peculiarities of the EHT biomechanics and the influence of the mechanical forces exerted by blood flow.
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http://dx.doi.org/10.7554/eLife.37355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6105311PMC
August 2018

TIM-1 Ubiquitination Mediates Dengue Virus Entry.

Cell Rep 2018 05;23(6):1779-1793

INSERM U944-CNRS 7212, Laboratoire de Pathologie et Virologie Moléculaire, Institut Universitaire d'Hématologie, Université Paris Diderot Sorbonne Paris Cité, Hôpital St. Louis, 75475 Paris Cedex 10, France. Electronic address:

Dengue virus (DENV) is a major human pathogen causing millions of infections yearly. Despite intensive investigations, a DENV receptor that directly participates in virus internalization has not yet been characterized. Here, we report that the phosphatidylserine receptor TIM-1 is an authentic DENV entry receptor that plays an active role in virus endocytosis. Genetic ablation of TIM-1 strongly impaired DENV infection. Total internal reflection fluorescence microscopy analyses of live infected cells show that TIM-1 is mostly confined in clathrin-coated pits and is co-internalized with DENV during viral entry. TIM-1 is ubiquitinated at two lysine residues of its cytoplasmic domain, and this modification is required for DENV endocytosis. Furthermore, STAM-1, a component of the ESCRT-0 complex involved in intracellular trafficking of ubiquitinated cargos, interacts with TIM-1 and is required for DENV infection. Overall, our results show that TIM-1 is the first bona fide receptor identified for DENV.
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http://dx.doi.org/10.1016/j.celrep.2018.04.013DOI Listing
May 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

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

MUB Binds to Lactoferrin and Stands as a Specific Neutrophil Marker.

Cell Chem Biol 2018 04 22;25(4):483-493.e9. Epub 2018 Feb 22.

Institut Pasteur, Unité de Pathogénie Microbienne Moléculaire, 28 rue du Dr Roux, 75724 Paris Cedex 15, France; INSERM Unité 1202, 28 rue du Dr Roux, 75724 Paris Cedex 15, France; Institut Gustave Roussy, Laboratoire de Thérapie Cellulaire, 114 rue Edouard Vaillant, 94800 Villejuif, France. Electronic address:

Neutrophils represent the most abundant immune cells recruited to inflamed tissues. A lack of dedicated tools has hampered their detection and study. We show that a synthesized peptide, MUB, binds to lactoferrin, the most abundant protein stored in neutrophil-specific and tertiary granules. Lactoferrin is specifically produced by neutrophils among other leukocytes, making MUB a specific neutrophil marker. Naive mammalian neutrophils (human, guinea pig, mouse, rabbit) were labeled by fluorescent MUB conjugates (-Cy5, Dylight405). A peptidase-resistant retro-inverso MUB (RI-MUB) was synthesized and its lactoferrin-binding property validated. Neutrophil lactoferrin secretion during in vitro Shigella infection was assessed with RI-MUB-Cy5 using live cell microscopy. Systemically administered RI-MUB-Cy5 accumulated at sites of inflammation in a mouse arthritis inflammation model in vivo and showed usefulness as a potential tool for inflammation detection using non-invasive imaging. Improving neutrophil detection with the universal and specific MUB marker will aid the study of broad ranges of inflammatory diseases.
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http://dx.doi.org/10.1016/j.chembiol.2018.01.014DOI Listing
April 2018

The infectious hypoxia: occurrence and causes during Shigella infection.

Microbes Infect 2017 Mar 21;19(3):157-165. Epub 2016 Nov 21.

Institut Pasteur, Unité de Pathogénie Microbienne Moléculaire, 28 rue du Dr Roux, 75724 Paris Cedex 15, France; INSERM Unité 1202, 28 rue du Dr Roux, 75724 Paris Cedex 15, France; Gustave Roussy Cancer Campus, Laboratoire de Thérapie Cellulaire, 114 Rue Edouard Vaillant, 94800 Villejuif, France. Electronic address:

Hypoxia is defined as a tissue oxygenation status below physiological needs. During Shigella infection, an infectious hypoxia is induced within foci of infection. In this review, we discuss how Shigella physiology and virulence are modulated and how the main recruited immune cells, the neutrophils, adapt to this environment.
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http://dx.doi.org/10.1016/j.micinf.2016.10.011DOI Listing
March 2017

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

Object Segmentation and Ground Truth in 3D Embryonic Imaging.

PLoS One 2016 22;11(6):e0150853. Epub 2016 Jun 22.

Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.

Many questions in developmental biology depend on measuring the position and movement of individual cells within developing embryos. Yet, tools that provide this data are often challenged by high cell density and their accuracy is difficult to measure. Here, we present a three-step procedure to address this problem. Step one is a novel segmentation algorithm based on image derivatives that, in combination with selective post-processing, reliably and automatically segments cell nuclei from images of densely packed tissue. Step two is a quantitative validation using synthetic images to ascertain the efficiency of the algorithm with respect to signal-to-noise ratio and object density. Finally, we propose an original method to generate reliable and experimentally faithful ground truth datasets: Sparse-dense dual-labeled embryo chimeras are used to unambiguously measure segmentation errors within experimental data. Together, the three steps outlined here establish a robust, iterative procedure to fine-tune image analysis algorithms and microscopy settings associated with embryonic 3D image data sets.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0150853PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4917178PMC
July 2017

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells.

J Vis Exp 2015 Oct 10(104). Epub 2015 Oct 10.

Human Genetics and Cognitive Functions, Institut Pasteur; CNRS URA 2182 'Genes, synapses and cognition', Institut Pasteur; Human Genetics and Cognitive Functions, Université Paris Diderot, Sorbonne Paris Cité;

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis (1). The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.
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http://dx.doi.org/10.3791/53197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4692647PMC
October 2015

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

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

Msx1 and Msx2 act as essential activators of Atoh1 expression in the murine spinal cord.

Development 2014 Apr;141(8):1726-36

Institut Pasteur, Morphogenesis Molecular Genetics, CNRS URA 2578, 75015 Paris, France.

Dorsal spinal neurogenesis is orchestrated by the combined action of signals secreted from the roof plate organizer and a downstream transcriptional cascade. Within this cascade, Msx1 and Msx2, two homeodomain transcription factors (TFs), are induced earlier than bHLH neuralizing TFs. Whereas bHLH TFs have been shown to specify neuronal cell fate, the function of Msx genes remains poorly defined. We describe dramatic alterations of neuronal patterning in Msx1/Msx2 double-mutant mouse embryos. The most dorsal spinal progenitor pool fails to express the bHLH neuralizing TF Atoh1, which results in a lack of Lhx2-positive and Barhl2-positive dI1 interneurons. Neurog1 and Ascl1 expression territories are dorsalized, leading to ectopic dorsal differentiation of dI2 and dI3 interneurons. In proportion, the amount of Neurog1-expressing progenitors appears unaffected, whereas the number of Ascl1-positive cells is increased. These defects occur while BMP signaling is still active in the Msx1/Msx2 mutant embryos. Cell lineage analysis and co-immunolabeling demonstrate that Atoh1-positive cells derive from progenitors expressing both Msx1 and Msx2. In vitro, Msx1 and Msx2 proteins activate Atoh1 transcription by specifically interacting with several homeodomain binding sites in the Atoh1 3' enhancer. In vivo, Msx1 and Msx2 are required for Atoh1 3' enhancer activity and ChIP experiments confirm Msx1 binding to this regulatory sequence. These data support a novel function of Msx1 and Msx2 as transcriptional activators. Our study provides new insights into the transcriptional control of spinal cord patterning by BMP signaling, with Msx1 and Msx2 acting upstream of Atoh1.
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http://dx.doi.org/10.1242/dev.099002DOI Listing
April 2014

TNF and IL-1 exhibit distinct ubiquitin requirements for inducing NEMO-IKK supramolecular structures.

J Cell Biol 2014 Jan;204(2):231-45

Unité de Signalisation Moléculaire et Activation Cellulaire and 2 Laboratoire Trafic Membranaire et Division Cellulaire, Institut Pasteur, Centre National de la Recherche Scientifique URA 2582, Paris 75015, France.

Nuclear factor κB (NF-κB) essential modulator (NEMO), a regulatory component of the IκB kinase (IKK) complex, controls NF-κB activation through its interaction with ubiquitin chains. We show here that stimulation with interleukin-1 (IL-1) and TNF induces a rapid and transient recruitment of NEMO into punctate structures that are anchored at the cell periphery. These structures are enriched in activated IKK kinases and ubiquitinated NEMO molecules, which suggests that they serve as organizing centers for the activation of NF-κB. These NEMO-containing structures colocalize with activated TNF receptors but not with activated IL-1 receptors. We investigated the involvement of nondegradative ubiquitination in the formation of these structures, using cells deficient in K63 ubiquitin chains or linear ubiquitin chain assembly complex (LUBAC)-mediated linear ubiquitination. Our results indicate that, unlike TNF, IL-1 requires K63-linked and linear ubiquitin chains to recruit NEMO into higher-order complexes. Thus, different mechanisms are involved in the recruitment of NEMO into supramolecular complexes, which appear to be essential for NF-κB activation.
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http://dx.doi.org/10.1083/jcb.201307172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3897181PMC
January 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 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

Crumbs affects protein dynamics in anterior regions of the developing Drosophila embryo.

PLoS One 2013 21;8(3):e58839. Epub 2013 Mar 21.

Max-Planck-Institute of Molecular Cell Biology and Genetics, Dresden, Germany.

Maintenance of apico-basal polarity is essential for epithelial integrity and requires particular reinforcement during tissue morphogenesis, when cells are reorganised, undergo shape changes and remodel their junctions. It is well established that epithelial integrity during morphogenetic processes depends on the dynamic exchange of adherens junction components, but our knowledge on the dynamics of other proteins and their dynamics during these processes is still limited. The early Drosophila embryo is an ideal system to study membrane dynamics during morphogenesis. Here, morphogenetic activities differ along the anterior-posterior axis, with the extending germband showing a high degree of epithelial remodelling. We developed a Fluorescence Recovery After Photobleaching (FRAP) assay with a higher temporal resolution, which allowed the distinction between a fast and a slow component of recovery of membrane proteins during the germband extension stage. We show for the first time that the recovery kinetics of a general membrane marker, SpiderGFP, differs in the anterior and posterior parts of the embryo, which correlates well with the different morphogenetic activities of the respective embryonic regions. Interestingly, absence of crumbs, a polarity regulator essential for epithelial integrity in the Drosophila embryo, decreases the fast component of SpiderGFP and of the apical marker Stranded at Second-Venus specifically in the anterior region. We suggest that the defects in kinetics observed in crumbs mutant embryos are the first signs of tissue instability in this region, explaining the earlier breakdown of the head epidermis in comparison to that of the trunk, and that diffusion in the plasma membrane is affected by the absence of Crumbs.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0058839PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3605435PMC
October 2013

A simple model for the fate of the cytokinesis midbody remnant: implications for remnant degradation by autophagy.

Bioessays 2013 May 1;35(5):472-81. Epub 2013 Mar 1.

Institut Pasteur, Membrane Traffic and Cell Division Lab, Paris, France.

When a cell divides, it produces two daughter cells initially connected by a cytokinesis bridge, which is eventually cut through abscission. One of the two daughter cells inherits a bridge "remnant", which has been proposed to be degraded by autophagy. The fate and function of remnants is attracting increasing attention, as their accumulation appears to influence proliferation versus differentiation of the daughter cells. Here, we present a simple model for bridge and remnant turnover in a dynamic cell population. We demonstrate that remnant proportions depend on the ratio of remnant and bridge lifetimes to the cell population doubling time. Our results yield new alternative interpretations for published experimental data, leading us to believe that autophagy-independent pathways for remnant degradation may exist. In addition, using the model, we determined experimentally inaccessible parameters such as remnant lifetime. Our model proves to be a useful tool for studying bridge and remnant populations.
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http://dx.doi.org/10.1002/bies.201200132DOI Listing
May 2013

Fiji: an open-source platform for biological-image analysis.

Nat Methods 2012 Jun 28;9(7):676-82. Epub 2012 Jun 28.

Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.

Fiji is a distribution of the popular open-source software ImageJ focused on biological-image analysis. Fiji uses modern software engineering practices to combine powerful software libraries with a broad range of scripting languages to enable rapid prototyping of image-processing algorithms. Fiji facilitates the transformation of new algorithms into ImageJ plugins that can be shared with end users through an integrated update system. We propose Fiji as a platform for productive collaboration between computer science and biology research communities.
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http://dx.doi.org/10.1038/nmeth.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3855844PMC
June 2012

A quantitative method for measuring phototoxicity of a live cell imaging microscope.

Methods Enzymol 2012 ;506:291-309

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

Fluorescence-based imaging regimes require exposure of living samples under study to high intensities of focused incident illumination. An often underestimated, overlooked, or simply ignored fact in the design of any experimental imaging protocol is that exposure of the specimen to these excitation light sources must itself always be considered a potential source of phototoxicity. This can be problematic, not just in terms of cell viability, but much more worrisome in its more subtle manifestation where phototoxicity causes anomalous behaviors that risk to be interpreted as significant, whereas they are mere artifacts. This is especially true in the case of microbial pathogenesis, where host-pathogen interactions can prove especially fragile to light exposure in a manner that can obscure the very processes we are trying to observe. For these reasons, it is important to be able to bring the parameter of phototoxicity into the equation that brings us to choose one fluorescent imaging modality, or setup, over another. Further, we need to be able to assess the risk that phototoxicity may occur during any specific imaging experiment. To achieve this, we describe here a methodological approach that allows meaningful measurement, and therefore relative comparison of phototoxicity, in most any variety of different imaging microscopes. In short, we propose a quantitative approach that uses microorganisms themselves to reveal the range over which any given fluorescent imaging microscope will yield valid results, providing a metrology of phototoxic damage, distinct from photobleaching, where a clear threshold for phototoxicity is identified. Our method is widely applicable and we show that it can be adapted to other paradigms, including mammalian cell models.
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http://dx.doi.org/10.1016/B978-0-12-391856-7.00039-1DOI Listing
August 2012
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