ABSTRACT
Single molecule localization microscopy (SMLM) with a dichroic image splitter can provide invaluable multi-color information regarding colocalization of individual molecules, but it often suffers from technical limitations. Classical demixing algorithms tend to give suboptimal results in terms of localization precision and correction of chromatic errors. Here we present an image splitter based multi-color SMLM method (splitSMLM) that offers much improved localization precision and drift correction, compensation of chromatic distortions, and optimized performance of fluorophores in a specific buffer to equalize their reactivation rates for simultaneous imaging. A novel spectral demixing algorithm, SplitViSu, fully preserves localization precision with essentially no data loss and corrects chromatic errors at the nanometer scale. Multi-color performance is further improved by using optimized fluorophore and filter combinations. Applied to three-color imaging of the nuclear pore complex (NPC), this method provides a refined positioning of the individual NPC proteins and reveals that Pom121 clusters act as NPC deposition loci, hence illustrating strength and general applicability of the method.
Subject(s)
Microscopy , Nuclear Pore , Algorithms , Fluorescent Dyes/metabolism , Microscopy/methods , Nuclear Pore/metabolism , Single Molecule Imaging/methodsABSTRACT
Automating the extraction of meaningful temporal information from sequences of microscopy images represents a major challenge to characterize dynamical biological processes. So far, strong limitations in the ability to quantitatively analyze single-cell trajectories have prevented large-scale investigations to assess the dynamics of entry into replicative senescence in yeast. Here, we have developed DetecDiv, a microfluidic-based image acquisition platform combined with deep learning-based software for high-throughput single-cell division tracking. We show that DetecDiv can automatically reconstruct cellular replicative lifespans with high accuracy and performs similarly with various imaging platforms and geometries of microfluidic traps. In addition, this methodology provides comprehensive temporal cellular metrics using time-series classification and image semantic segmentation. Last, we show that this method can be further applied to automatically quantify the dynamics of cellular adaptation and real-time cell survival upon exposure to environmental stress. Hence, this methodology provides an all-in-one toolbox for high-throughput phenotyping for cell cycle, stress response, and replicative lifespan assays.
Subject(s)
Deep Learning , Cell Division , Cell Tracking , Image Processing, Computer-Assisted/methods , Saccharomyces cerevisiae , Software , Survival AnalysisABSTRACT
3D imaging in animal models, during development or in adults, facilitates the identification of structural morphological changes that cannot be achieved with traditional 2D histological staining. Through the reconstruction of whole embryos or a region-of-interest, specific changes are better delimited and can be easily quantified. We focused here on high-resolution episcopic microscopy (HREM), and its potential for visualizing and quantifying the organ systems of normal and genetically altered embryos and adult organisms. Although the technique is based on episcopic images, these are of high resolution and are close to histological quality. The images reflect the tissue structure and densities revealed by histology, albeit in a grayscale color map. HREM technology permits researchers to take advantage of serial 2D aligned stacks of images to perform 3D reconstructions. Three-dimensional visualization allows for an appreciation of topology and morphology that is difficult to achieve with classical histological studies. The nature of the data lends itself to novel forms of computational analysis that permit the accurate quantitation and comparison of individual embryos in a manner that is impossible with histology. Here, we have developed a new HREM prototype consisting of the assembly of a Leica Biosystems Nanocut rotary microtome with optics and a camera. We describe some examples of applications in the prenatal and adult lifestage of the mouse to show the added value of HREM for phenotyping experimental cohorts to compare and quantify structure volumes. At prenatal stages, segmentations and 3D reconstructions allowed the quantification of neural tissue and ventricular system volumes of normal brains at E14.5 and E16.5 stages. 3D representations of normal cranial and peripheric nerves at E15.5 and of the normal urogenital system from stages E11.5 to E14.5 were also performed. We also present a methodology to quantify the volume of the atherosclerotic plaques of ApoEtm1Unc/tm1Unc mutant mice and illustrate a 3D reconstruction of knee ligaments in adult mice.
ABSTRACT
Many areas of biological research demand the combined use of different imaging modalities to cover a wide range of magnifications and measurements or to place fluorescent patterns into an ultrastructural context. A technically difficult problem is the efficient specimen transfer between different imaging modalities without losing the coordinates of the regions-of-interest (ROI). Here, we report a new and highly sensitive integrated system that combines a custom designed microscope with an ultramicrotome for in-resin-fluorescence detection in blocks, ribbons and sections on EM-grids. Although operating with long-distance lenses, this system achieves a very high light sensitivity. Our instrumental set-up and operating workflow are designed to investigate rare events in large tissue volumes. Applications range from studies of individual immune, stem and cancer cells to the investigation of non-uniform subcellular processes. As a use case, we present the ultrastructure of a single membrane repair patch on a muscle fiber in intact muscle in a whole animal context.
ABSTRACT
Opioid receptors are G protein-coupled receptors (GPCRs) that modulate brain function at all levels of neural integration, including autonomic, sensory, emotional and cognitive processing. Mu (MOR) and delta (DOR) opioid receptors functionally interact in vivo, but whether interactions occur at circuitry, cellular or molecular levels remains unsolved. To challenge the hypothesis of MOR/DOR heteromerization in the brain, we generated redMOR/greenDOR double knock-in mice and report dual receptor mapping throughout the nervous system. Data are organized as an interactive database offering an opioid receptor atlas with concomitant MOR/DOR visualization at subcellular resolution, accessible online. We also provide co-immunoprecipitation-based evidence for receptor heteromerization in these mice. In the forebrain, MOR and DOR are mainly detected in separate neurons, suggesting system-level interactions in high-order processing. In contrast, neuronal co-localization is detected in subcortical networks essential for survival involved in eating and sexual behaviors or perception and response to aversive stimuli. In addition, potential MOR/DOR intracellular interactions within the nociceptive pathway offer novel therapeutic perspectives.
Subject(s)
Brain/metabolism , Nerve Net/metabolism , Neurons/metabolism , Receptors, Opioid, delta/analysis , Receptors, Opioid, mu/analysis , Animals , Female , Gene Knock-In Techniques , Male , Mice , Mice, Inbred C57BLABSTRACT
BACKGROUND: Tumor necrosis factor-alpha (TNF-α) is a pro-inflammatory cytokine today identified as a key mediator of several chronic inflammatory diseases. TNF-α, initially synthesized as a membrane-anchored precursor (pro-TNF-α), is processed by proteolytic cleavage to generate the secreted mature form. TNF-α converting enzyme (TACE) is currently the first and single protease described as responsible for the inducible release of soluble TNF-α. METHODOLOGY/PRINCIPAL FINDINGS: Here, we demonstrated the presence on THP-1 cells as on human monocytes of a constitutive proteolytical activity able to cleave pro-TNF-α. Revelation of the cell surface TACE protein expression confirmed that the observed catalytic activity is due to TACE. However, further studies using effective and innovative TNF-α inhibitors, as well as a highly selective TACE inhibitor, support the presence of a catalytically different sheddase activity on LPS activated THP-1 cells. It appears that this catalytically different TACE protease activity might have a significant contribution to TNF-α release in LPS activated THP-1 cells, by contrast to human monocytes where the TACE activity remains catalytically unchanged even after LPS activation. CONCLUSIONS/SIGNIFICANCE: On the surface of LPS activated THP-1 cells we identified a releasing TNF-α activity, catalytically different from the sheddase activity observed on human monocytes from healthy donors. This catalytically-modified TACE activity is different from the constitutive shedding activity and appears only upon stimulation by LPS.
Subject(s)
ADAM Proteins/metabolism , Lipopolysaccharides/metabolism , Monocytes/cytology , Tumor Necrosis Factor-alpha/metabolism , ADAM17 Protein , Animals , Benzoquinones/pharmacology , Cell Line , Cell Membrane/metabolism , Chronic Disease , Enzyme-Linked Immunosorbent Assay/methods , Flow Cytometry/methods , Humans , Hydroxamic Acids/pharmacology , Inflammation , Inhibitory Concentration 50 , Mice , Microscopy, Confocal/methods , Pentacyclic Triterpenes , Peptide Hydrolases/metabolism , Recombinant Proteins/metabolism , Sulfonamides/pharmacology , Triterpenes/pharmacologyABSTRACT
BACKGROUND: In cell biology, the study of proteins and organelles requires the combination of different imaging approaches, from live recordings with light microscopy (LM) to electron microscopy (EM). METHODOLOGY: To correlate dynamic events in adherent cells with both ultrastructural and 3D information, we developed a method for cultured cells that combines confocal time-lapse images of GFP-tagged proteins with electron microscopy. With laser micro-patterned culture substrate, we created coordinates that were conserved at every step of the sample preparation and visualization processes. Specifically designed for cryo-fixation, this method allowed a fast freezing of dynamic events within seconds and their ultrastructural characterization. We provide examples of the dynamic oligomerization of GFP-tagged myotubularin (MTM1) phosphoinositides phosphatase induced by osmotic stress, and of the ultrastructure of membrane tubules dependent on amphiphysin 2 (BIN1) expression. CONCLUSION: Accessible and versatile, we show that this approach is efficient to routinely correlate functional and dynamic LM with high resolution morphology by EM, with immuno-EM labeling, with 3D reconstruction using serial immuno-EM or tomography, and with scanning-EM.
Subject(s)
Electron Microscope Tomography/methods , Microscopy, Confocal/methods , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/ultrastructure , Animals , COS Cells , Chlorocebus aethiops , Freezing , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Green Fluorescent Proteins/ultrastructure , Humans , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microscopy, Electron, Scanning , Molecular Dynamics Simulation , Pressure , Protein Tyrosine Phosphatases, Non-Receptor/genetics , Protein Tyrosine Phosphatases, Non-Receptor/metabolism , Protein Tyrosine Phosphatases, Non-Receptor/ultrastructure , Recombinant Fusion Proteins/genetics , Reproducibility of ResultsABSTRACT
Crohn's disease (CD) is a multifactorial chronic inflammatory bowel disease of unknown cause. The aim of the present study was to explore if mRNA over-expression of SSTR5 and CCR7 found in CD patients could be correlated to respective protein expression. When compared to healthy donors, SSTR5 was over-expressed 417 +/- 71 times in CD peripheral blood mononuclear cells (PBMCs). Flow cytometry experiments showed no correlation between mRNA and protein expression for SSTR5 in PBMCs. In an attempt to find a reason of such a high mRNA expression, SSTR5 present on CD PBMCs were tested and found as biologically active as on healthy cells. In biopsies of CD intestinal tissue, SSTR5 was not over-expressed but CCR7, unchanged in PBMCs, was over-expressed by 10 +/- 3 times in the lamina propria. Confocal microscopy showed a good correlation of CCR7 mRNA and protein expression in CD intestinal biopsies. Our data emphasize flow and image cytometry as impossible to circumvent in complement to molecular biology so to avoid false interpretation on receptor expressions. Once confirmed by further large-scale studies, our preliminary results suggest a role for SSTR5 and CCR7 in CD pathogenesis.
Subject(s)
Crohn Disease/metabolism , Gene Expression Regulation , RNA, Messenger/metabolism , Receptors, CCR7/metabolism , Receptors, Somatostatin/metabolism , Adult , Female , Flow Cytometry , Humans , Leukocytes, Mononuclear/metabolism , Male , Microscopy, Confocal/methods , Models, Biological , Mucous Membrane/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Prostanoids, bioactive lipids derived from arachidonic acid (AA), are important for vascular homeostasis. Among them, prostaglandin E2 (PGE2) enhances aggregation of platelets submaximally stimulated in vitro. This results from activation of EP3, one of the four PGE2 receptors, which decreases the threshold at which agonists activate platelets to aggregate. Although PGE2 altered venous thrombosis induced by administration of AA, its role in pathophysiopathological conditions has remained speculative. We report that arterial walls subjected to inflammatory stimuli produce PGE2. In several models, we show that PGE2 produced by the arterial wall facilitates arterial thrombosis. Next, we detected PGE2 in mouse atherosclerotic plaques. We demonstrate that this plaque-produced PGE2 is not altered and is still able to activate EP3. In addition, we present evidence that PGE2 can leave the plaque and activate EP3 on blood platelets. Consistent with these findings, we observed that atherothrombosis induced in vivo by mechanical rupture of the plaque was drastically decreased when platelets lacked EP3. In conclusion, PGE2 facilitates the initiation of arterial thrombosis and, hence, contributes to atherothrombosis. Inhibition of the platelet EP3 receptor should improve prevention of atherothrombosis.
Subject(s)
Arteries/immunology , Atherosclerosis/immunology , Blood Platelets/metabolism , Carotid Artery Thrombosis/physiopathology , Dinoprostone/metabolism , Receptors, Prostaglandin E/metabolism , Animals , Apolipoproteins E/genetics , Arachidonic Acid/toxicity , Arteries/metabolism , Atherosclerosis/physiopathology , Carotid Artery Thrombosis/chemically induced , Immunoenzyme Techniques , Mice , Mice, Knockout , Platelet Aggregation/immunology , Receptors, Prostaglandin E/genetics , Receptors, Prostaglandin E, EP3 Subtype , Statistics, NonparametricABSTRACT
Retinoic acid (RA) activity plays sequential roles during the development of the ventral spinal cord. Here, we have investigated the functions of local RA synthesis in the process of motoneuron specification and early differentiation using a conditional knockout strategy that ablates the function of the retinaldehyde dehydrogenase 2 (Raldh2) synthesizing enzyme essentially in brachial motoneurons, and later in mesenchymal cells at the base of the forelimb. Mutant (Raldh2L-/-) embryos display an early embryonic loss of a subset of Lim1+ brachial motoneurons, a mispositioning of Islet1+ neurons and inappropriate axonal projections of one of the nerves innervating extensor limb muscles, which lead to an adult forepaw neuromuscular defect. The molecular basis of the Raldh2L-/- phenotype relies in part on the deregulation of Hoxc8, which in turn regulates the RA receptor RARbeta. We further show that Hoxc8 mutant mice, which exhibit a similar congenital forepaw defect, display at embryonic stages molecular defects that phenocopy the Raldh2L-/- motoneuron abnormalities. Thus, interdependent RA signaling and Hox gene functions are required for the specification of brachial motoneurons in the mouse.