1,721,166 research outputs found
Myelinated mouse nerves studied by X-ray phase contrast zoom tomography
No full textWe have used X-ray phase contrast tomography to resolve the structure of uncut, entire myelinated optic, saphenous and sciatic mouse nerves. Intrinsic electron density contrast suffices to identify axonal structures. Specific myelin labeling by an osmium tetroxide stain enables distinction between axon and surrounding myelin sheath. Utilization of spherical wave illumination enables zooming capabilities which enable imaging of entire sciatic intemodes as well as identification of sub-structures such as nodes of Ranvier and Schmidt-Lanterman incisures. (C) 2015 Elsevier Inc. All rights reserved
Gangliosides are Transported from the Plasma Membrane to Intralysosomal Membranes as Revealed by Immuno-Electron Microscopy
No full textA biotin-labeled derivative of the ganglioside GM1 (biotin-GM1) was used to study its transport along the endocytic pathway of cultured fibroblasts by immuno-electron microscopy. Using electron dense endocytic tracers we could demonstrate that late endosomes and lysosomes of these cells are long living organelles with a high content of internal membranes. Our studies show that during endocytosis the biotin-GM1 was transported to these intraendosomal and intralysosomal membranes. These observations support the hypothesis that glycosphingolipids (GSL) are preferentially degraded in intralysosomal vesicles.A biotin-labeled derivative of the ganglioside GM1 (biotin-GM1) was used to study its transport along the endocytic pathway of cultured fibroblasts by immuno-electron microscopy. Using electron dense endocytic tracers we could demonstrate that late endosomes and lysosomes of these cells are long living organelles with a high content of internal membranes. Our studies show that during endocytosis the biotin-GM1 was transported to these intraendosomal and intralysosomal membranes. These observations support the hypothesis that glycosphingolipids (GSL) are preferentially degraded in intralysosomal vesicles
Biological sample preparation by high-pressure freezing, microwave-assisted contrast enhancement, and minimal resin embedding for volume imaging
Full text linkThe described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data
Mechanics of Individual Keratin Bundles in Living Cells
Full text linkAbstractAlong with microtubules and microfilaments, intermediate filaments are a major component of the eukaryotic cytoskeleton and play a key role in cell mechanics. In cells, keratin intermediate filaments form networks of bundles that are sparser in structure and have lower connectivity than, for example, actin networks. Because of this, bending and buckling play an important role in these networks. Buckling events, which occur due to compressive intracellular forces and cross-talk between the keratin network and other cytoskeletal components, are measured here in situ. By applying a mechanical model for the bundled filaments, we can access the mechanical properties of both the keratin bundles themselves and the surrounding cytosol. Bundling is characterized by a coupling parameter that describes the strength of the linkage between the individual filaments within a bundle. Our findings suggest that coupling between the filaments is mostly complete, although it becomes weaker for thicker bundles, with some relative movement allowed
Sugar and ice: Immunoelectron microscopy using cryosections according to the Tokuyasu method
No full textSince the pioneering work of Kiyoteru Tokuyasu in the 70ths the use of thawed cryosections prepared according to the “Tokuyasu-method” for immunoelectron microscopy did not lose popularity. We owe this method a whole subcellular world described by discrete gold particles pointing at cargo, receptors and organelle markers on delicate images of the inner life of a cell. Here we explain the procedure of sample preparation, sectioning and immunolabeling in view of recent developments and the reasoning behind protocols including some historical perspective. Cryosections are prepared from chemically fixed and sucrose infiltrated samples and labeled with affinity probes and electron dense markers. These sections are ideal substrates for immunolabeling, since antigens are not exposed to organic solvent dehydration or masked by resin. Instead, the structures remain fully hydrated throughout the labeling procedure. Furthermore, target molecules inside dense intercellular structural elements, cells and organelles are accessible to antibodies from the section surface. For the validation of antibody specificity several approaches are recommended including knock-out tissue and reagent controls. Correlative light and electron microscopy strategies involving correlative probes are possible as well as correlation of live imaging with the underlying ultrastructure. By applying stereology, gold labeling can be quantified and evaluated for specificity
Neodymium acetate as a contrast agent for X-ray phase-contrast tomography
Full text linkPurposeX-ray phase-contrast tomography (XPCT) is a non-destructive, three-dimensional imaging modality that provides higher contrast in soft tissue than absorption-based CT and allows one to cover the cytoarchitecture from the centi- and millimeter scale down to the nanoscale. To further increase contrast and resolution of XPCT, for example, in view of addressing connectivity issues in the central nervous system (CNS), metal staining is indispensable. However, currently used protocols, for example, based on osmium and/or uranium are less suited for XPCT, due to an excessive β / δ-ratio. In this work, we explore the suitability of different staining agents for XPCT. Particularly, neodymium(III)-acetate (NdAc), which has recently been proposed as a non-toxic, non-radioactive easy to use alternative contrast agent for uranyl acetate (UAc) in electron microscopy, is investigated. Due to its vertical proximity to UAc in the periodic table, similar chemical but better suited optical properties for phase contrast can be expected.ApproachDifferently stained whole eye samples of wild type mouse and tissues of the CNS are embedded into EPON epoxy resin and scanned using synchrotron as well as with laboratory radiation. Phase retrieval is performed on the projection images, followed by tomographic reconstruction, which enables a quantitative analysis based on the reconstructed electron densities. Segmentation techniques and rendering software is used to visualize structures of interest in the sample.ResultsWe show that staining neuronal samples with NdAc enhances contrast, in particular for laboratory scans, allowing high-resolution imaging of biological soft tissue in-house. For the example of murine retina, specifically rods and cones as well as the sclera and the Ganglion cell layer seem to be targeted by the stain. A comparison of electron density by the evaluation of histograms allowed to determine quantitative measures to describe the difference between the examined stains.ConclusionThe results suggest NdAc to be an effective stain for XPCT, with a preferential binding to anionic groups, such as phosphate and carboxyl groups at cell surfaces, targeting certain layers of the retina with a stronger selectivity compared to other staining agents. Due to the advantageous X-ray optical properties, the stain seems particularly well-suited for phase contrast, with a comparably small number density and an overall superior image quality at laboratory sources
New species can broaden myelin research: suitability of little skate, Leucoraja erinacea
Full text link© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Moebius, W., Huemmert, S., Ruhwedel, T., Kuzirian, A., & Gould, R. New species can broaden myelin research: suitability of little skate, Leucoraja erinacea. Life, 11(2), (2021): 136, https://doi.org/10.3390/life11020136.Although myelinated nervous systems are shared among 60,000 jawed vertebrates, studies aimed at understanding myelination have focused more and more on mice and zebrafish. To obtain a broader understanding of the myelination process, we examined the little skate, Leucoraja erinacea. The reasons behind initiating studies at this time include: the desire to study a species belonging to an out group of other jawed vertebrates; using a species with embryos accessible throughout development; the availability of genome sequences; and the likelihood that mammalian antibodies recognize homologs in the chosen species. We report that the morphological features of myelination in a skate hatchling, a stage that supports complex behavioral repertoires needed for survival, are highly similar in terms of: appearances of myelinating oligodendrocytes (CNS) and Schwann cells (PNS); the way their levels of myelination conform to axon caliber; and their identity in terms of nodal and paranodal specializations. These features provide a core for further studies to determine: axon–myelinating cell communication; the structures of the proteins and lipids upon which myelinated fibers are formed; the pathways used to transport these molecules to sites of myelin assembly and maintenance; and the gene regulatory networks that control their expressions.This research received no external funding
Phylogeny of proteolipid proteins : divergence, constraints, and the evolution of novel functions in myelination and neuroprotection
No full textThe protein composition of myelin in the central nervous system (CNS) has changed at the evolutionary transition from fish to tetrapods, when a lipid-associated transmembrane-tetraspan (proteolipid protein, PLP) replaced an adhesion protein of the immunoglobulin superfamily (P0) as the most abundant constituent. Here, we review major steps of proteolipid evolution. Three paralog proteolipids (PLP/DM20/DMα, M6B/DMγ and the neuronal glycoprotein M6A/DMβ) exist in vertebrates from cartilaginous fish to mammals, and one (M6/CG7540) can be traced in invertebrate bilaterians including the planktonic copepod Calanus finmarchicus that possess a functional myelin equivalent. In fish, DMα and DMγ are coexpressed in oligodendrocytes but are not major myelin components. PLP emerged at the root of tetrapods by the acquisition of an enlarged cytoplasmic loop in the evolutionary older DMα/DM20. Transgenic experiments in mice suggest that this loop enhances the incorporation of PLP into myelin. The evolutionary recruitment of PLP as the major myelin protein provided oligodendrocytes with the competence to support long-term axonal integrity. We suggest that the molecular shift from P0 to PLP also correlates with the concentration of adhesive forces at the radial component, and that the new balance between membrane adhesion and dynamics was favorable for CNS myelination.The protein composition of myelin in the central nervous system (CNS) has changed at the evolutionary transition from fish to tetrapods, when a lipid-associated transmembrane-tetraspan (proteolipid protein, PLP) replaced an adhesion protein of the immunoglobulin superfamily (P0) as the most abundant constituent. Here, we review major steps of proteolipid evolution. Three paralog proteolipids (PLP/DM20/DMα, M6B/DMγ and the neuronal glycoprotein M6A/DMβ) exist in vertebrates from cartilaginous fish to mammals, and one (M6/CG7540) can be traced in invertebrate bilaterians including the planktonic copepod Calanus finmarchicus that possess a functional myelin equivalent. In fish, DMα and DMγ are coexpressed in oligodendrocytes but are not major myelin components. PLP emerged at the root of tetrapods by the acquisition of an enlarged cytoplasmic loop in the evolutionary older DMα/DM20. Transgenic experiments in mice suggest that this loop enhances the incorporation of PLP into myelin. The evolutionary recruitment of PLP as the major myelin protein provided oligodendrocytes with the competence to support long-term axonal integrity. We suggest that the molecular shift from P0 to PLP also correlates with the concentration of adhesive forces at the radial component, and that the new balance between membrane adhesion and dynamics was favorable for CNS myelination
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