The Experts below are selected from a list of 159 Experts worldwide ranked by ideXlab platform
Nicholas J Severs - One of the best experts on this subject based on the ideXlab platform.
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topography of lipid droplet associated proteins insights from Freeze Fracture replica immunogold labeling
Journal of Lipids, 2011Co-Authors: Horst Robenek, Insa Buers, Oliver Hofnagel, David Troyer, Mirko J Robenek, Anneke Ruebel, Nicholas J SeversAbstract:Lipid droplets are not merely storage depots for superfluous intracellular lipids in times of hyperlipidemic stress, but metabolically active organelles involved in cellular homeostasis. Our concepts on the metabolic functions of lipid droplets have come from studies on lipid droplet-associated proteins. This realization has made the study of proteins, such as PAT family proteins, caveolins, and several others that are targeted to lipid droplets, an intriguing and rapidly developing area of intensive inquiry. Our existing understanding of the structure, protein organization, and biogenesis of the lipid droplet has relied heavily on microscopical techniques that lack resolution and the ability to preserve native cellular and protein composition. Freeze-Fracture replica immunogold labeling overcomes these disadvantages and can be used to define at high resolution the precise location of lipid droplet-associated proteins. In this paper illustrative examples of how Freeze-Fracture immunocytochemistry has contributed to our understanding of the spatial organization in the membrane plane and function of PAT family proteins and caveolin-1 are presented. By revisiting the lipid droplet with Freeze-Fracture immunocytochemistry, new perspectives have emerged which challenge prevailing concepts of lipid droplet biology and may hopefully provide a timely impulse for many ongoing studies.
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lipid droplet growth by fusion insights from Freeze Fracture imaging
Journal of Cellular and Molecular Medicine, 2009Co-Authors: Horst Robenek, Nicholas J SeversAbstract:An understanding of how lipid droplets grow in the cell is important to current human health issues. Homotypic fusion of small lipid droplets to create larger ones is one proposed mechanism though the evidence for this process continues to be debated. By applying the technique of Freeze-Fracture electron microscopy to cells that have been stimulated to accumulate lipid droplets, we here present images which suggest that at least some large lipid droplets may indeed result from amalgamation of multiple smaller ones. These visual data add significantly to the notion that fusion contributes to lipid droplet growth.
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Recent advances in Freeze-Fracture electron microscop: the replica immunolabeling technique
Biological procedures online, 2008Co-Authors: Horst Robenek, Nicholas J SeversAbstract:Freeze-Fracture electron microscopy is a technique for examining the ultrastructure of rapidly frozen biological samples by transmission electron microscopy. Of a range of approaches to Freeze-Fracture cytochemistry that have been developed and tried the most successful is the technique termed Freeze-Fracture replica immunogold labeling (FRIL). In this technique samples are frozen Fractured and replicated with platinum-carbon as in standard Freeze Fracture and then carefully treated with sodium dodecylsulphate to remove all the biological material except a fine layer of molecules attached to the replica itself. Immunogold labeling of these molecules permits their distribution to be seen superimposed upon high resolution planar views of membrane structure. Examples of how this technique has contributed to our understanding of lipid droplet biogenesis and function are discussed.
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Freeze-Fracture cytochemistry in cell biology.
Methods in cell biology, 2008Co-Authors: Nicholas J Severs, Horst RobenekAbstract:The term Freeze-Fracture cytochemistry embraces a series of techniques which share the goal of chemical identification of the structural components viewed in Freeze-Fracture replicas. As one of the major features of Freeze Fracture is its ability to provide planar views of membranes, a major emphasis in Freeze-Fracture cytochemistry is to identify integral membrane proteins, study their spatial organization in the membrane plane, and examine their role in dynamic cellular processes. Effective techniques in Freeze-Fracture cytochemistry, of wide application in cell biology, are now available. These include Fracture-label, label Fracture, and the Freeze-Fracture replica immunolabeling technique (FRIL). In Fracture-label, samples are frozen and Fractured, thawed for labeling, and finally processed for viewing either by critical-point drying and platinum-carbon replication or by thin-section electron microscopy. Label-Fracture involves immunogold labeling a cell suspension, processing as for standard Freeze-Fracture replication, and then examining the replica without removal of the cellular components. Of greatest versatility, however, is the FRIL technique, in which samples are frozen, Fractured, and replicated with platinum-carbon as in standard Freeze Fracture, and then carefully treated with sodium dodecylsulphate (SDS) to remove all the biological material except a fine layer of molecules attached to the replica itself. Immunogold labeling of these molecules permits the distribution of identified components to be viewed superimposed upon high resolution planar views of replicated membrane structure, for both the plasma membrane and intracellular membranes in cells and tissues. Examples of how these techniques have contributed to our understanding of cardiovascular cell function in health and disease are discussed.
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Freeze-Fracture electron microscopy
Nature Protocols, 2007Co-Authors: Nicholas J SeversAbstract:The Freeze-Fracture technique consists of physically breaking apart (fracturing) a frozen biological sample; structural detail exposed by the Fracture plane is then visualized by vacuum-deposition of platinum–carbon to make a replica for examination in the transmission electron microscope. The four key steps in making a Freeze-Fracture replica are (i) rapid freezing, (ii) fracturing, (iii) replication and (iv) replica cleaning. In routine protocols, a pretreatment step is carried out before freezing, typically comprising fixation in glutaraldehyde followed by cryoprotection with glycerol. An optional etching step, involving vacuum sublimation of ice, may be carried out after fracturing. Freeze Fracture is unique among electron microscopic techniques in providing planar views of the internal organization of membranes. Deep etching of ultrarapidly frozen samples permits visualization of the surface structure of cells and their components. Images provided by Freeze Fracture and related techniques have profoundly shaped our understanding of the functional morphology of the cell.
Dori C Woods - One of the best experts on this subject based on the ideXlab platform.
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a method for Freeze Fracture and scanning electron microscopy of isolated mitochondria
MethodsX, 2018Co-Authors: Julie A Macdonald, William H Fowle, Ellie Shin, Dori C WoodsAbstract:Abstract Electron microscopy as a methodology for the study of mitochondria based on morphological features is a standard technique that has experienced little evolution over the course of several decades. This technology has identified heterogeneity of mitochondria populations across both whole tissues, as well between individual cells, using primarily ultrathin sections for transmission electron microscopy (TEM). However, this technique constrains the evaluation of a sample to a single two-dimensional plane. To overcome this limitation, scanning electron microscopy (SEM) has been successfully utilized to observe three-dimensional mitochondria structures within the complex microenvironment containing total cellular components. In response to these dual technical caveats of existing electron microscopy protocols, we developed a methodology to evaluate the three-dimensional ultrastructure of isolated mitochondria, utilizing a Freeze-Fracture step and rigorous preservation of sample morphology. This protocol allows for a more high-throughput analysis of mitochondria populations from a specimen of interest, as the sample has been previously purified, as well as a finer resolution of complex intra-mitochondrial structures, using the depth of field created by SEM. • Protocol designed for SEM of isolated mitochondria samples. • SEM visualizes mitochondria ultrastructure in 3-D. • Freeze-Fracture creates cross-sectional plane for view of interior organelle structures.
David M Shotton - One of the best experts on this subject based on the ideXlab platform.
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Rapid Freezing of Biological Specimens for Freeze Fracture and Deep Etching
Cell Biology, 2006Co-Authors: Nicholas J Severs, David M ShottonAbstract:Publisher Summary Stabilization of the biological structure by the physical process of freezing (cryofixation) forms the starting point for Freeze Fracture and deep etching and for Freeze substitution, cryoultramicrotomy, and cryoelectron microscopy. For Freeze Fracture and deep etching, the requirement for a larger specimen size precludes true vitrification throughout the specimen because the maximal cooling rate possible within the sample is limited by the rate of heat conduction through it. Ice crystal damage can be avoided at relatively slow cooling rates if the specimen is first infiltrated with a buffered cryoprotectant. To enable processing through the various subsequent steps of Freeze Fracture or deep etching, specimens are first mounted on specially designed supports. Standard specimen supports are made from metals of high thermal conductivity and are as small as is compatible with ease of handling. Various techniques have been developed for mounting cultured cells for Freeze Fracture. The traditional alternative method for freezing specimens is to immerse them manually into a secondary cryogenic liquid cooled to near its freezing point using liquid nitrogen as the primary cryogen.
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Freeze Fracture and Freeze Etching
Cell Biology, 2006Co-Authors: David M ShottonAbstract:Publisher Summary The technique of Freeze Fracture is unique among electron microscopic (EM) methods in that it gives enface views of the internal organization of biological membranes. Although Freeze Fracture can be undertaken using very simple equipment that can be constructed in any workshop, in conjunction with a standard vacuum coating unit, or using a commercial attachment to such a coating unit, it is normally performed within a specialized high vacuum Freeze-Fracture apparatus, with a temperature-controlled, liquid nitrogen-cooled holder for specimens. Integral proteins, which span the membrane, may partition with one or the other half of the membrane, from which they will protrude to form small Freeze-Fracture intra-membrane particles (IMPs), leaving complementary pits in the other half membrane from which they were wrenched. A quartz crystal replica thickness (thin film) monitor, positioned adjacent to the specimen stage in the path of the shadowing beams, is usually used to monitor replica deposition rates and amounts. Sequentially, operate the platinum and carbon guns according to the manufacturer's instructions to deposit the desired thickness of material, with the specimen stage stationary or rotating, as required.
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rapid freezing Freeze Fracture and deep etching
1995Co-Authors: Nicholas John Severs, David M ShottonAbstract:Partial table of contents: An Introduction to Freeze Fracture and Deep Etching (D. Shotton & N. Severs). A Guide to Equipment for Production of Freeze--Fracture Replicas (T. Newman). Freeze--Fracture Artifacts: How to Recognize and Avoid Them (R. Abeysekera & A. Robards). High Pressure Freezing (J. Kiss & L. Staehelin). Time--Resolved Analysis of Rapid Events (G. Knoll). Freeze--Fracture Cytochemistry: An Explanatory Survey of Methods (N. Severs). Lipid Localization by Membrane Perturbation: A Cautionary Tale (N. Severs). Membrane Lipids and Model Membrane Systems (M. Hope & W. Rodrigueza). Visualization of Basement Membrane Molecules and Supramolecular Assemblies (P. Yurchenco & G. Ruben). Index.
Toyoshi Fujimoto - One of the best experts on this subject based on the ideXlab platform.
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High-Resolution Molecular Localization by Freeze-Fracture Replica Labeling
Methods in molecular biology (Clifton N.J.), 2010Co-Authors: Akikazu Fujita, Toyoshi FujimotoAbstract:The Freeze-Fracture technique splits the frozen lipid bilayer membrane into two halves and immobilizes membrane proteins and lipids by the vacuum evaporation of platinum and carbon. After a treatment by SDS to remove extramembrane materials, the specimen is subjected to immunogold labeling, which gives information on the two-dimensional distribution of membrane molecules and their relationship to various differentiated structures. In combination with rapid freezing, the Freeze-Fracture technique has an advantage over other methods using conventional chemical fixation because the distribution of lipids as well as proteins can be observed at the mesoscale in a wide area of the membrane.
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transmembrane phospholipid distribution revealed by Freeze Fracture replica labeling
Journal of Cell Science, 1996Co-Authors: Kazushi Fujimoto, Masato Umeda, Toyoshi FujimotoAbstract:We propose the use of membrane splitting by Freeze-Fracture for differential phospholipid analysis of protoplasmic and exoplasmic membrane leaflets (halves). Unfixed cells or tissues are quick-frozen, Freeze-Fractured, and platinum-carbon (Pt/C) shadowed. The Pt/C replicas are then treated with 2.5% sodium dodecyl sulfate (SDS) to solubilize unFractured membranes and to release cytoplasm or contents. While the detergent dissolves unFractured membranes, it would not extract lipids from split membranes, as their apolar domains are stabilized by their Pt/C replicas. After washing, the Pt/C replicas, along with attached protoplasmic and exoplasmic membrane halves, are processed for immunocytochemical labeling of phospholipids with antibody, followed by electron microscopic observation. Here, we present the application of the SDS-digested Freeze-Fracture replica labeling (SDS-FRL) technique to the transmembrane distribution of a major membrane phospholipid, phosphatidylcholine (PC), in various cell and intracellular membranes. Immunogold labeling revealed that PC is exclusively localized on the exoplasmic membrane halves of the plasma membranes, and the intracellular membranes of various organelles, e.g. nuclei, mitochondria, endoplasmic reticulum, secretory granules, and disc membranes of photoreceptor cells. One exception to this general scheme was the plasma membrane forming the myelin sheath of neurons and the Ca(2+)-treated erythrocyte membranes. In these cell membranes, roughly equal amounts of immunogold particles for PC were seen on each outer and inner membrane half, implying a symmetrical transmembrane distribution of PC. Initial screening suggests that the SDS-FRL technique allows in situ analysis of the transmembrane distribution of membrane lipids, and at the same time opens up the possibility of labeling membranes such as intracellular membranes not normally accessible to cytochemical labels without the distortion potentially associated with membrane isolation procedures.
Thaddée Gulik-krzywicki - One of the best experts on this subject based on the ideXlab platform.
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Freeze-Fracture transmission electron microscopy
Current Opinion in Colloid & Interface Science, 1997Co-Authors: Thaddée Gulik-krzywickiAbstract:Freeze-Fracture electron microscopy is one of the oldest methods of a large family of valuable cryoelectron microscopic techniques, based on initial cryofixation of the specimens. Its main advantage for low- and medium-resolution structural studies is the very large spectrum of different materials (solid, liquid, suspension, etc.) and experimental conditions (composition, temperature, pressure, etc.) that may be used. Most of the corresponding procedures require, nevertheless, a careful evaluation of the effects of cryofixation, Fracture and replication upon the sample structure. Among the methods that might be used for such evaluation, X-ray diffraction is the most straightforward. The largest advances in Freeze-Fracture transmission electron microscopy are, indeed, due to the combined use of X-ray scattering and Freeze-Fracture, particularly when applied, together with image analysis, to the determination of the structure of three-dimensionally ordered specimens.
