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Three-dimensional reconstruction of the membrane skeleton at the plasma membrane interface by electron tomography

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      Abstract

      Three-dimensional images of the undercoat structure on the cytoplasmic surface of the upper cell membrane of normal rat kidney fibroblast (NRK) cells and fetal rat skin keratinocytes were reconstructed by electron tomography, with 0.85-nm–thick consecutive sections made ∼100 nm from the cytoplasmic surface using rapidly frozen, deeply etched, platinum-replicated plasma membranes. The membrane skeleton (MSK) primarily consists of actin filaments and associated proteins. The MSK covers the entire cytoplasmic surface and is closely linked to clathrin-coated pits and caveolae. The actin filaments that are closely apposed to the cytoplasmic surface of the plasma membrane (within 10.2 nm) are likely to form the boundaries of the membrane compartments responsible for the temporary confinement of membrane molecules, thus partitioning the plasma membrane with regard to their lateral diffusion. The distribution of the MSK mesh size as determined by electron tomography and that of the compartment size as determined from high speed single-particle tracking of phospholipid diffusion agree well in both cell types, supporting the MSK fence and MSK-anchored protein picket models.

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      RADIOAUTOGRAPHIC STUDIES OF CHOLINE INCORPORATION INTO PERIPHERAL NERVE MYELIN

      This radioautographic study was designed to localize the cytological sites involved in the incorporation of a lipid precursor into the myelin and the myelin-related cell of the peripheral nervous system. Both myelinating and fully myelinated cultures of rat dorsal root ganglia were exposed to a 30-min pulse of tritiated choline and either fixed immediately or allowed 6 or 48 hr of chase incubation before fixation. After Epon embedding, light and electron microscopic radioautograms were prepared with Ilford L-4 emulsion. Analysis of the pattern of choline incorporation into myelinating cultures indicated that radioactivity appeared all along the length of the internode, without there being a preferential site of initial incorporation. Light microscopic radioautograms of cultures at varying states of maturity were compared in order to determine the relative degree of myelin labeling. This analysis indicated that the myelin-Schwann cell unit in the fully myelinated cultures incorporated choline as actively as did this unit in the myelinating cultures. Because of technical difficulties, it was not possible to determine the precise localization of the incorporated radioactivity within the compact myelin. These data are related to recent biochemical studies indicating that the mature myelin of the central nervous system does incorporate a significant amount of lipid precursor under the appropriate experimental conditions. These observations support the concept that a significant amount of myelin-related metabolic activity occurs in mature tissue; this activity is considered part of an essential and continuous process of myelin maintenance and repair.
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        Computer visualization of three-dimensional image data using IMOD.

        We have developed a computer software package, IMOD, as a tool for analyzing and viewing three-dimensional biological image data. IMOD is useful for studying and modeling data from tomographic, serial section, and optical section reconstructions. The software allows image data to be visualized by several different methods. Models of the image data can be visualized by volume or contour surface rendering and can yield quantitative information.
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          Caveolin, a protein component of caveolae membrane coats.

          Caveolae have been implicated in the transcytosis of macromolecules across endothelial cells and in the receptor-mediated uptake of 5-methyltetrahydrofolate. Structural studies indicate that caveolae are decorated on their cytoplasmic surface by a unique array of filaments or strands that form striated coatings. To understand how these nonclathrin-coated pits function, we performed structural analysis of the striated coat and searched for the molecular component(s) of the coat material. The coat cannot be removed by washing with high salt; however, exposure of membranes to cholesterol-binding drugs caused invaginated caveolae to flatten and the striated coat to disassemble. Antibodies directed against a 22 kd substrate for v-src tyrosine kinase in virus-transformed chick embryo fibroblasts decorated the filaments, suggesting that this molecule is a component of the coat. We have named the molecule caveolin. Caveolae represent a third type of coated membrane specialization that is involved in molecular transport.
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            Author and article information

            Affiliations
            [1 ]Kusumi Membrane Organizer Project, Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology Agency, Nagoya 460-0012, Japan
            [2 ]Department of Cell Biology and Anatomy, Graduate School of Medicine, Nagoya University, Nagoya 466-8550, Japan
            [3 ]Department of Ultrastructural Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo 187-8502, Japan
            [4 ]Membrane Mechanisms Project, International Cooperative Research Project (ICORP), Japan Science and Technology Agency, Institute for Frontier Medical Sciences, Kyoto University, Shougoin, Kyoto 606-8507, Japan
            Author notes

            Correspondence to Akihiro Kusumi: akusumi@ 123456frontier.kyoto-u.ac.jp

            Journal
            J Cell Biol
            JCB
            The Journal of Cell Biology
            The Rockefeller University Press
            0021-9525
            1540-8140
            11 September 2006
            : 174
            : 6
            : 851-862
            2064339
            200606007
            10.1083/jcb.200606007
            16954349
            Copyright © 2006, The Rockefeller University Press
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