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Quantitative electron microscopy for the nanoscale analysis of membrane lipid distribution

Nature Protocols volume 5, pages 661–669 (2010)Cite this article

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Abstract

An important goal of membrane biology is to define the local heterogeneity of membrane lipid composition. Here we describe a quantitative electron microscopic method that enables the localization of specific membrane lipids at the nanometer scale. The method involves freezing cells rapidly to halt the molecular motion, physically stabilizing membrane molecules in the freeze-fracture replica by the deposition of evaporated platinum and carbon layers and labeling with specific probes for electron microscopic observation. Lipids in both the outer and inner membrane leaflets can thus be labeled, and their distributions can be analyzed quantitatively by statistical methods. A major advantage of this method is that it does not require the expression of artificial probes. Therefore, this method can be applied to any cell in vitro or in vivo, and the whole procedure can be completed in 1–2 d.

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Figure 1: The outline of the procedure.
Figure 2: A schematic diagram of freeze fracture and molecular partitioning.
Figure 3: Replica preparation and treatment procedure.
Figure 4: GM1 distribution in a fibroblast cell membrane.
Figure 5: Caveolae and PI(4,5)P2 distribution.

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Acknowledgements

This work was supported by Grants-in-Aid for Scientific Research and the Global COE Program 'Integrated Molecular Medicine for Neuronal and Neoplastic Disorders' of the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government.

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Authors and Affiliations

  1. Department of Anatomy and Molecular Cell Biology, Nagoya University Graduate School of Medicine, Nagoya, Japan

    Akikazu Fujita, Jinglei Cheng & Toyoshi Fujimoto

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  1. Akikazu Fujita
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  2. Jinglei Cheng
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  3. Toyoshi Fujimoto
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Contributions

A.F. performed the experiments and prepared the manuscript. J.C. technically assisted. T.F. designed the experiments and prepared the manuscript.

Corresponding author

Correspondence to Toyoshi Fujimoto.

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Fujita, A., Cheng, J. & Fujimoto, T. Quantitative electron microscopy for the nanoscale analysis of membrane lipid distribution. Nat Protoc 5, 661–669 (2010). https://doi.org/10.1038/nprot.2010.20

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