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Membrane molecules mobile even after chemical fixation

Nature Methods volume 7, pages 865–866 (2010)Cite this article

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With the frequently used fixation conditions of 4% paraformaldehyde at 25 °C for 30 min, contrary to general expectation glycosylphosphatidylinositol (GPI)-anchored proteins were difficult to immobilize and at least ∼40% of H-ras, ∼70% of transferrin receptor (TfR) and ∼2% of Cbp diffused laterally (Fig. 1b and Supplementary Figs. 3–5). Including 0.1% glutaraldehyde in addition to 4% paraformaldehyde did not immobilize Halo-GPI (51% immobilized) but immobilized ≥90% of monomeric (m)GFP-GPI, Lyn, H-ras, Cbp and TfR (we observed ∼70% immobilization of GPI-anchored proteins, on average, depending strongly on the protein; Fig. 1b and Supplementary Figs. 3–5). Increasing glutaraldehyde concentration to 0.2% or methanol (100%) fixation resulted in immobilization of >80% GPI-anchored proteins but not of phospholipids or cholesterol.

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Figure 1: Single-molecule mobility under different fixation conditions.

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Acknowledgements

We thank R. Bittman of the City University of New York for providing Bodipy488-cholesterol, and the members of the Kusumi laboratory for helpful discussions.

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Author notes

  1. Kenji A K Tanaka and Kenichi G N Suzuki: These authors contributed equally to this work.

Authors and Affiliations

  1. Membrane Mechanisms Project, International Cooperative Research Project, Japan Science and Technology Agency (JST), Institute for Integrated Cell-Material Sciences (iCeMS) and Research Center for Nano Medical Engineering, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan

    Kenji A K Tanaka, Yuki M Shirai, Shusaku T Shibutani, Manami S H Miyahara, Hisae Tsuboi, Miyako Yahara & Akihiro Kusumi

  2. Precursory Research for Embryonic Science and Technology, JST, iCeMS, Kyoto University, Kyoto, Japan

    Kenichi G N Suzuki

  3. Department of Microbiology and Immunology, School of Medicine, Keio University and Core Research for Evolutional Science and Technology, JST, Tokyo, Japan

    Akihiko Yoshimura

  4. National Centre for Biological Sciences, Bangalore, India

    Satyajit Mayor

  5. Center for Meso-Bio Single-Molecule Imaging, iCeMS, Kyoto University, Kyoto, Japan

    Takahiro K Fujiwara & Akihiro Kusumi

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  1. Kenji A K Tanaka
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Correspondence to Akihiro Kusumi.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Methods (PDF 6422 kb)

Supplementary Video 1

A typical video clip showing the diffusion of single Halo-GPI molecules on the apical plasma membrane at 37 °C. Before chemical cross-linking (untreated, left), and after treatment with 4% PFA for 30 min (center) or with 4% PFA + 0.1% GA for 30 min (right). (MOV 2481 kb)

Supplementary Video 2

A typical video clip showing the diffusion of single Halo–H-Ras molecules on the apical plasma membrane at 37 °C. Before chemical cross-linking (untreated, left), and after treatment with 4% PFA for 30 min (center) or with 4% PFA + 0.1% GA for 30 min (right). (MOV 2811 kb)

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Tanaka, K., Suzuki, K., Shirai, Y. et al. Membrane molecules mobile even after chemical fixation. Nat Methods 7, 865–866 (2010). https://doi.org/10.1038/nmeth.f.314

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