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Transient GPI-anchored protein homodimers are units for raft organization and function

Nature Chemical Biology volume 8, pages 774–783 (2012)Cite this article

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Abstract

Advanced single-molecule fluorescent imaging was applied to study the dynamic organization of raft-associated glycosylphosphatidylinositol-anchored proteins (GPI-APs) in the plasma membrane and their stimulation-induced changes. In resting cells, virtually all of the GPI-APs are mobile and continually form transient (∼200 ms) homodimers (termed homodimer rafts) through ectodomain protein interactions, stabilized by the presence of the GPI-anchoring chain and cholesterol. Heterodimers do not form, suggesting a fundamental role for the specific ectodomain protein interaction. Under higher physiological expression conditions , homodimers coalesce to form hetero– and homo–GPI-AP oligomer rafts through raft-based lipid interactions. When CD59 was ligated, it formed stable oligomer rafts containing up to four CD59 molecules, which triggered intracellular Ca2+ responses that were dependent on GPI anchorage and cholesterol, suggesting a key part played by transient homodimer rafts. Transient homodimer rafts are most likely one of the basic units for the organization and function of raft domains containing GPI-APs.

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Figure 1: Model for the MAC-induced CD59 cluster raft and signaling and transient homocolocalization of GPI-APs in steady-state cells.
Figure 2: Single-molecule FRET between ACP-CD59 molecules.
Figure 3: Two-color single-molecule tracking did not detect prolonged colocalization of hetero-GPI-APs.
Figure 4: GPI-AP homodimers are stabilized by raft-lipid interactions, forming GPI-AP homodimer rafts, whereas raft-lipid interaction alone without ectodomain protein interaction can hardly induce GPI-AP heterodimer rafts.
Figure 5: The fractions of transient (apparent) homo-oligomers of ACP-CD59 increased with increased expression.
Figure 6: MAC induced stable CD59-oligomer rafts (MAC-rafts) dependent on dynamic CD59 homodimers.
Figure 7: MAC-rafts are stable, undergo repeated diffusion-STALL cycles and induce an intracellular Ca2+ response.

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Acknowledgements

We thank V. Horejsi (Institute of Molecular Genetics, Academy of Sciences of the Czech Republic), M. Tone (University of Oxford) and M. Maio (Istituto Nazionale di Ricovero e Cura a Carattere Scientifico) for the hybridoma lines that produce the CD59- and DAF-specific antibodies, the cDNA construct for human CD59, and that for CD59TM, respectively. This work was supported in part by Grants-in-Aid for Scientific Research (A12020366) and Specific Research on Innovative Areas (23110002, 233306) from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese government and by the Development of Systems and Technology for Advanced Measurement and Analysis program of the Japan Science and Technology Agency (10101506).

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Authors and Affiliations

  1. Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto, Japan

    Kenichi G N Suzuki, Koichiro M Hirosawa, Yuri L Nemoto, Munenori Ishibashi, Takahiro K Fujiwara & Akihiro Kusumi

  2. Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan

    Rinshi S Kasai & Akihiro Kusumi

  3. Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Saitama, Japan

    Kenichi G N Suzuki

  4. Department of Pharmacology, Institute of Basic Medical Sciences, University of Tsukuba, Ibaraki, Japan

    Yoshihiro Miwa

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Contributions

K.G.N.S. performed experiments. T.K.F. and A.K. developed the TIRF microscope system. Y.M. developed the pOsTet15T3 vector. K.M.H., Y.L.N. and M.I. helped the data analysis. K.G.N.S., R.S.K. and A.K. formulated the project. K.G.N.S. and A.K. wrote the manuscript.

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Correspondence to Akihiro Kusumi.

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Suzuki, K., Kasai, R., Hirosawa, K. et al. Transient GPI-anchored protein homodimers are units for raft organization and function. Nat Chem Biol 8, 774–783 (2012). https://doi.org/10.1038/nchembio.1028

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