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Raft-based interactions of gangliosides with a GPI-anchored receptor

Abstract

Gangliosides, glycosphingolipids containing one or more sialic acid(s) in the glyco-chain, are involved in various important physiological and pathological processes in the plasma membrane. However, their exact functions are poorly understood, primarily because of the scarcity of suitable fluorescent ganglioside analogs. Here, we developed methods for systematically synthesizing analogs that behave like their native counterparts in regard to partitioning into raft-related membrane domains or preparations. Single-fluorescent-molecule imaging in the live-cell plasma membrane revealed the clear but transient colocalization and codiffusion of fluorescent ganglioside analogs with a fluorescently labeled glycosylphosphatidylinisotol (GPI)-anchored protein, human CD59, with lifetimes of 12 ms for CD59 monomers, 40 ms for CD59's transient homodimer rafts in quiescent cells, and 48 ms for engaged-CD59-cluster rafts, in cholesterol- and GPI-anchoring-dependent manners. The ganglioside molecules were always mobile in quiescent cells. These results show that gangliosides continually and dynamically exchange between raft domains and the bulk domain, indicating that raft domains are dynamic entities.

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Figure 1
Figure 2: Partitioning of fluorescent GM1 and GM3 analogs into Lo- and Ld-like domains in the GPMVs of RBL-2H3 cells at 10 °C.
Figure 3: Single molecules of 594-GM3 and 594-GM1, but not 594-DOPE, are recruited to micron-scale CD59 patches.
Figure 4: Single molecules of 594-GM3 and 594-GM1 were frequently and transiently recruited to CD59 monomers (CD59-monomer-associated rafts) and CD59-homodimer rafts in quiescent cells in the time scale of 12–40 ms.
Figure 5: Single molecules of 594-GM3 and 594-GM1 were frequently and transiently recruited to CD59-cluster signaling rafts for 48 ms.

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Acknowledgements

We thank M. Sokabe of Nagoya University, B. Baird of Cornell University, S. Ohnishi of Kyoto University, and V. Horejsi of Academy of Sciences of the Czech Republic for their kind gifts of human epithelial T24 cells, RBL-2H3 cells, NRK cells, and a mouse hybridoma cell line MEM43/5 which produces anti CD59 monoclonal antibody, respectively, as well as R.S. Kasai, K.A.K. Tanaka, and K.M. Hirosawa for setting up the single-molecule tracking station and useful discussions. This work was supported in part by Grants-in-Aid for scientific research from the Japan Society for the Promotion of Science (Wakate A to H.A. (23688014), Kiban B to K.G.N.S. (24370055 and 15H04351), H.A. (15H04495), K.F. (24390078), and M.K. (22380067), and Kiban A to A.K. (24247029)); by Grants-in-Aid for Innovative Areas (Deciphering sugar chain-based signals regulating integrative neuronal functions) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan to H.A. (24110505 and 26110704), K.G.N.S. (23110001), K.F. (23110008), and T.K.F. (25113515 and 26115707); by a grant from Core Research for Evolutional Science and Technology (CREST project of “Creation of Fundamental Technologies for Understanding and Control of Biosystem Dynamics) of Japan Science and Technology Agency (JST) to A.K.; and by a grant awarded by the NIH to W.C. (GM68849). WPI-iCeMS of Kyoto University is supported by the World Premiere Research Center Initiative (WPI) of the MEXT.

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Contributions

K.G.N.S., H.A., A.K., and M. Kiso conceived this research and designed experiments; N.K., H.A., M. Konishi, M. Koikeda, A.I., and M. Kiso developed organic synthesis methods and performed organic synthesis; R.S. and W.C. performed SPR experiments and analysis; K.G.N.S. examined ganglioside partitioning into DRM and Lo/Ld domains, studied colocalizations of CD59 patches with ganglioside analogues using epifluorescence, and performed all of the single-molecule tracking experiments; R.C. conducted most of the Lo/Ld partitioning measurements; T.K.F. set up the single-molecule tracking instrument and developed software for single-molecule colocalization detection; H.T. examined colocalizations of CD59 patches with ganglioside analogues using a confocal fluorescence microscope; F. Koichi, F. Keiko, and Y.Y. prepared GM3-depleted cell lines; K.G.N.S., H.A., A.K., and M. Kiso wrote the paper; and all authors participated in the revisions of the manuscript.

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Correspondence to Akihiro Kusumi or Makoto Kiso.

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Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–5 and Supplementary Figures 1–6. (PDF 2068 kb)

Supplementary Note

Synthetic Procedures (PDF 452 kb)

41589_2016_BFnchembio2059_MOESM69_ESM.mov

A representative simultaneous two-color single fluorescent-molecule imaging video clip of a 594-GM1 molecule (red) diffusing in the PM containing CD59 patches (green) (the original video for the still image in Fig. 3a, left). (MOV 1393 kb)

As for Supplementary Video 1, except that the observed single molecule was 594-GM3 (Fig. 3a, middle). (MOV 4980 kb)

As for Supplementary Video 1, except that the observed single molecule was 594-DOPE (Fig. 3a, right). (MOV 2571 kb)

A typical video clip of single 594-GM1 molecules diffusing in the T24-cell PM at 23°C. (MOV 842 kb)

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Komura, N., Suzuki, K., Ando, H. et al. Raft-based interactions of gangliosides with a GPI-anchored receptor. Nat Chem Biol 12, 402–410 (2016). https://doi.org/10.1038/nchembio.2059

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