A Typical superimposed video-rate sequences of a CD59 cluster and single molecules of 594-GM3, including a colocalization period (frame 6-9)B Schematic figure showing the transient association for 594-DOPE in each of the four cell types. We conclude that there are no tiny membrane domains which trap the ganglioside probes for longer than 5 ms3).References1) Hakomori, SI. The glycosynapse. Proc Natl Acad Sci USA (99): 225-232, 2002.2) Tanaka, KA, Suzuki, KG, Shirai, YM, Shibutani, ST, Miyahara, MS, Tsuboi, H, Yahara, M, Yoshimura, A, Mayor, S, Fujiwara, TK & Kusumi, A. Membrane molecules mobile even after chemical fixation. Nat Methods (7): 865-866, 2010.3) Komura, N, Suzuki, KGN, Ando, H, Konishi, M, Koikeda, M, Imamura, A, Chadda, R, Fujiwara, TK, Tsuboi, H, Sheng, R, Cho, W, Furukawa, K, Furukawa, K, Yamauchi, Y, Ishida, H, Kusumi, A & Kiso, M. Raft-based interactions of gangliosides with a GPI-anchored receptor. Nat Chem Biol (12):402-410, 2016.4) Suzuki, KGN, Ando, H, Komura, N, Fujiwara, TK, Kiso, M & Kusumi, A. Development of new ganglioside probes and unraveling of raft domain structure by single-molecule imaging. Biochim Biophys Acta (1861):2494-2506, 2017.5) Suzuki, KGN, Ando, H, Komura, N, Konishi, M, Imamura, A, Ishida, H, Kiso, M, Fujiwara, TK & Kusumi, A. Revealing the raft domain organization in the plasma membrane by single-molecule imaging of fluorescent ganglioside analogs. Methods Enzymol (598):267-282, 2018.6) Konishi, M, Komura, N, Hirose, Y, Suganuma, Y, Tanaka, HN, Imamura, A, Ishida, H, Suzuki, KGN & Ando, H. Development of fluorescent ganglioside GD3 and GQ1b analogs for elucidation of raft-associated interactions. J Org Chem (85):15998-16013, 2020.7) Asano, S, Pal, R, Tanaka, HN, Imamura, A, Ishida, H, Suzuki, KGN & Ando H. Development of fluorescently labeled SSEA-3, SSEA-4, and Globo-H glycosphingolipids for elucidating molecular interactions in the cell membrane. Int J Mol Sci (20):6187, 2019.Development of other glycosphingolipid probesAccording to these findings, we concluded that ganglioside probes can be used for studies to unravel dynamic behaviors of gangliosides in living cell PMs. Next, based on these results, other ganglioside probes, including GD3 and GQ1b6), and globoseries glycosphingolipids, such as SSEA-3, SSEA-4, Figure 3. Single molecules of 594-GM3 and 594-GM1 were frequently and transiently recruited to CD59-clusters and CD59 homodimersand Globo-H7), have been synthesized through conjugation with ATTO594 at the terminal sugar residues. All of these glycosphingolipid probes preferentially partitioned into raft fractions. Now, studies to unravel the mechanism of regulation of receptor activity by glycosphingolpids are ongoing by single-molecule imaging. Especially, we focus on the structural change of receptors by association with gangliosides in living cell PMs. Furthermore, we are investigating how rafts regulate the interactions between receptors and gangliosides.of gangliosides with CD59 monomers, transient homodimers and clusters.49
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