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Sci Rep. 2017 Dec 1;7(1):16801. doi: 10.1038/s41598-017-16361-x.

Emphatic visualization of sphingomyelin-rich domains by inter-lipid FRET imaging using fluorescent sphingomyelins.

Author information

1
JST-ERATO Lipid Active Structure Project, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka, 560-0043, Japan. kinoshi@chem.kyushu-univ.jp.
2
Project Research Center for Fundamental Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka, 560-0043, Japan. kinoshi@chem.kyushu-univ.jp.
3
Department of Chemistry, Faculty of Science, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395, Japan. kinoshi@chem.kyushu-univ.jp.
4
JST-ERATO Lipid Active Structure Project, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka, 560-0043, Japan.
5
Project Research Center for Fundamental Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka, 560-0043, Japan.
6
Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka, 560-0043, Japan.
7
Department of Biology, Faculty of Science, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395, Japan.
8
JST-ERATO Lipid Active Structure Project, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka, 560-0043, Japan. matsmori@chem.kyushu-univ.jp.
9
Department of Chemistry, Faculty of Science, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395, Japan. matsmori@chem.kyushu-univ.jp.
10
Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka, 560-0043, Japan. matsmori@chem.kyushu-univ.jp.

Abstract

Imaging the distribution of sphingomyelin (SM) in membranes is an important issue in lipid-raft research. Recently we developed novel fluorescent SM analogs that exhibit partition and dynamic behaviors similar to native SM, and succeeded in visualizing lateral domain-segregation between SM-rich liquid-ordered (Lo) and SM-poor liquid-disordered (Ld) domains. However, because the fluorescent contrast between these two domains depends directly on their partition ratio for the fluorescent SMs, domain-separation becomes indeterminate when the distribution difference is not great enough. In this study, we propose the use of inter-lipid Förster resonance energy transfer (FRET) imaging between fluorescent SMs to enhance the contrast of the two domains in cases in which the inter-domain difference in SM distribution is inadequate for conventional monochromic imaging. Our results demonstrate that inter-lipid FRET intensity was significantly higher in the Lo domain than in the Ld domain, resulting in a clear and distinguishable contrast between the two domains even in poorly phase-separated giant unilamellar vesicles. In addition, we show that inter-lipid FRET imaging is useful for selective visualization of highly condensed assemblies and/or clusters of SM molecules in living cell membranes. Thus, the inter-lipid FRET imaging technique can selectively emphasize the SM-condensed domains in both artificial and biological membranes.

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