Raft-based sphingomyelin interactions revealed by new fluorescent sphingomyelin analogs.
Sphingomyelin (SM) has been proposed to form cholesterol-dependent raft domains and sphingolipid domains in the plasma membrane (PM). How SM contributes to the formation and function of these domains remains unknown, primarily because of the scarcity of suitable fluorescent SM analogs. We developed new fluorescent SM analogs by conjugating a hydrophilic fluorophore to the SM choline headgroup without eliminating its positive charge, via a hydrophilic nonaethylene glycol linker. The new analogs behaved similarly to the native SM in terms of their partitioning behaviors in artificial liquid order-disorder phase-separated membranes and detergent-resistant PM preparations. Single fluorescent molecule tracking in the live-cell PM revealed that they indirectly interact with each other in cholesterol- and sphingosine backbone-dependent manners, and that, for ∼10-50 ms, they undergo transient colocalization-codiffusion with a glycosylphosphatidylinositol (GPI)-anchored protein, CD59 (in monomers, transient-dimer rafts, and clusters), in CD59-oligomer size-, cholesterol-, and GPI anchoring-dependent manners. These results suggest that SM continually and rapidly exchanges between CD59-associated raft domains and the bulk PM.
Lipid Active Structure Project, Exploratory Research for Advanced Technology Organization, Japan Science and Technology Agency, Osaka University, Osaka 560-0043, Japan.|Project Research Center for Fundamental Science, Osaka University, Osaka 560-0043, Japan.
Masanao
M
Kinoshita
Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto 606-8507, Japan.|The Institute for Stem Cell Biology and Regenerative Medicine, The National Centre for Biological Sciences, Bangalore 560065, India.
Kenichi G N
KG
Suzuki
Lipid Active Structure Project, Exploratory Research for Advanced Technology Organization, Japan Science and Technology Agency, Osaka University, Osaka 560-0043, Japan matsmori@chem.kyushu-univ.jp akihiro.kusumi@oist.jp.|Project Research Center for Fundamental Science, Osaka University, Osaka 560-0043, Japan.|Department of Chemistry, Graduate School of Science, Osaka University, Osaka 560-0043, Japan.
Nobuaki
N
Matsumori
Department of Chemistry, Graduate School of Science, Osaka University, Osaka 560-0043, Japan.
Misa
M
Takada
Lipid Active Structure Project, Exploratory Research for Advanced Technology Organization, Japan Science and Technology Agency, Osaka University, Osaka 560-0043, Japan.|Project Research Center for Fundamental Science, Osaka University, Osaka 560-0043, Japan.
Hikaru
H
Ano
Research Center for Environmental Genomics, Kobe University, Kobe 657-8501, Japan.
Kenichi
0000-0002-2454-6513
K
Morigaki
Cellular Informatics Laboratory, Institute of Physical and Chemical Research, Wako, Saitama 351-0198, Japan.
Mitsuhiro
M
Abe
Cellular Informatics Laboratory, Institute of Physical and Chemical Research, Wako, Saitama 351-0198, Japan.
Asami
A
Makino
UMR 7213 Centre National de la Recherche Scientifique, University of Strasbourg, Illkirch 67401, France.
Toshihide
T
Kobayashi
Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto 606-8507, Japan.
Koichiro M
KM
Hirosawa
Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto 606-8507, Japan.
Takahiro K
TK
Fujiwara
Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto 606-8507, Japan matsmori@chem.kyushu-univ.jp akihiro.kusumi@oist.jp.|Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan.|Membrane Cooperativity Unit, Okinawa Institute of Science and Technology, Okinawa 904-0412, Japan.
Akihiro
0000-0002-9558-6950
A
Kusumi
Lipid Active Structure Project, Exploratory Research for Advanced Technology Organization, Japan Science and Technology Agency, Osaka University, Osaka 560-0043, Japan.|Project Research Center for Fundamental Science, Osaka University, Osaka 560-0043, Japan.|Department of Chemistry, Graduate School of Science, Osaka University, Osaka 560-0043, Japan.
Michio
M
Murata
D000818:Animals; D018957:CD59 Antigens / Q000378:metabolism; D016466:CHO Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D003412:Cricetulus; D003902:Detergents / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D021962:Membrane Microdomains / Q000378:metabolism; D013107:Sphingolipids / Q000378:metabolism; D013109:Sphingomyelins / Q000378:metabolism*; D013552:Swine
D018957:CD59 Antigens; D003902:Detergents; D005456:Fluorescent Dyes; D017261:Glycosylphosphatidylinositols; D013107:Sphingolipids; D013109:Sphingomyelins; C051590:CD59 protein, human; D002784:Cholesterol