title: Sphingomyelin regulates the transbilayer movement of diacylglycerol in the plasma membrane of Madin-Darby canine kidney cells.
abstract: Diacylglycerol (DAG) is a key component in lipid metabolism and signaling. Previous model membrane studies using DAG analogs suggest their rapid membrane transbilayer movement. However, little is known about the DAG distribution and dynamics in cell membranes. Using live-cell fluorescence microscopy, we monitored the transbilayer movement of DAG with the yellow fluorescent protein-tagged C1AB domain from protein kinase C-γ (EYFP-C1AB), which selectively binds DAG. When HeLa cells were treated with Bacillus cereus phospholipase C (Bc-PLC) to produce DAG on the outer leaflet of the plasma membrane, intracellularly expressed EYFP-C1AB probe accumulated at the plasma membrane, indicating the transbilayer movement of the outer leaflet DAG to the inner leaflet. This Bc-PLC-induced translocation of EYFP-C1AB probe to the plasma membrane was not observed in the sphingolipid-enriched plasma membrane of Madin-Darby canine kidney cells, but was recovered after cell treatment with sphingomyelinase or preincubation with an inhibitor of sphingolipid biosynthesis. The inhibitory effect of sphingomyelin (SM) on the transbilayer movement of DAG was reproduced in model membranes using a fluorescent short-chain DAG analog. These results demonstrate that the SM content on the outer leaflet regulates the transbilayer movement of DAG in the plasma membrane, thus providing new insights into the dynamics of DAG in cell pathophysiology.
authors.affiliation: Lipid Biology Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
authors.forename: Yoshibumi
authors.initials: Y
authors.lastname: Ueda
authors.forename: Asami
authors.initials: A
authors.lastname: Makino
authors.forename: Kotono
authors.initials: K
authors.lastname: Murase-Tamada
authors.forename: Shota
authors.initials: S
authors.lastname: Sakai
authors.forename: Takehiko
authors.initials: T
authors.lastname: Inaba
authors.forename: Françoise
authors.initials: F
authors.lastname: Hullin-Matsuda
authors.forename: Toshihide
authors.initials: T
authors.lastname: Kobayashi
mesh_terms: D000818:Animals; D001409:Bacillus cereus / Q000201:enzymology; D001665:Binding Sites / Q000235:genetics; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D003016:Clostridium perfringens / Q000201:enzymology; D004075:Diglycerides / Q000378:metabolism*; D004285:Dogs; D004910:Erythrocyte Membrane / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008563:Membrane Lipids / Q000378:metabolism; D018613:Microscopy, Confocal; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism; D013109:Sphingomyelins / Q000378:metabolism*; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: C011439:1,2-diacylglycerol; D004075:Diglycerides; D008051:Lipid Bilayers; D008164:Luminescent Proteins; D008563:Membrane Lipids; D013109:Sphingomyelins; C087857:protein kinase C gamma; D011493:Protein Kinase C; D010738:Type C Phospholipases; D013108:Sphingomyelin Phosphodiesterase
keywords: C1AB domain; ceramides; flip-flop; lipid probe