Asymmetrical diacylglycerol dynamics on the cytosolic and lumenal sides of a single endomembrane in living cells.
The elucidation of lipid dynamics on the cytosolic and lumenal sides of a single endomembrane has been challenging in living cells because of the lack of appropriate methods. Diacylglycerol (DAG) is a lipid second messenger that is produced by enzymes that reside on both the cytosolic and lumenal sides of the endomembrane. In the present study, we attempted to observe both the cytosolic and lumenal DAG dynamics at endomembranes including the Golgi apparatus and the endoplasmic reticulum in Madin-Darby canine kidney (MDCK) cells. We developed a Förster resonance energy transfer (FRET)-based probe to detect DAG at the luminal side (lumenal DAG) of endomembranes. In combination with the FRET-based cytosolic DAG probe that has already been established, it was found that lumenal DAG is generated in a calcium-dependent manner by thapsigargin, which increases cytosolic calcium concentrations. In contrast, DAG production at the cytosolic side of endomembranes did not occur under the same experimental conditions. The thapsigargin-induced DAG generation was abolished by treatment with an inhibitor of sphingomyelin synthase (SMS) and phosphatidylcholine-specific phospholipase C (PC-PLC), which produce lumenal DAG. Thus, we have established a successful method for monitoring both cytosolic and lumenal DAG dynamics at the endomembrane in living cells.
1] Lipid Biology Laboratory, RIKEN, 2-1, Hirosawa, Wako-shi, Saitama 351-0198, Japan [2] Department of Hematology and Immunology, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Kahoku, Ishikawa 920-0265, Japan.
Yoshibumi
Y
Ueda
Department of Hematology and Immunology, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Kahoku, Ishikawa 920-0265, Japan.
Hideo
H
Ogiso
Department of General Systems Studies, School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan.
Moritoshi
M
Sato
Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan.
Yoshio
Y
Umezawa
Department of Hematology and Immunology, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Kahoku, Ishikawa 920-0265, Japan.
Toshiro
T
Okazaki
1] Lipid Biology Laboratory, RIKEN, 2-1, Hirosawa, Wako-shi, Saitama 351-0198, Japan [2] INSERM U1060-Université Lyon1, 69621 Villeurbanne, France.
Toshihide
T
Kobayashi
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism*; D004075:Diglycerides / Q000378:metabolism*; D004285:Dogs; D031541:Fluorescence Resonance Energy Transfer; D061985:Madin Darby Canine Kidney Cells
D004075:Diglycerides