title: Complementary probes reveal that phosphatidylserine is required for the proper transbilayer distribution of cholesterol.
abstract: Cholesterol is an essential component of metazoan cellular membranes and it helps to maintain the structural integrity and fluidity of the plasma membrane. Here, we developed a cholesterol biosensor, termed D4H, based on the fourth domain of Clostridium perfringens theta-toxin, which recognizes cholesterol in the cytosolic leaflet of the plasma membrane and organelles. The D4H probe disassociates from the plasma membrane upon cholesterol extraction and after perturbations in cellular cholesterol trafficking. When used in combination with a recombinant version of the biosensor, we show that plasmalemmal phosphatidylserine is essential for retaining cholesterol in the cytosolic leaflet of the plasma membrane. In vitro experiments reveal that 1-stearoy-2-oleoyl phosphatidylserine can induce phase separation in cholesterol-containing lipid bilayers and shield cholesterol from cholesterol oxidase. Finally, the altered transbilayer distribution of cholesterol causes flotillin-1 to relocalize to endocytic organelles. This probe should be useful in the future to study pools of cholesterol in the cytosolic leaflet of the plasma membrane and organelles.
authors: Keenan Research Centre for Biomedical Science, St. Michael's Hospital, 209 Victoria Street, Toronto, ON, M5S 1T8, Canada.
authors: Masashi
authors: M
authors: Maekawa
authors: Keenan Research Centre for Biomedical Science, St. Michael's Hospital, 209 Victoria Street, Toronto, ON, M5S 1T8, Canada Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada Department of Surgery, University of Toronto, Toronto, ON M5T 1P5, Canada Institute of Medical Science, Faculty of Medicine, University of Toronto, Toronto, ON M5S 1A8, Canada Institute for Biomedical Engineering and Science Technology (iBEST) at Ryerson University and St. Michael's Hospital, Toronto, ON M5B 2K3, Canada fairng@smh.ca.
authors: Gregory D
authors: GD
authors: Fairn
mesh_terms: D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D015335:Molecular Probes / Q000737:chemistry* / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism*
chemical_list: D008051:Lipid Bilayers; D015335:Molecular Probes; D010718:Phosphatidylserines; D002784:Cholesterol
keywords: Biosensor; Cholesterol; Phosphatidylserine; Plasma membrane