title: Lysosome fusion to the cell membrane is mediated by the dysferlin C2A domain in coronary arterial endothelial cells.
abstract: Dysferlin has recently been reported to participate in cell membrane repair in muscle and other cells through lysosome fusion. Given that lysosome fusion is a crucial mechanism that leads to membrane raft clustering, the present study attempted to determine whether dysferlin is involved in this process and its related signalling, and explores the mechanism underlying dysferlin-mediated lysosome fusion in bovine coronary arterial endothelial cells (CAECs). We found that dysferlin is clustered in membrane raft macrodomains after Fas Ligand (FasL) stimulation as detected by confocal microscopy and membrane fraction flotation. Small-interfering RNA targeted to dysferlin prevented membrane raft clustering. Furthermore, the translocation of acid sphingomyelinase (ASMase) to membrane raft clusters, whereby local ASMase activation and ceramide production--an important step that mediates membrane raft clustering--was attenuated. Functionally, silencing of the dysferlin gene reversed FasL-induced impairment of endothelium-dependent vasodilation in isolated small coronary arteries. By monitoring fluorescence quenching or dequenching, silencing of the dysferlin gene was found to almost completely block lysosome fusion to plasma membrane upon FasL stimulation. Further studies to block C2A binding and silencing of AHNAK (a dysferlin C2A domain binding partner), showed that the dysferlin C2A domain is required for FasL-induced lysosome fusion to the cell membrane, ASMase translocation and membrane raft clustering. We conclude that dysferlin determines lysosome fusion to the plasma membrane through its C2A domain and it is therefore implicated in membrane-raft-mediated signaling and regulation of endothelial function in coronary circulation.
authors.affiliation: Department of Pharmacology & Toxicology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298,  USA.
authors.forename: Wei-Qing
authors.initials: WQ
authors.lastname: Han
authors.forename: Min
authors.initials: M
authors.lastname: Xia
authors.forename: Ming
authors.initials: M
authors.lastname: Xu
authors.forename: Krishna M
authors.initials: KM
authors.lastname: Boini
authors.forename: Joseph K
authors.initials: JK
authors.lastname: Ritter
authors.forename: Ning-Jun
authors.initials: NJ
authors.lastname: Li
authors.forename: Pin-Lan
authors.initials: PL
authors.lastname: Li
mesh_terms: D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000502:physiology*; D002518:Ceramides / Q000378:metabolism; D003331:Coronary Vessels / Q000166:cytology* / Q000502:physiology; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D053222:Fas Ligand Protein / Q000378:metabolism; D008247:Lysosomes / Q000502:physiology*; D021962:Membrane Microdomains / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D021381:Protein Transport; D034622:RNA Interference; D034741:RNA, Small Interfering; D015398:Signal Transduction
chemical_list: D002518:Ceramides; D053222:Fas Ligand Protein; D008565:Membrane Proteins; D009124:Muscle Proteins; D018129:Phosphatidylinositol Phosphates; D034741:RNA, Small Interfering; C037178:phosphatidylinositol 4-phosphate; D019255:NADPH Oxidases