Line tension at lipid phase boundaries regulates formation of membrane vesicles in living cells |
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Authors: | Dina Vind-Kezunovic Claus Hélix Nielsen Urszula Wojewodzka |
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Institution: | a Department of Dermatology, Copenhagen University Hospital, Bispebjerg, DK-2400 Copenhagen NV, Denmark b Quantum Protein Centre, Department of Physics, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark c Department of Cell Ultrastructure, Polish Academy of Sciences Medical Research Center, 02-106 Warsaw, Poland |
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Abstract: | Ternary lipid compositions in model membranes segregate into large-scale liquid-ordered (Lo) and liquid-disordered (Ld) phases. Here, we show μm-sized lipid domain separation leading to vesicle formation in unperturbed human HaCaT keratinocytes. Budding vesicles in the apical portion of the plasma membrane were predominantly labelled with Ld markers 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate, 1,1′-dilinoleyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate, 1,1′-didodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate and weakly stained by Lo marker fluorescein-labeled cholera toxin B subunit which labels ganglioside GM1 enriched plasma membrane rafts. Cholesterol depletion with methyl-β-cyclodextrin enhanced DiI vesiculation, GM1/DiI domain separation and was accompanied by a detachment of the subcortical cytoskeleton from the plasma membrane. Based on these observations we describe the energetic requirements for plasma membrane vesiculation. We propose that the decrease in total ‘Lo/Ld’ boundary line tension arising from the coalescence of smaller Ld-like domains makes it energetically favourable for Ld-like domains to bend from flat μm-sized surfaces to cap-like budding vesicles. Thus living cells may utilize membrane line tension energies as a control mechanism of exocytic events. |
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Keywords: | BSA bovine serum albumin CTB-FITC cholera toxin B subunit-conjugated fluorescein isothiocyanate DiI-C12:0 1 1&prime -didodecyl-3 3 3&prime 3&prime -tetramethylindocarbocyanine perchlorate DiI-C18:0 1 1&prime -dioctadecyl-3 3 3&prime 3&prime -tetramethylindocarbocyanine perchlorate DiI-C18:2 1 1&prime -dilinoleyl-3 3 3&prime 3&prime -tetramethylindocarbocyanine perchlorate DMEM Dulbecco's modified Eagle's medium EFEM embedment-free electron microscopy GFP green fluorescent protein GUV giant unilamellar vesicle Ld liquid-disordered Lo liquid-ordered MβCD methyl-β-cyclodextrin PBS phosphate buffered saline PH pleckstrin homology PIP2 phosphatidylinositol 4 5-bisphosphate PLC phospholipase C SEM scanning electron microscopy TEM transmission electron microscopy |
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