Line tension at lipid phase boundaries regulates formation of membrane vesicles in living cells

Ternary lipid compositions in model membranes segregate into large-scale liquid-ordered (L-o) and liquid-disordered (L-d) phases. Here, we show mu m-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 L-d 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 L-o marker fluorescein-labeled cholera toxin B subunit which labels ganglioside GM(1) enriched plasma membrane rafts. Cholesterol depletion with methyl-beta-cyclodextrin enhanced DiI vesiculation, GM(1)/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 'L-o/L-d' boundary line tension arising from the coalescence of smaller L-d-like domains makes it energetically favourable for L-d-like domains to bend from flat mu m-sized surfaces to cap-like budding vesicles. Thus living cells may utilize membrane line tension energies as a control mechanism of exocytic events.