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

AbstractTernary 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

Diffusion Coefficient of Fluorescent Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane of Cells

Phosphatidylinositol 4,5-bisphosphate (PIP2) controls a surprisingly large number of processes in cells. Thus, many investigators have suggested that there might be different pools of PIP2 on the inner leaflet of the plasma membrane. If a significant fraction of PIP2 is bound electrostatically to unstructured clusters of basic residues on membrane proteins, the PIP2 diffusion constant, D, should be reduced. We microinjected micelles of Bodipy TMR-PIP2 into cells, and we measured D on the inner leaflet of fibroblasts and epithelial cells by using fluorescence correlation spectroscopy. The average ± SD value from all cell types was D = 0.8 ± 0.2 μm2/s (n = 218; 25°C). This is threefold lower than the D in blebs formed on Rat1 cells, D = 2.5 ± 0.8 μm2/s (n = 26). It is also significantly lower than the D in the outer leaflet or in giant unilamellar vesicles and the diffusion coefficient for other lipids on the inner leaflet of these cell membranes. The simplest interpretation is that approximately two thirds of the PIP2 on inner leaflet of these plasma membranes is bound reversibly