Membrane fusion: studies with a calcium-sensitive dye, arsenazo III, in liposomes.

Abstract

Fusion between vesicles, cells or organelles may be defined as confluence of 2 membrane-bound compartments without access of their solutes to external milieu. To study fusion by this criterion, the metallochromic Ca-sensitive dye, arsenazo III (AIII), partially Ca-saturated (AIII.cntdot.Ca) was trapped in one population of liposomes (phosphatidylcholine 90:dicetylphosphate 10), and ethylene glycol-bis(.beta.-aminoethyl ether)-N,N''-tetraacetate (EGTA) in a second. In such mixtures, interaction of EGTA with AIII.cntdot.Ca was measured by a large color shift from blue .fwdarw. red (decreased absorbance at 660 nm). Fusion of liposomes (but also lysis and diffusion across the membranes) was proportional to these decrements. The exogenous fusogens, lysolecithin and retinol, were added to liposomes for 5-24 h at 37.degree. C; after rechromatography, measurements were made of total dye, fraction of dye converted from AIII.cntdot.Ca to AIII and total lipid. After correction for lysis and diffusion, lysolecithin (200 .mu.g/ml) induced 23% fusion (volume of AIII liposomes confluent with EGTA liposomes) and retinol (300 .mu.g/ml) induced 15%. With 1 molar percent cortisol (a membrane stabilizer) in the liposome membranes, fusion induced by fusogens was reduced 2-fold. Neither multi- nor unilamellar liposomes fused with each other in the absence of exogenous fusogens, despite wide variations in molar lipid ratios. Results suggest that liposome-liposome fusion is a slow process requiring exogenous fusogens, which may depend on contributions of other membrane constituents to mimic closely the fusion of natural membranes.