Calcium-induced lateral phase separation in phosphatidic acid/phosphatidylcholine monolayers as revealed by fluorescence microscopy

Abstract

Phase separation in mixed monolayers of phosphatidylcholine (PC) and pyrene-labeled phosphatidic acid (PA) was observed by fluorescence microscopy on an air/water interface as a function of subphase Ca2+ concentration and lateral packing pressure of the film. Below 45 mN m-1 and in the absence of Ca2+ no indications of phase immiscibility were observed. Addition of 1 mM Ca2+ caused extensive phase separation, which was evident immediately after spreading of the film. Further increase in Ca2+ concentration up to 30 mM increased the pyrene excimer intensity of the separated phosphatidic acid enriched domains. In the presence of Ca2+ (1-30 mM) and at surface pressures below 10 mN m-1 phase separation was always evident. However, as surface pressure exceeded 10 mN m-1, mixing of PC and PA occurred. Upon decompression of the film, phase separation reappeared at surface pressures close to 10 mN m-1. The surface textures of the film before and after the compression and subsequent relaxation were different. Inclusion of 30 mol% cholesterol increased the number and decreased the size of the PA domains. In films containing 50 mol% cholesterol no phase separation could be detected at the resolution available.

Absence of lipid gel-phase domains in seven mammalian cell lines and in four primary cell types

“…The behaviour vs. wavelength is qualitatively similar to that observed in the liquid-crystalline phase and quite different from that observed in the presence of coexisting domains of the two phases. Several recent articles reported on the progressive removal of phospholipid phase transition by cholesterol addition [32][33][34]. Many groups are concerned with the occurrence of phospholipid domains containing different cholesterol concentrations in model systems [21][22][23][24]35,36].…”

Binding of Adriamycin to Liposomes as a Probe for Membrane Lateral Organization

“…Ca 2ϩ ions bind to acidic phospholipids and are known to have a strong influence on their phase behavior and organization (Galla and Sackmann, 1975;Hartmann et al, 1977;Eklund et al, 1988Eklund et al, , 1991. Binding of adriamycin to LUVs (X PG ϭ 0.25) was subsequently measured at increasing [CaCl 2 ].…”

“…There is evidence of lateral heterogeneity in lipid membranes being induced by alcohol (Rowe, 1987;Mou et al, 1994). In binary membranes containing charged (anionic or cationic) species, phase separation can be induced by Ca 2ϩ (Galla and Sackmann, 1975;Eklund et al, 1988), polycations (Hartmann et al, 1977;Eklund and Kinnunen, 1986), DNA , electric fields (Lee et al, 1994;Lee and McConnell, 1995;Söderlund et al, 1997), and pH (Tilcock and Cullis, 1981). Likewise, several cationic peripheral proteins induce the segregation of acidic phospholipids .…”

Atomic View of Calcium-Induced Clustering of Phosphatidylserine in Mixed Lipid Bilayers

“…To date, most investigations of divalent cation- and protein-induced PS microdomains have either relied on perturbative methods or focused on bulk characteristics such as surface pressure (15–17). For instance, it has been demonstrated that membrane-binding proteins, such as annexin A5 and 2, in concert with plasma concentrations of Ca 2+ induce the lateral organization of anionic phospholipids such as PS, phosphatidic acid (PA) and phosphatidylinositol (4,5)-bisphosphate (PIP 2 ) (16, 18, 19). Recently, fluorescence microscopy has revealed that plasma Ca 2+ concentrations induce clustering of PIP 2 into sub-micrometer domains, but no such effect was observed for PS (20).…”