Morphometric Image Analysis of Giant Vesicles: A New Tool for Quantitative Thermodynamics Studies of Phase Separation in Lipid Membranes

Abstract

We have developed a strategy to determine lengths and orientations of tie lines in the coexistence region of liquid-ordered and liquid-disordered phases of cholesterol containing ternary lipid mixtures. The method combines confocal-fluorescence-microscopy image stacks of giant unilamellar vesicles (GUVs), a dedicated 3D-image analysis, and a quantitative analysis based in equilibrium thermodynamic considerations. This approach was tested in GUVs composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-palmitoyl-sn-glycero-3-phosphocholine/cholesterol. In general, our results show a reasonable agreement with previously reported data obtained by other methods. For example, our computed tie lines were found to be nonhorizontal, indicating a difference in cholesterol content in the coexisting phases. This new, to our knowledge, analytical strategy offers a way to further exploit fluorescence-microscopy experiments in GUVs, particularly retrieving quantitative data for the construction of three lipid-component-phase diagrams containing cholesterol.

Compositional and structural characterization of monolayers and bilayers composed of native pulmonary surfactant from wild type mice

“…Removal of cholesterol changes the bilayer structural pattern [6,14] and modification of its molar ratio to higher or lower values has been shown to negatively affect the functional activity of PulS [36]. Interestingly, the molar ratio between saturated, unsaturated phospholipids and cholesterol (39:43:18 mol) in MPulS coincides with a region in the DOPC/DPPC/cholesterol phase diagram where ld/lo-like phase coexistence occurs [37][38][39]. This suggests that at quasi-equilibrium conditions, these key major lipid classes in the complex biological mixture can produce the same scaffold observed in a canonical (compositionally simpler) mixture.…”

Preparing giant unilamellar vesicles (GUVs) of complex lipid mixtures on demand: Mixing small unilamellar vesicles of compositionally heterogeneous mixtures

“…It was reported by Husen et. al [27] that the electroformation based GUVs preparation method involving dissolution of lipids in an organic solvent produce a system of GUVs with small variability in composition. Electrofusion of a calculable composition of single or two-component GUVs prepared by electroformation to prepare single GUV of three lipid components has been also reported [29], which shows improved compositional homogeneity.…”

“…al that a high yield of giant unilamellar vesicles is expedited if rehydration of dried lipids plaque is assisted by an AC electric field [26,27,16,28], the process is known as electroformation. It was reported by Husen et.…”

“…This allows for a controlled tuning of the lipid environment in GUVs without any need to prepare new SUVs samples. This particular mixture was chosen since it is the one that most model membrane studies use for investigating putative raft formation [27]. The special feature of this ternary mixture is that it represents a fluid-fluid phase separation region (liquid-ordered / liquid-disordered, l o /l d ).…”

Reconstituição da Anexina V em sistemas de lipossomos: associação com a fosfatase alcalina e correlação com estudos de biomineralização

“…propriedades físicas e biológicas de membranas lipídicas tais como elasticidade e mudanças na morfologia das vesículas (Tamba e Yamazaki, 2005;Husen et al, 2012;Dezi et al, 2013), além de possibilitar a observação de mudanças no formato e propriedades físicas em tempo real induzidas por peptídeos, proteínas e pequenas moléculas como detergentes (Sudbrack et al, 2011;Alam et al, 2012;Shimanouchi et al, 2013).…”