Morphology, biophysical properties and protein-mediated fusion of archaeosomes

Abstract

As variance from standard phospholipids of eubacteria and eukaryotes, archaebacterial diether phospholipids contain branched alcohol chains (phytanol) linked to glycerol exclusively with ether bonds. Giant vesicles (GVs) constituted of different species of archaebacterial diether phospholipids and glycolipids (archaeosomes) were prepared by electroformation and observed under a phase contrast and/or fluorescence microscope. Archaebacterial lipids and different mixtures of archaebacterial and standard lipids formed GVs which were analysed for size, yield and ability to adhere to each other due to the mediating effects of certain plasma proteins. GVs constituted of different proportions of archaeal or standard phosphatidylcholine were compared. In nonarchaebacterial GVs (in form of multilamellar lipid vesicles, MLVs) the main transition was detected at T(m) = 34. 2°C with an enthalpy of ΔH = 0.68 kcal/mol, whereas in archaebacterial GVs (MLVs) we did not observe the main phase transition in the range between 10 and 70°C. GVs constituted of archaebacterial lipids were subject to attractive interaction mediated by beta 2 glycoprotein I and by heparin. The adhesion constant of beta 2 glycoprotein I-mediated adhesion determined from adhesion angle between adhered GVs was in the range of 10(-8) J/m(2). In the course of protein mediated adhesion, lateral segregation of the membrane components and presence of thin tubular membranous structures were observed. The ability of archaebacterial diether lipids to combine with standard lipids in bilayers and their compatibility with adhesion-mediating molecules offer further evidence that archaebacterial lipids are appropriate for the design of drug carriers.

Figure 1. Basic archaeal lipid constituents.

Archaeol and caldarchaeaol.

Figure 2. Structural characterisation of archaeosomes.

Measurement of GVs’ size (A,B), effective angle of contact between adhered GVs (C) and GVs’ yield (D).

Figure 3. Determination of the adhesion angle.

Geometrical parameters of adhered GVs which are needed to assess the adhesion constant as described in the text.

Figure 4. Phase contrast microscope images of archaeosomes composed of different archaebacterial lipids.

GVs composed of pure archaebacterial lipids as indicated in individual panels. The arrows in panel C show relatively small crystal-like structures found at the bottom of the chamber. The lower panel D shows a larger crystal-like structure found at the bottom of the observation chamber.

Figure 5. Phase contrast microscope images of archaeosomes composed of different mixtures of archaeabacterial lipids.

Archaeosomes composed of different mixtures of archaebacterial lipids as indicated in individual panels. Panel A shows different regions in the same observation chamber.

Figure 6. Effect of lipid composition on GVs size.

Average size of GVs composed of pure archaebacterial lipids and mixtures of different archaebacterial lipids (A) and of mixtures of non-archaebacterial and archaebacterial lipids (B). Numbers of GVs in each experimet are indicated. Dependence of the average GVs size on the proportion of phosphatidylcholine in the lipid mixture (C). Lines represent best fits of data. Bars represent standard deviations.

Figure 7. Effect of lipid composition on GVs electroformation yield.

Yield of GVs composed of pure archaebacterial lipids and mixtures of different archaebacterial lipids (A) and of mixtures of non-archaebacterial and archaebacterial lipids (B). Dependence of yield on the proportion of phosphatidylcholine in the lipid mixture (C). Lines represent best fits of data.

Figure 8. The excess specific heat of MLVs composed of archaebacterial DPPC (aPC) and of non-archebacterial DPPC.

Red curve pertains to aPC while black curve pertains to non-archaebacterial DPPC. The inset shows an enhanced view on the peak pertaining to aPC.

Figure 9. Heparin-induced adhesion of archaebacterial GVs.

A sequence of images taken at different times showing gradual adhesion of archaebacterial GVs (composed of 25%BPG and 75% aPC) after the addition of fraxiparine to the suspension of GVs.

Figure 10. β2-GPI-induced effects on archaebacterial GVs.

The effects of β2-GPI on GVs: adhesion (A and B) and lateral segregation of membrane constituents of GVs composed of archaebacterial lipids (S-DGD-5, PGP-Me and PG in proportions 55∶30∶15) (C, marked by arrows). Due to binding of proteins to the membrane, tubular protrusions of GVs (composed of S-DGD-5, aPC, PGP-Me and PG in proportions 25∶30∶30∶15), which are otherwise too thin to be observed by the phase contrast microscope, become visible (D, marked by an arrow). The lengths of all bars are 20 µm.

Figure 11. β2-GPI-induced adhesion of non-archaebacterial GVs.

Effective angle of contact between the adhering GVs (Y) as a function of β2-GPI concentration.