Non-Polar Lipids as Regulators of Membrane Properties in Archaeal Lipid Bilayer Mimics

Abstract

The modification of archaeal lipid bilayer properties by the insertion of apolar molecules in the lipid bilayer midplane has been proposed to support cell membrane adaptation to extreme environmental conditions of temperature and hydrostatic pressure. In this work, we characterize the insertion effects of the apolar polyisoprenoid squalane on the permeability and fluidity of archaeal model membrane bilayers, composed of lipid analogues. We have monitored large molecule and proton permeability and Laurdan generalized polarization from lipid vesicles as a function of temperature and hydrostatic pressure. Even at low concentration, squalane (1 mol%) is able to enhance solute permeation by increasing membrane fluidity, but at the same time, to decrease proton permeability of the lipid bilayer. The squalane physicochemical impact on membrane properties are congruent with a possible role of apolar intercalants on the adaptation of Archaea to extreme conditions. In addition, such intercalant might be used to cheaply create or modify chemically resistant liposomes (archeaosomes) for drug delivery.

Keywords: archaea; cell membrane; fluorescence; permeability; polyisoprenoids; squalane.

Figure 1

Skeletal formula of the archaeal lipids 1,2-di-O-phytanyl-sn-glycero-3-phosphocholine (DoPhPC), 1,2-di-O-phytanyl-sn-glycero-3-phosphoethanolamine (DoPhPE) and 2,6,10,15,19,23-Hexamethyl-tetracosane (squalane).

Figure 2

(a) Schematic representation of the CF efflux method, inside liposomes. CF is self-quenched but the fluorescence intensity emerges once it is released from liposomes due to a concentration decrease. The size of probes is not at scale. (b) The % CF efflux as a function of temperature and (c) CF normalized intensity versus high hydrostatic pressure applied on liposomes composed of DoPhPC:DoPhPE (9:1) in absence (black squares) or in presence of different squalane percentages: 1 mol% (red spheres), 2.5 mol% (green diamonds), 5 mol% (blue left triangles) and 10 mol% (cyan left triangles). Data represent triplicate (temperature) or duplicate (high hydrostatic pressure) measurements. No data points were excluded from the analyses.

Figure 3

(a) Schematic representation of the pyranine technique to measure pH inside vesicles. Initially, pyranine is encapsulated in the liposomes and the entrance of protons will cause a decrease on pyranine fluorescent intensity. The size of probes is not at scale. (b) Pyranine normalized intensity inside vesicles as a function of temperature and (c) hydrostatic pressure applied on liposomes of DoPhPC:DoPhPE (9:1) in absence (black squares) or in presence of diverse squalane percentages: 1 mol% (red spheres), 2.5 mol% (green diamonds), 5 mol% (blue left triangles) and 10 mol% (cyan left triangles). Data represent triplicate (temperature) or duplicate (high hydrostatic pressure) measurements. No data points were excluded from the analyses.

Figure 4

(a) Schematic representation of Laurdan position in a lipid bilayer composed by conventional diester phospholipids. While lipids are highly ordered in a gel phase, Laurdan emits in the blue region. A phase transition to a more disordered phase, the liquid-crystalline phase, induces an emission shift to green values. (b) Laurdan GP values from liposomes of DoPhPC:DoPhPE (9:1) in absence (black squares) or in presence of different squalane percentages: 1 mol% (red spheres), 2.5 mol% (green diamonds), 5 mol% (blue left triangles) and 10 mol% (cyan left triangles). (c) Schematic representation of Laurdan emplacement suggested for bilayers composed of archaeal-like diether lipids: Up to 50 °C, the fluorescent dye may adopt an “L-shape” form which would be consistent with the low Laurdan GP values. However, above 50 °C, there is a probe relocation to a deeper position that gives a change on dye emission and an increase on Laurdan GP values. Data represent triplicate measurements. No data points were excluded from the analyses. Dashed lines on (a,c) sketches represent Laurdan depth in the lipid layer.