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It has been proposed that adaptation to high temperature involved the synthesis of monolayer-forming ether phospholipids. Recently, a novel membrane architecture was proposed to explain the membrane stability in polyextremophiles unable to synthesize such lipids, in which apolar polyisoprenoids populate the bilayer midplane and modify its physico-chemistry, extending its stability domain. Here, we have studied the effect of the apolar polyisoprenoid squalane on a model membrane analogue using neutron diffraction, SAXS and fluorescence spectroscopy. We show that squalane resides inside the bilayer midplane, extends its stability domain, reduces its permeability to protons but increases that of water, and induces a negative curvature in the membrane, allowing the transition to novel non-lamellar phases. This membrane architecture can be transposed to early membranes and could help explain their emergence and temperature tolerance if life originated near hydrothermal vents. Transposed to the archaeal bilayer, this membrane architecture could explain the tolerance to high temperature in hyperthermophiles which grow at temperatures over 100 °C while having a membrane bilayer. The induction of a negative curvature to the membrane could also facilitate crucial cell functions that require high bending membranes., By studying the properties of a synthetic membrane mimicking an archaeal membrane and containing the apolar polyisoprenoid squalane, Salvador-Castell et al. show that lipid bilayer stability and functionality are shifted to higher temperatures under high pressure. These findings support the view that apolar lipids may constitute an adaptative route to high-temperature tolerance in archaeal hyperthermophiles.

International audience; Archaea are known to inhabit some of the most extreme environments on Earth. The ability of archaea possessing membrane bilayers to adapt to high temperature (>85 • C) and high pressure (>1,000 bar) environments is proposed to be due to the presence of apolar polyisoprenoids at the midplane of the bilayer. In this work, we study the response of this novel membrane architecture to both high temperature and high hydrostatic pressure using neutron diffraction. A mixture of two diether, phytanyl chain lipids (DoPhPC and DoPhPE) and squalane was used to model this novel architecture. Diffraction data indicate that at high temperatures a stable coexistence of fluid lamellar phases exists within the membrane and that stable coexistence of these phases is also possible at high pressure. Increasing the amount of squalane in the membrane regulates the phase separation with respect to both temperature and pressure, and also leads to an increase in the lamellar repeat spacing. The ability of squalane to regulate the ultrastructure of an archaea-like membrane at high pressure and temperature supports the hypothesis that archaea can use apolar lipids as an adaptive mechanism to extreme conditions.

Most of Earth’s biotopes are under extreme environmental conditions, e.g. distant from the optimal life conditions for humans. Nevertheless, a large biological diversity of organisms, the extremophiles, inhabit such environments. For instance, organisms living at deep-sea hydrothermal vents experience temperatures above 100°C, high concentrations of reduced metals, the absence of oxygen and high hydrostatic pressures. To date, there is no understanding of the molecular mechanisms enabling them to sustain such extreme conditions. The cell membrane is particularly sensitive to external conditions, but at the same time must maintain specific physical properties, such as fluidity and permeability, to preserve the integrity and functionality of the cell. This thesis work seeks to understand how the lipid bilayer can remain functional at high temperatures and high pressures and thus can allow life under extreme conditions. This work establishes novel membrane components, apolar lipids from the polyisoprenoid’s family, which play the role of membrane regulators and confer stability to the lipid bilayer, along with dynamism and heterogeneity, which are essential properties for an optimal function of the membrane. Apolar lipids are localized in the midplane of the lipid bilayer and adjust membrane curvature and permeability. Because of such specific localization, apolar lipids reduce proton membrane permeability at high hydrostatic pressures. They establish functional domains with differentiated functions that could favour the insertion of specific membrane proteins and hence could enable essential cell functions that require highly curved membrane domains, such as fusion and fission. All these results demonstrate experimentally a novel cell membrane architecture for extremophiles in which the presence and the quantity of polyisoprenoids play a key role in cell membrane adaptation and constitute a novel adaptation route to extreme conditions applicable to the origin of life., La majorité des biotopes terrestres se trouve sous des conditions environnementales dites extrêmes, c'est-à-dire éloignées des conditions optimales de vie des humains. Néanmoins, une grande variété biologique d’organismes biologiques y habite, i.e. les extrémophiles. Par exemple, il existe des organismes vivants adaptés aux conditions des sources hydrothermales de l'océan profond: températures supérieures à 100°C, fortes concentrations en métaux réduits, absence d'oxygène, hautes pressions hydrostatiques, sans que l'on comprenne les mécanismes moléculaires leur permettant de résister à de telles conditions. La membrane cellulaire est un des constituants cellulaires le plus sensible aux conditions externes, mais en même temps, elle doit maintenir des caractéristiques physicochimiques très spécifiques, en terme de rigidité et de perméabilité, pour rester fonctionnelle. Ce travail de thèse cherche à comprendre comment la bicouche lipidique peut demeurer fonctionnelle sous hautes températures et hautes pressions, et permettre la vie en conditions extrêmes. Ce travail identifie de nouveau constituants de la membrane, les lipides apolaires de type polyisoprènes, qui jouent le rôle de régulateurs membranaires, et confèrent à la bicouche lipidique la stabilité, mais aussi le dynamisme et l'hétérogénéité, indispensables pour une fonctionnalité optimale de la membrane. Ces lipides apolaires se placent au centre de la bicouche lipidique et modifient la courbure et la perméabilité de la membrane. En raison de leur emplacement, ils diminuent la perméabilité aux protons sous hautes pressions hydrostatiques. Ils créent des îlots fonctionnels différenciés pouvant faciliter l’insertion de certaines protéines membranaires ou l’accomplissement des fonctions essentielles au cycle cellulaire comme la fusion et la fission qui requièrent une forte courbure. L'ensemble des résultats valide expérimentalement une nouvelle architecture membranaire chez les extrémophiles où la présence et la quantité de lipides apolaires jouent un rôle fondamental, et représente une voie nouvelle d'adaptation aux conditions extrêmes applicable à l'origine de la vie.

Most of Earth’s biotopes are under extreme environmental conditions, e.g. distant from the optimal life conditions for humans. Nevertheless, a large biological diversity of organisms, the extremophiles, inhabit such environments. For instance, organisms living at deep-sea hydrothermal vents experience temperatures above 100°C, high concentrations of reduced metals, the absence of oxygen and high hydrostatic pressures. To date, there is no understanding of the molecular mechanisms enabling them to sustain such extreme conditions. The cell membrane is particularly sensitive to external conditions, but at the same time must maintain specific physical properties, such as fluidity and permeability, to preserve the integrity and functionality of the cell. This thesis work seeks to understand how the lipid bilayer can remain functional at high temperatures and high pressures and thus can allow life under extreme conditions. This work establishes novel membrane components, apolar lipids from the polyisoprenoid’s family, which play the role of membrane regulators and confer stability to the lipid bilayer, along with dynamism and heterogeneity, which are essential properties for an optimal function of the membrane. Apolar lipids are localized in the midplane of the lipid bilayer and adjust membrane curvature and permeability. Because of such specific localization, apolar lipids reduce proton membrane permeability at high hydrostatic pressures. They establish functional domains with differentiated functions that could favour the insertion of specific membrane proteins and hence could enable essential cell functions that require highly curved membrane domains, such as fusion and fission. All these results demonstrate experimentally a novel cell membrane architecture for extremophiles in which the presence and the quantity of polyisoprenoids play a key role in cell membrane adaptation and constitute a novel adaptation route to extreme conditions applicable to the origin of life.; La majorité des biotopes terrestres se trouve sous des conditions environnementales dites extrêmes, c'est-à-dire éloignées des conditions optimales de vie des humains. Néanmoins, une grande variété biologique d’organismes biologiques y habite, i.e. les extrémophiles. Par exemple, il existe des organismes vivants adaptés aux conditions des sources hydrothermales de l'océan profond: températures supérieures à 100°C, fortes concentrations en métaux réduits, absence d'oxygène, hautes pressions hydrostatiques, sans que l'on comprenne les mécanismes moléculaires leur permettant de résister à de telles conditions. La membrane cellulaire est un des constituants cellulaires le plus sensible aux conditions externes, mais en même temps, elle doit maintenir des caractéristiques physicochimiques très spécifiques, en terme de rigidité et de perméabilité, pour rester fonctionnelle. Ce travail de thèse cherche à comprendre comment la bicouche lipidique peut demeurer fonctionnelle sous hautes températures et hautes pressions, et permettre la vie en conditions extrêmes. Ce travail identifie de nouveau constituants de la membrane, les lipides apolaires de type polyisoprènes, qui jouent le rôle de régulateurs membranaires, et confèrent à la bicouche lipidique la stabilité, mais aussi le dynamisme et l'hétérogénéité, indispensables pour une fonctionnalité optimale de la membrane. Ces lipides apolaires se placent au centre de la bicouche lipidique et modifient la courbure et la perméabilité de la membrane. En raison de leur emplacement, ils diminuent la perméabilité aux protons sous hautes pressions hydrostatiques. Ils créent des îlots fonctionnels différenciés pouvant faciliter l’insertion de certaines protéines membranaires ou l’accomplissement des fonctions essentielles au cycle cellulaire comme la fusion et la fission qui requièrent une forte courbure. L'ensemble des résultats valide expérimentalement une nouvelle architecture membranaire chez les extrémophiles où la présence et la quantité de lipides apolaires jouent un rôle fondamental, et représente une voie nouvelle d'adaptation aux conditions extrêmes applicable à l'origine de la vie.

Archaea, the most extremophilic domain of life, contain ether and branched lipids which provide extraordinary bilayer properties. We determined the structural characteristics of diether archaeal-like phospholipids as functions of hydration and temperature by neutron diffraction. Hydration and temperature are both crucial parameters for the self-assembly and physicochemical properties of lipid bilayers. In this study, we detected non-lamellar phases of archaeal-like lipids at low hydration levels, and lamellar phases at levels of 90% relative humidity or more exclusively. Moreover, at 90% relative humidity, a phase transition between two lamellar phases was discernible. At full hydration, lamellar phases were present up to 70ᵒC and no phase transition was observed within the temperature range studied (from 25 &deg, C to 70 &deg, C). In addition, we determined the neutron scattering length density and the bilayer&rsquo, s structural parameters from different hydration and temperature conditions. At the highest levels of hydration, the system exhibited rearrangements on its corresponding hydrophobic region. Furthermore, the water uptake of the lipids examined was remarkably high. We discuss the effect of ether linkages and branched lipids on the exceptional characteristics of archaeal phospholipids.