Invisible molecules show the truth: cells don't break even

The lipid membranes surrounding human cells are known to be asymmetric in terms of their molecular composition. The localisation of cholesterol within these membranes has, however, been difficult to pinpoint until recently. Using neutron scattering and selective deuteration, researchers were able to tackle this challenge and to provide deep insights into the complexity of biological membranes. This knowledge can help design articificial membranes for drug delivery and facilitate the handling of demanding proteins.

The human body is made up of a breathtaking 30 trillion cells. In order to protect the complex metabolic processes taking place in each one of them, the cells are surrounded by two layers consisting of a variety of fatty-acid containing molecules (also referred to as a "lipid bilayer membrane"). While one of these layers faces the inner side of the cells, the other is exposed to their surroundings. The layers are asymmetric: some of the fatty molecules are found preferentially in the inner bilayer and others are predominantly found in the outer one.

This asymmetry is highly physiologically relevant: the presence of certain fats in the outer layer plays a crucial role in processes such as blood coagulation and immune responses. In the human body, the asymmetry is actively maintained by biologically active proteins (enzymes) and by passive movement of the lipids within the lipid layers. While the concept of membrane asymmetry has been known for decades, the localisation of a certain lipid - cholesterol, an essential determinant of cell membrane fluidity - has been more difficult to assess.

This question was tackled by researchers from the University of Illinois at Chicago, the University of Wisconsin at Madison and the ILL. The team produced small, spherical assemblies of lipid bilayers ("vesicles") which are often used to mimic living cells. In a first step, the researchers investigated vesicles made of an unsaturated fat and cholesterol by nuclear magnetic resonance (NMR), observing an even (symmetrical) distribution of cholesterol between the two bilayers. When a saturated fat was added to the mixture, the team found an overwhelming majority - 90% - of the cholesterol to be located within the inner leaflet. Moreover, they showed that unsaturated and saturated lipids^* distributed unevenly between the two leaflets, with the saturated lipids preferentially occupying the same leaflet as cholesterol.

This surprising result was further investigated by small-angle neutron scattering (SANS) on the ILL instrument D22. "In SANS, we direct a beam of neutrons at our samples and detect how the neutrons are scattered as a function of the scattering angle", explains Lionel Porcar, the main responsible of D22 and the ILL local contact for this study. "The angle-dependent scattering provides us with information on the structure and the molecular properties of the samples."

In order to selectively study the location of the different lipids in the vesicles, the team relied on a sophisticated technique: selective deuteration of the molecules involved. "By exchanging some of the hydrogen atoms in our lipids by deuterium, its heavier isotopic "twin", we can make them more or less visible to neutrons", explains Krishna C. Batchu, one of the ILL's deuteration experts. "Here, this technique allowed us to highlight cholesterol while making the other fatty molecules invisible (and vice versa), thereby identifying their exact locations." SANS confirmed the asymmetry of cholesterol in the presence of saturated lipids. Furthermore, in combination with NMR, a detailed quantification of the partitioning of some of the lipid molecules was possible.

"The exciting part about our work is that we revealed an asymmetric partitioning of lipids at physiological temperatures, where we actually expected all components to be symmetrical across the membrane. Previously, it was thought that the active transport of lipids by proteins was the sole mechanism responsible for this asymmetric arrangement", says Ursula Perez-Salas, the corresponding author of the publication. "We also showed that the passive diffusion time of cholesterol between membrane leaflets is slower than what had been found by all-atom simulations by at least two orders of magnitude. Our findings contradict current working hypotheses in the field of lipid research, which may lead to a revision of some notions."

(Text by Olga Matsarskaia)

This study highlights the unique strength of neutrons and selective deuteration in studying intricate biological processes and the importance of these techniques in advancing biomedical science. In particular, the results presented here will allow to design membranes with specific lipid asymmetries that may be of use for the encapsulation of drugs. Such membranes can also be useful in the handling of membrane proteins with asymmetric domains.

"The ILL's diverse and state-of-the-art instrument suite and the excellent on-site combination of neutron science and cutting-edge laboratory and deuteration expertise make it a fantastic environment for such complex experiments", says Ursula Perez-Salas, the corresponding author of the study. "Despite the large distance, we therefore very much cherish our long-standing transatlantic collaboration."

* Unsaturated lipids contain at least one double bond between neighboring carbon atoms; saturated lipids do not contain double bonds.