Hydration layer of few molecules controls lateral diffusion of lipids in biomembranes

The cellular membranes are chemically highly heterogeneous structures, whose components work in a concerted manner, facilitating a broad range of important cell functions. The organization of the cell membrane involves not only assembly into microstructure, but also intricate interactions with the hydrating water. Since defects in membrane organization translate into dysregulation in their activity, understanding the interactions of membrane components with their hydration layer is of utmost importance. As lipid bilayers curl up to form vesicles and aggregates during dehydration, preparation of membranes with decreased hydration has been very challenging. We established an unprecedented method of obtaining desiccation-tolerant supported lipid bilayer (SLB) by a slow and gradual decrease of relative humidity (RH) of the sample, which enabled us to study lipid dynamics at varying hydration conditions. Using confocal imaging and fluorescence recovery after photobleaching (FRAP) technique, we studied single component as well as phase-separated SLBs prepared in 10 mM HEPES-150 mM NaCl buffer at varying local hydration states. Our study showed that the lateral mobility of PC lipids varies accordingly with the change in local hydration state reversibly and repeatably during dehydration and rehydration cycles. A six-fold decrease in diffusion coefficient values was observed from a fully hydrated membrane to a membrane equilibrated to 0% relative humidity condition. The diffusion coefficient of PC lipid decreases steadily in the range of 90-50% RH, whereas, below ~50% RH, lipid mobility remains almost constant. The break at ~50% RH is related to the breaking of the water clathrate cage around the phosphocholine moiety of the lipid head groups which facilitates lateral diffusion of lipids acting as a lubricant. Arrhenius plots for fully hydrated and dehydrated samples revealed almost a twofold increase in activation energy of diffusion as a result of dehydration indicating the role of water molecules in the clathrate cage screening the electrostatic repulsion between the adjacent lipid head groups. Moreover, as the measured diffusion coefficient at different hydration conditions can be precisely correlated to the number of water molecules per lipid, the strong correlation between the lateral diffusion of lipids and the availability of water molecules per lipid can be used as a novel method of sensing the local hydration state of lipids in biomembranes down to 2-3 water molecules per lipid. Thus our study not only provides a clear picture of the dehydration process of lipid membranes, but the hydration dependence of lipid mobility possesses a huge potential to be utilized to investigate local hydration heterogeneity on a molecular level in biomimetic systems.

Audience take-away:

• I shall explain the importance of investigating the water-lipid interactions at lower hydrationconditions. There are several biological phenomena including cell fusion, adsorption of macromolecules, viral entry, fertilization etc., where local and transient dehydration is an important intermediate step. Understanding how lipids behave at low water availability is essential to understand the mechanism of such processes. Knowing about this motivation of my work, the audience may conceptualize their own research projects related to this.
• Preparation of desiccation-tolerant membrane has been very challenging as lipid bilayer curls up when water is removed. I shall discuss how slow and gradual dehydration keeps the structure of the membrane intact at lower hydration condition. Understanding this procedure of successful preparation of a dehydrated lipid bilayer will offer the audience huge possibilities for performing various experiments on lipid membranes at dehydrated condition.
• I shall also explain how lateral diffusion of lipid is controlled by the hydration level of the membrane. A clear molecular picture of hydration dependence of lipid mobility will help the audience to understand the chemistry behind such behavior of PC lipids. I believe that it will be very interesting for any membrane scientist.
• From my presentation, the audience will also learn about our novel approach of using the diffusion coefficient of lipids as a local hydration sensing tool in biomembranes. Scientists working on cell fusion or other biological processes where two lipid bilayer merge, will find it very helpful as they can try to use this approach in their research.
• My presentation will provide a deep insight on the hydration dependence of lipid mobility in phase-separated supported lipid bilayer. Understanding the chemistry behind hydration dependence of lipid mobility, many scientists may get interested in this topic, formulate new research questions and plan to start their own research projects on related topics. Moreover, they might be interested to collaborate with us. Faculty members can also use this knowledge for teaching their students.
• Dehydrating a lipid membrane while keeping its structure intact is very challenging. Knowing about our method of slow and gradual membrane dehydration, the audience will be able to prepare a dehydrated membrane without the addition of any external chemical and physical modifications. This will solve the problem of membrane dehydration and allow them to perform various interesting experiments on dehydrated lipid bilayers.
• My talk will enable the audience to use the diffusion coefficient of lipids to sense the hydration level in their biomembrane samples down to 2-3 water molecules per lipid. The audience may design new research experiments using the hydration sensing approach to solve their research problems.