Passive and active ion permeation through lipid bilayers investigated by second harmonic water imaging

Lipid membranes are complex and dynamic systems which are known to mediate signaling processes between cells and their environment. To do this multiple ion channels and pumps are involved in controlling the in- and out-flux of various ions (K+, Na+, Mg2+, Ca2+, etc.) and help maintain and regulate a concentration gradient of ions across the membrane. Additionally, membranes are semi-permeable for some species including water and even ions. However, molecular level information about passive and active transport of ions across lipid membranes is either missing or incomplete and ignores membrane hydration without which a membrane would not self-assemble.In this project, we use high-throughput wide-field second harmonic (SH) microscopy to learn about the molecular level structure of membrane interfaces during those processes by imaging the non-resonant response of interfacial water. We show that this technique is extremely sensitive to the transmembrane distribution of interfacial ions which disturb membrane hydration and thus change SH contrast. We apply this technique to probe the molecular structure(orientational order of water, strength of ion binding) at the interfaces of freestanding lipid bilayers and giant unilamellar vesicles (GUVs). We start with SH imaging of operational voltage-gated alamethicin ion channels in freestanding lipid membranes surrounded by an aqueous solution on either side. We observe a change in SH intensity upon channel activation that is traced back to a change in the orientational distribution of water molecules caused by transported ions. Of the transported ions, a fraction of 10-4 arrives at the hydrated membrane interface, leading to interfacial electrostatic changes on the time scale of a second. Later on, in order to improve SH contrast from low-emitting samples (such as interfacial water) and thus be able to study even more fundamental and delicate surface processes, we develop and build a femtosecond optical parametric amplifier. This laser system generates a tunable output in the range from 670 nm to 1000 nm at a 1 MHz repetition rate with a minimum pulse duration of 23 fs at 900 nm. Comparing our current femtosecond laser system with the new one at equal fluences we experimentally observe a 30 times improvement in SH contrast of low-emitting samples.Then, we present a new approach of label-free second harmonic imaging of GUV hydration which directly reports on the cross-membrane distribution of divalent cations bound to negatively charged lipid headgroups. We use this property to study passive transport of Ca2+ ions through lipid membranes. By varying the hydrophobic core of the bilayer, we observe Ca2+ translocation for mono-unsaturated (DOPC:DOPA) membranes which is reduced upon adding cholesterol. A complete inhibition of translocation is observed for branched (DPhPC:DPhPA) and poly-unsaturated (SLPC:SLPA) lipid membranes. In order to gain more insight into the mechanism of divalent cation transport, we perform a series of experiments with different ions (Mg2+, Ba2+, Ca2+and Cu2+). Surprisingly, we observe translocation of Ba2+, Ca2+ and Cu2+ ions through unsaturated DOPC:DOPA membranes which fully neutralize the inner membrane leaflet. Translocation time increases in the order Cu2+< Ca2+< Ba2+, while Mg2+ ions do not show any permeation. The observed trend in passive transport correlates with a trend in ion binding strength measured on impermeable GUVs with the same headgroup composition.

Water as a contrast agent for imaging interfacial structure and ion transport in giant vesicles

David Roesel

Hydrated lipid bilayer membranes and their asymmetry play a fundamental role in living cells by maintaining and regulating concentration gradients between cells, their environment, and their compartments. They achieve this not only through various channels and transporters, but also by being inherently semi-permeable to various molecules, including water and ions. However, molecular information about passive ion transport, as well as the complex structure of membrane hydration remain elusive, mainly due to a lack of experimental methods that can access this information, and relate it to micro- and macroscopic membrane properties. In this work, we study the molecular structure of lipid bilayer membranes and ion transport across them with high throughput wide-field second harmonic (SH) microscopy by utilizing water as the contrast agent. We show that by detecting small amounts of asymmetry in the structure of interfacial water, it is possible to measure the distribution of charged species across the membrane with high sensitivity. We apply this surface-selective technique to measure ion-lipid-water interactions, track ion transport, and quantify electro-chemical surface properties at the interfaces of lipid bilayer membranes in the form of giant unilamellar vesicles (GUVs). We start by improving the throughput of wide field SH microscopy in order to probe low-asymmetry interfaces with high contrast in a label-free manner. To do this, we design a custom optical parametric amplifier (OPA) with a tunable output in the 670-1000 nm range, up to a 1 MHz repetition rate, and an ultra-short 23 fs pulse duration. We then experimentally demonstrate the achieved throughput improvement. Next, we establish a way to probe interfacial hydration of GUVs. We quantify the surface properties of vesicles composed of different ratios of zwitterionic and anionic lipids, and show that only a few percent of anionic headgroups are ionized. We also observe spatial and temporal fluctuations in surface properties, and demonstrate that these fluctuations are universally found in lipid bilayer systems.Following that, we demonstrate a direct link between membrane potential fluctuations and divalent ion transport. Molecular dynamics simulations reveal that these fluctuations reduce the free energy cost of transient pore formation and increase the ion flux across an open pore. These transient pores can act as conduits for ion transport, which we SH image for a series of divalent cations (Cu2+, Ca2+, Ba2+, Mg2+) passing through GUV membranes. Combining the experimental and computational results, we show that permeation through pores formed via an ion-induced electrostatic field is a viable mechanism for unassisted ion transport.Then, we focus on unassisted Ca2+ translocation in more detail. We vary the hydrophobic core of bilayer membranes and observe different types of behavior in high throughput wide-field SH images. Ca2+ translocation is observed through mono-unsaturated membranes, significantly reduced upon adding cholesterol, and completely inhibited for branched and poly-unsaturated membranes. We propose, using molecular dynamics simulations, that ion transport occurs through ion induced transient pores, which require non-equilibrium membrane restructuring. This results in different transport rates at different locations and suggests that the hydrophobic structure of lipids plays a much more sophisticated regulating role than previously thought.

EPFL2022

Dynamic Imaging of Lipid Membranes by Means of Water

Orly Bagunu Tarun

Lipid membranes are self-assembled structures whose composition determines the properties of membranes of cells and organelles. The molecular level understanding of lipid membranes is based on spectroscopy and MD simulations of lipid monolayer systems. As spectroscopy rely on spatial and temporal averaging and are necessarily linked to mean field models, information about the molecular interactions and their spatiotemporal evolution in real membranes is currently unavailable. In this thesis, we use high throughput wide-field second harmonic (SH) microscopy to image water-membrane interactions at sub-second time scale to follow the spatiotemporal evolution of membranes in freestanding membranes. We improve the throughput of nonlinear SH microscope by 2-3 orders and integrate the apparatus of freestanding membranes for simultaneous electrical and optical characterization of membranes. We show that the non-resonant response of water can be SH imaged on sub-second time scales and use this response as a contrast mechanism to image membrane dynamics. We investigate how oil redistributes within bilayers after formation. Using SH imaging, there is less oil present within bilayers prepared with hexadecane versus squalene. Diffusion of excess hexadecane droplets within bilayer follow directed diffusion whereas squalene show no directed motion. Hexadecane can diffuse within a single leaflet whereas squalene span both leaflets and more branched, couples to both leaflets and moves slower. We probe the diffusion of charged lipid domains at sub-second time scale and construct electrostatic potential maps of asymmetric membranes. The average membrane potential is quadratic to an applied external bias, modeled by nonlinear optical theory. We observe fluctuations in the membrane potential of ~100 mV implying that membranes are dynamic in structure and in their potential landscape. We probe the interactions of divalent cations with water and negatively charged membranes and show that Ca2+, Ba2+ and Mg2+ induce short-lived ( Mg2+ > Ba2+, for all quantities. We quantify KD and observe domain values that deviate up to 4 orders of magnitude from average KD. The transient domains exhibit potential fluctuations of up to -386 mV (dG = 28.6 kT), induce strain in the membrane resulting to fluctuations in membrane curvature. We SH image the opening and closing of voltage-gated alamethicin ion channels in freestanding membranes. The SH intensity is due to changes in the orientational distribution of water molecules induced by electric field gradients. Only 0.01 % of the transported ions arrives at the membrane interface, leading to interfacial electrostatic changes on the time scale of a second. We quantify the distribution of ion channels and observe that regions with high ion channel density exhibit a lower rate of interfacial charge build-up, likely caused by crowding. Ion transport along the membrane, thought to be involved in the propagation of action potential, is taking place over seconds. The observation suggests a more complex mechanism for the propagation of action potentials. On a fundamental level, structural and temporal heterogeneity needs to be included in biochemical, physical and molecular models of membranes.

EPFL2019

Soft matter probed by nonlinear scattering: self-assembly, interfaces, hydration, and long-range order

Jan Dedic

All organisms are made to a large extent of soft matter - macromolecules such as proteins and polysaccharides, or assemblies of small molecules such as lipids embedded in an aqueous environment. Understanding the role of order in soft matter presents a challenge for experimentalists because such systems are neither completely disordered nor perfectly crystalline. This is especially the case for the aqueous environment itself whose order fluctuates on femto- and pico-second time scales. In this thesis, we attempt to elucidate the presence, extent, changes, and implications of orientational order in several soft matter systems: from polyelectrolyte solutions to lipid membrane interfaces and supramolecular structures. To perform this task, we employ nonlinear optical techniques: second-harmonic and sum-frequency scattering. In the first two chapters, we explore the structure of water in aqueous polyelectrolyte solutions. Polyelectrolytes of high molecular mass are shown to induce orientational order in water that spans hundreds of nanometers. Using an adapted model of second-harmonic scattering from charged particles, we can explain the observed ordering in terms of weakly screened long-range electrostatic interactions that perturb the structure of water via charge-dipole interactions. Upon exchanging ordinary (light) water for heavy water, we observe a significant nuclear quantum effect in the induced order, indicating that bulk water-water correlations are enhanced as well by polyelectrolytes. In the following chapter, we exploit the same nuclear quantum effect in polyelectrolyte solutions to show that the polyelectrolyte-induced orientational order correlates with an anomalous increase in the reduced viscosity. We propose that the viscosity anomaly arises from enhanced order in a solvent which hinders the viscous flow. This observation represents a rare direct link between a microscopic property and a macroscopic observable. In the last two chapters, we change focus from homogeneous solutions to dispersions of liposomes which serve as models of biological membranes. In Chapter 5 we explore the binding of the protein alpha-synuclein to anionic vesicles from the perspective of interfacial water. We show that the adsorption of the protein to a vesicle decreases the order in the interfacial water by 30% but does not affect the surface nor the zeta potential. We propose that the changes in the structure of interfacial water can serve as a label-free probe of protein binding. In the last chapter, we investigate the supramolecular chemistry of inclusion complexes of methyl-Î²-cyclodextrin with various lipids. By combining multiple nonlinear scattering techniques, we show that the complexes self-assemble into micrometer-long directed fibers. Based on measurements of different lipids, we propose that the self-assembly process is driven by hydrogen bonding between the lipid headgroup and cyclodextrin. Lastly, we show that the long-range order of the self-assembled structure is transmitted into the structure of the hydrating water.

EPFL2020