Imaging Cu2+ binding to charged phospholipid membranes by high-throughput second harmonic wide-field microscopy

Seonwoo Lee, David Roesel, Sylvie Roke
AIP Publishing, 2021
Journal paper

Abstract

The interaction of divalent copper ions (Cu2+) with cell membranes is crucial for a variety of physiological processes of cells, such as hormone synthesis and cellular energy production. These interactions would not be possible without membrane hydration. However, the role of water has not received a lot of attention in membrane studies. Here, we use high-throughput wide-field second harmonic (SH) microscopy to study the interaction between Cu2+ and hydrated freestanding Montal-Muller lipid membranes. The symmetric lipid membranes are composed of 1,2-diphytanoyl-sn-glycero-3-phosphocholine and either 1,2-diphytanoyl-sn-glycero-3-phosphate or 1,2-diphytanoyl-sn-glycero-3-phospho L-serine and are brought into contact with divalent Cu2+, which are added to one leaflet while maintaining the ionic strength balance. We observe transient domains of high SH intensity. In these areas, Cu2+ ions bind to the charged head groups, leading to charge neutralization on one side of the membrane. This exposes the ordered water at the non-interacting side of the membrane interface, which can be used to compute the interfacial membrane potential difference. We find that the domains of lipids with phosphatidic acid head groups display a higher interfacial membrane potential than those with phosphatidylserine head groups, which converts into higher dynamic electrostatic free energies and binding constants.& nbsp;(C) 2021 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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Seonwoo Lee, David Roesel, Sylvie Roke
AIP Publishing, 2021
Journal paper

Abstract

Official source

https://infoscience.epfl.ch/record/290445?ln=en

About this result

Related concepts (13)

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Related publications (15)

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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.

EPFL2021

Water is one of the most abundant molecules in the universe. It forms a hydrogen bond network with unique structure and dynamics. Hydrogen bonding is highly relevant to many biological processes including membrane formation, protein folding, adsorption, and lubrication. However, the underlying molecular interactions within water and between water and membranes are difficult to study and hence not fully understood. In this thesis, we utilize second harmonic scattering (SHS) to investigate inhomogeneities in bulk water, probe structural changes in lipid bilayer membranes, and detect and characterize protein-membrane interactions. Firstly, we explore the structure of bulk water in comparison to a common model liquid, carbon tetrachloride. Combining SHS measurements and molecular dynamics simulations, we report on the nanoscale inhomogeneities in bulk water that occur on femtosecond timescales. We attribute the rise in the coherent second harmonic (SH) intensity to charge density fluctuations with enhanced nanoscale polarizabilities around transient voids. Even though the transient voids also exist in carbon tetrachloride, they do not show polarizability fluctuations and the SH response of carbon tetrachloride thus corresponds to that of a model isotropic liquid. Then, we study the structure of water around large unilamellar vesicles (LUVs), which serve as a model for cell membranes. We focus on the role of water during the lipid melting phase transition. Using SHS, differential scanning calorimetry, and temperature-dependent zeta-potential measurements, we investigate the structural changes of hydration shells around the vesicle membrane. We use lipids with phosphoserine, phosphocholine, and phosphate headgroups and observe different hydration behavior upon the lipid melting transition depending on the membrane composition. Next, we characterize the interface of membranes composed of lipids containing phosphocholine and phosphate headgroups in solutions with different CaCl2 concentrations. We observe that the SH intensity is decreasing with the addition of CaCl2, which we attribute to the formation of a condensed layer of Na+ counterions on the inner leaflet of the LUVs. Additionally, we extract the vesicle surface potential and second-order surface susceptibility from SHS data for membranes with different content of lipids with phosphate headgroups, and observe composition dependent behaviour which suggests structural membrane reorganization. We continue by exploring protein-membrane interactions using water as a contrast agent. We study a pore-forming toxin, perfringolysin O (PFO), known for selective insertion into cholesterol-rich membranes. Combining SHS measurements and cryo-electron microscopy, we compare the interaction between PFO and LUV membranes with a high and low cholesterol content. By obtaining a sub-picomolar insertion constant of PFO, we show that SHS can be used to determine protein-membrane binding constants in concentration ranges not accessible by conventional methods.Finally, we investigate the interaction of protein aerolysin and its mutants with lipid membranes. We observe changes in surface charge in the cap region of membrane-bound aerolysin at different pH of the surrounding solution. By combining SHS with single-channel measurements, we obtain the membrane binding constant of wild-type aerolysin and its mutants, and we dissect their pore-forming mechanism.

EPFL2022

Transient domains of ordered water induced by divalent ions lead to lipid membrane curvature fluctuations

Halil Ibrahim Okur, Sylvie Roke, Orly Bagunu Tarun

Cations influence the electrostatic, chemical and mechanical properties of lipid bilayers. Here, label-free second harmonic microscopy shows that cation-induced transient ordering of water also plays a role in driving membrane curvature fluctuations, linking molecular hydration to macroscopic properties. Cell membranes are composed of a hydrated lipid bilayer that is molecularly complex and diverse, and the link between molecular hydration structure and membrane macroscopic properties is not well understood, due to a lack of technology that can probe and relate molecular level hydration information to micro- and macroscopic properties. Here, we demonstrate a direct link between lipid hydration structure and macroscopic dynamic curvature fluctuations. Using high-throughput wide-field second harmonic (SH) microscopy, we observe the formation of transient domains of ordered water at the interface of freestanding lipid membranes. These domains are induced by the binding of divalent ions and their structure is ion specific. Using nonlinear optical theory, we convert the spatiotemporal SH intensity into maps of membrane potential, surface charge density, and binding free energy. Using an electromechanical theory of membrane bending, we show that transient electric field gradients across the membrane induce spatiotemporal membrane curvature fluctuations.

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