Supported membranes

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Dynamic patterns in a supported lipid bilayer driven by standing surface acoustic waves

M. Hennig, J. Neumann, A. Wixforth, J. O. Rädler & M. F. Schneider
Lab on a Chip (2009)

In the past decades supported lipid bilayers (SLBs) have been an important tool in order to study the physical properties of biological membranes and cells. So far, controlled manipulation of SLBs is very limited. Here we present a new technology to create lateral patterns in lipid membranes controllable in both space and time. Surface acoustic waves (SAWs) are used to generate lateral standing waves on a piezoelectric substrate which create local ‘‘traps’’ in the lipid bilayer and lead to a lateral modulation in lipid concentration and membrane density. We demonstrate that pattern formation is reversible and does not affect the integrity of the lipid bilayer as shown by extracting the diffusion constant of fluid membranes. The described method could possibly be used to design switchable interfaces for the lateral transport and organization of membrane bound macromolecules to create dynamic bioarrays and control biofilm formation.

Transport, Separation, and Accumulation of Proteins on Supported Lipid Bilayers

Jürgen Neumann, Martin Hennig, Achim Wixforth, Stefan Manus, Joachim Rädler, Matthias Schneider
Nano Letters (2010)

Transport, separation, and accumulation of proteins in their natural environment are central goals in protein biotechnology. Miniaturized assays of supported lipid bilayers (SLBs) have been proposed as promising candidates to realize such technology on a chip, but a modular system for the controlled transport of membrane proteins does not exist. We demonstrate that standing surface acoustic waves drive the in-plane redistribution of proteins on planar SLBs over macroscopic distances (3.5 mm). Accumulation of proteins in periodic patterns of about 10-fold protein concentration difference is accomplished and shown to relax into the homogeneous state by diffusion. Different proteins separate in individual fractions from a homogeneous distribution and are transported and accumulated into clusters using beats. The modular planar setup has the potential of integrating other lab-on-a-chip tools, for monitoring the membrane-protein integrity or adding microfluidic features for blood screening or DNA analysis.

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Alignment and Deformation of Lipid Bilayer Domains in Vesicles Adhering to Microstructured Substrates

T. Stögbauer, M. Hennig, J. O. Rädler
Biophysical Reviews and Letters (2010)

In heterogeneous lipid membranes, the lateral organization is coupled to local curvature as the membrane bending energies depend on composition. We investigate phase separated vesicles of ternary lipid composition in contact with structured surfaces, where the distinct elastic properties of the Lo (liquid ordered) and Ld (liquid disordered) phases come into play. We show that fused silica substrates with microstructured grooves induce sorting, alignment and deformation of Lo domains in adhering vesicles. The same phenomenon is observed on flat, chemically modified substrates with alternating stripes of rough and smooth regions. In both cases it is the Lo phase which accumulates over the smooth substrate and membrane spanned groove regions respectively. Deformation of Lo domains occurs when domain diameters grow beyond the width of the microstructured stripes. Domain alignment was also observed in binary membranes featuring gel and fluid phase coexistence showing the generic character of domain sorting on microstructured surfaces.

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DNA-stretching

In the case of substrates with etched channels, we observe DNA stretching at the groove edges: The concave edges form attractive line potentials that bind the diffusing molecules after some time, thus polymer confinement to 1D.
The reason for the DNA stretching results from the geometry: Finally, molecules in a concave edge have closer contact with the positive charges within the membrane and are therefore bound tighter. Convex edges would have a contrary effect. The effect is strong enough to stretch DNA molecules to 95% of their contour length which could make applications like gene mapping possible.

Conformational dynamics of DNA electrophoresis on cationic membranes

V. Kahl, M. Hennig, B. Maier, J. O. Rädler
Electrophoresis (2009)

The conformational dynamics of DNA molecules undergoing electrophoresis on a fluid substrate-supported cationic lipid bilayer is investigated using fluorescence microscopy. At low electrophoretic velocities, drift of 2-D random coils is observed. In contrast, at velocities larger than 0.3 mm/s, the DNA molecules stretch out and assume branched configurations. The cross-over scenario is explained by the observation that cationic lipids segregate underneath the adsorbed DNA and confine the DNA to its counter charge imprint on time scales shorter than the relaxation time of the imprint. The concept of a tube-like confinement of the DNA is corroborated by the observed 1/N size dependence of the electrophoretic mobility in analogy to the biased reptation model in gels. The role of membrane defects and possible applications of membrane-based electrophoresis in microfluidic devices are discussed.

Macromolecular dynamics and adsorption

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Biopolymers have unique properties with respect to their self-assembly, conformational dynamics, transport and interactions with interfaces. Fluorescently labeled biopolymers and proteins can be studied by Quantitative Fluorescence Microscopy (QFM) and Fluorescence Correlations Spectroscopy (FCS).

We have studied the two-dimensional dynamics of DNA on supported lipid membranes using QFM and the conformational polymer dynamics of DNA in solution using FCS.One particular advantage of FCS is the fact that fluorescently labeled species can be followed in complex solution, e.g. solutions containing liposomes, blood proteins or full blood plasma. We used FCS to measure adsorption isotherms of peptides and proteins to liposomes.

Shear-Induced Unfolding and Enzymatic Cleavage of Full-Length VWF Multimers

S. Lippok, M. Radtke, T. Obser, L. Kleemeier, R. Schneppenheim, U. Budde, R. R. Netz, J. O. Rädler
Biophysical Journal (2016)

Proteolysis of the multimeric blood coagulation protein von Willebrand Factor (VWF) by ADAMTS13 is crucial for prevention of microvascular thrombosis. ADAMTS13 cleaves VWF within the mechanosensitive A2 domain, which is believed to open under shear flow. In this study, we combine fluorescence correlation spectroscopy (FCS) and a microfluidic shear cell to monitor real-time kinetics of full-length VWF proteolysis as a function of shear stress. For comparison, we also measure the Michaelis-Menten kinetics of ADAMTS13 cleavage of wild-type VWF in the absence of shear but partially denaturing conditions. Under shear, ADAMTS13 activity on full-length VWF arises without denaturing agent as evidenced by FCS and gel-based multimer analysis. In agreement with Brownian hydrodynamics simulations, we find a sigmoidal increase of the enzymatic rate as a function of shear at a threshold shear rate γ˙1/2 = 5522/s. The same flow-rate dependence of ADAMTS13 activity we also observe in blood plasma, which is relevant to predict hemostatic dysfunction.

Kinetics Models for Nanoparticles Interaction with Plasma Proteins

O. Vilanova, J. J. Mittag, P. M. Kelly, S. Milani, K. A. Dawson, J. O. Rädler and G. Franzese
ACS Nano (2016)

The interaction of nanoparticles with living cells is important with respect to both nanomedicine as well as nanotoxicity. We study various aspects of nanoparticles (NPs). The transport of NPs in biopolymer solutions, the adsorption of proteins to NPs, the uptake of NPs by cells and the interference of NPs with cellular signalling and cell death.

In a biological fluid, the surface of nanoparticles (NPs) is immediately modified by the adsorption of proteins, or other biomolecules, leading to the formation of a “protein corona”. This shell of biomolecules defines the real physicochemical properties of the nanoparticles: it determines the nanoparticles stability, and drives the uptake into the cells. Yet despite its role a comprehensive knowledge of the binding mechanisms and of the dependence of the protein corona on nanomaterial properties is still incomplete.

Here we use fluorescence correlation spectroscopy (FCS) to quantitatively analyse and model the adsorption of plasma proteins on different nanoparticle surfaces. Moreover, in order to understand the evolution of the protein corona as the nanoparticle moves from one biological environment to another, desorption kinetics in presence of competitive plasma proteins are also studied.

This work is funded by the EU-FP7 project: NanoTransKinetics

Size Distribution of the Mechanosensitive Blood Factor VWF

S. Lippok, T. Obser, J. P. Müller, V. K. Stierle, M. Benoit, U. Budde, R. Schneppenheim and J. O. Rädler
Biophysical Journal (2013)

Von Willebrand Factor (VWF) is a key protein in the force sensing cascade triggering primary hemostasis as it mediates the binding between thrombocytes and the injured vessel wall. VWF exists as multimers with variable number of dimeric subunits. Due to its shear flow sensitive structure, VWF functionality highly depends on its size. Although in recent years a general understanding of VWF function has emerged, its dynamic size regulation remains rather unexplored. We investigate the size distribution of recombinant eGFP-VWF using Fluorescence Correlation Spectroscopy (FCS). Following the size distribution of VWF in blood, we measure the in vivo and in vitro size regulation of VWF by the protease ADAMTS13, its most important size regulator. A two-focus FCS combined with a microfluidic device is established as a fast flow-through setup to investigate shear-induced changes of the VWF functionality.

This project is part of the DFG research unit FOR 1543 SHENC (Shear flow regulation of HEmostasis -bridging the gap between Nanomechanics and Clinical presentation) which is a collaboration between laboratories covering variable disciplines from medical research to theoretical biophysics: www.shenc.de