Dynamic stabilisation during the drainage of thin film polymer solutions

The drainage and rupture of polymer solutions was investigated using a dynamic thin film balance. The polymeric nature of the dissolved molecules leads to significant resistance to the deformation of...

Fabrication and electromechanical characterization of free-standing asymmetric membranes

ABSTRACTAll biological cell membranes maintain an electric transmembrane potential of around 100 mV, due in part to an asymmetric distribution of charged phospholipids across the membrane. This asymmetry is crucial to cell health and physiological processes such as intracell signaling, receptor-mediated endocytosis, and membrane protein function. Experimental artificial membrane systems incorporate essential cell membrane structures, such as the phospholipid bilayer, in a controllable manner where specific properties and processes can be isolated and examined. Here, we describe a new approach to fabricate and characterize planar, free-standing, asymmetric membranes and use it to examine the effect of headgroup charge on membrane stiffness. The approach relies on a thin film balance used to form a freestanding membrane by adsorbing aqueous phase lipids to an oil-water interface and subsequently thinning the oil to form a bilayer. We validate this lipid-in-aqueous approach by analyzing the thickness and compressibility of symmetric membranes with varying zwitterionic DOPC and anionic DOPG content as compared to previous lipid-in-oil methods. We find that as the concentration of DOPG increases, membranes become thicker and stiffer. By controlling the lipid composition in the aqueous compartments on either side of the oil within the thin film balance, asymmetric membranes are then examined. Membrane compositional asymmetry is qualitatively demonstrated using a fluorescence quenching assay and quantitatively characterized through voltage-dependent capacitance measurements. Stable asymmetric membranes with DOPC on one side and DOPC/DOPG mixtures on the other were created with transmembrane potentials ranging from 10 to 25 mV. Introducing membrane charge asymmetry also increases the stiffness of the membrane. These initial successes demonstrate a viable pathway to quantitatively characterize asymmetric bilayers that can be extended to accommodate more complex membrane processes in the future.SIGNIFICANCEA defining characteristic of the cell membrane is asymmetry in phospholipid composition between the interior and exterior bilayer leaflet. Although several methods have been used to artificially create membranes with asymmetry, there has not been extensive characterization of the impact of asymmetry on membrane material properties. Here, a technique to fabricate free-standing asymmetric membranes is developed which facilitates the visualization and electromechanical characterization of the bilayer. Asymmetry in anionic phospholipid concentration is quantified by measurements of membrane capacitance at varying voltages, which also allows for determination of the membrane compressibility. This method represents an advance in the development of artificial biomembranes by reliably creating phospholipid bilayers with asymmetry and facilitates the interrogation of more complex biological processes in the future.

Measuring Protein Insertion Areas in Lipid Monolayers by Fluorescence Correlation Spectroscopy

The insertion of protein domains into membranes is an important step in many membrane remodeling processes, for example in vesicular transport. The membrane area taken up by the protein insertion influences the protein binding affinity as well as the mechanical stress induced in the membrane and thereby its curvature. Total area changes in lipid monolayers can be measured on a Langmuir film balance. Finding the area per inserted protein however proves challenging for two reasons: The number of inserted proteins must be determined without disturbing the binding equilibrium and the change in the film area can be very small. Here we address both issues using Fluorescence Correlation Spectroscopy (FCS): Firstly, by labeling a fraction of the protein molecules fluorescently and performing FCS experiments directly on the monolayer, the number of inserted proteins is determined in situ without having to rely on invasive techniques, such as collecting the monolayer by aspiration. Secondly, by using another FCS color channel and adding a small fraction of fluorescent lipids, the reduction in fluorescent lipid density accompanying protein insertion can be monitored to determine the total area increase. Here, we use this method to determine the insertion area per molecule of Sar1, a protein of the COPII complex, which is involved in transport vesicle formation, in a lipid monolayer. Sar1 has an N-terminal amphipathic helix, which is responsible for membrane binding and curvature generation. An insertion area of (3.4 ± 0.8) nm2 was obtained for Sar1 in monolayers from a lipid mixture typically used in reconstitution, in good agreement with the expected insertion area of the Sar1 amphipathic helix. By using the two-color approach, determining insertion areas relies only on local fluorescence measurements. No macroscopic area measurements are needed, giving the method the potential to be applied also to laterally heterogeneous monolayers and bilayers.Statement of SignificanceWe show that two color Fluorescence Correlation Spectroscopy (FCS) measurements can be applied to the binding of a protein to a lipid monolayer on a Langmuir film balance in order to determine the protein insertion area. One labelling color was used to determine the number of bound proteins and the other one to monitor the area expansion of the lipid monolayer upon protein binding. A strategy for the FCS data analysis is provided, which includes focal area calibration by raster image correlation spectroscopy and a framework for applying z-scan FCS and including free protein in the aqueous subphase. This approach allows determining an area occupied by a protein in a quasi-planar model membrane from a local, non-invasive, optical measurement.

Mimicking coalescence using a pressure-controlled dynamic thin film balance

A novel modified version of the thin film balance is introduced, which allows the application of complex pressure profiles in free-standing films and the study of film dynamics during both drainage and retraction.

Structure–property relations of amphiphilic poly(furfuryl glycidyl ether)-block-poly(ethylene glycol) macromonomers at the air–water interface

Structure–property relations of poly(furfuryl glycidyl ether)-block-poly(ethylene glycol) macromonomers at the air–water interface are studied with a Langmuir film balance.