Markus Seitz

Polymer-Cushioned Bilayers. I. A Structural Study of Various Preparation Methods Using Neutron Reflectometry

This neutron reflectometry study evaluates the structures resulting from different methods of preparing polymer-cushioned lipid bilayers. Four different techniques to deposit a dimyristoylphosphatidylcholine (DMPC) bilayer onto a polyethylenimine (PEI)-coated quartz substrate were examined: 1) vesicle adsorption onto a previously dried polymer layer; 2) vesicle adsorption onto a bare substrate, followed by polymer adsorption; and 3, 4) Langmuir-Blodgett vertical deposition of a lipid monolayer spread over a polymer-containing subphase to form a polymer-supported lipid monolayer, followed by formation of the outer lipid monolayer by either 3) horizontal deposition of the lipid monolayer or 4) vesicle adsorption. We show that the initial conditions of the polymer layer are a critical factor for the successful formation of our desired structure, i.e., a continuous bilayer atop a hydrated PEI layer. Our desired structure was found for all methods investigated except the horizontal deposition. The interaction forces between these polymer-supported bilayers are investigated in a separate paper (Wong, J. Y., C. K. Park, M. Seitz, and J. Israelachvili. 1999. Biophys. J. 77:1458-1468), which indicate that the presence of the polymer cushion significantly alters the interaction potential. These polymer-supported bilayers could serve as model systems for the study of transmembrane proteins under conditions more closely mimicking real cellular membrane environments.

Structural Studies of Polymer-Cushioned Lipid Bilayers

The structure of softly supported polymer-cushioned lipid bilayers, prepared in two different ways at the quartz-solution interface, were determined using neutron reflectometry. The polymer cushion consisted of a thin layer of branched, cationic polyethyleneimine (PEI), and the bilayers were formed by adsorption of small unilamellar dimyristoylphosphatidylcholine (DMPC) vesicles. When vesicles were first allowed to adsorb to a bare quartz substrate, an almost perfect bilayer formed. When the polymer was then added to the aqueous solution, it appeared to diffuse beneath this bilayer, effectively lifting it from the substrate. In contrast, if the polymer layer is adsorbed first to the bare quartz substrate followed by addition of vesicles to the solution, there is very little interaction of the vesicles with the polymer layer, and the result is a complex structure most likely consisting of patchy multilayers or adsorbed vesicles.

Polymer-Cushioned Bilayers. II. An Investigation of Interaction Forces and Fusion Using the Surface Forces Apparatus

We have created phospholipid bilayers supported on soft polymer cushions which act as deformable substrates (see accompanying paper, Wong, J. Y., J. Majewski, M. Seitz, C. K. Park, J. N. Israelachvili, and G. S. Smith. 1999. Biophys. J. 77:1445-1457). In contrast to solid-supported membranes, such soft-supported membranes can exhibit more natural (higher) fluidity. Our bilayer system was constructed by adsorption of small unilamellar dimyristoylphosphatidylcholine (DMPC) vesicles onto polyethylenimine (PEI)-supported Langmuir-Blodgett lipid monolayers on mica. We used the surface forces apparatus (SFA) to investigate the long-range forces, adhesion, and fusion of two DMPC bilayers both above and below their main transition temperature (T(m) approximately 24 degrees C). Above T(m), hemi-fusion activation pressures of apposing bilayers were considerably smaller than for solid-supported bilayers, e.g., directly supported on mica. After separation, the bilayers naturally re-formed after short healing times. Also, for the first time, complete fusion of two fluid (liquid crystalline) phospholipid bilayers was observed in the SFA. Below T(m) (gel state), very high pressures were needed for hemi-fusion and the healing process became very slow. The presence of the polymer cushion significantly alters the interaction potential, e.g., long-range forces as well as fusion pressures, when compared to solid-supported systems. These fluid model membranes should allow the future study of integral membrane proteins under more physiological conditions.

Formation of tethered supported bilayers via membrane-inserting reactive lipids

Fluid phospholipid bilayers partially bound to a supporting polymer cushion (tethered supported membranes) have been widely discussed as model systems for studying biomembrane structure and function. We have synthesized a new isothiocyanate-functionalized lipid from dimyristoylphosphatidylethanolamine, which closely resembles naturally occurring membrane lipids. Monolayers containing the reactive lipid at the air‐water interface covalently bind to the amino functionalities of branched polyethylenimine (PEI) added to the water subphase which could be shown by infrared spectroscopy. At neutral pH, PEI is cationically charged which guarantees the transfer of a polymersupported lipid monolayer onto a mica substrate during Langmuir‐Blodgett deposition. A second layer of pure dimyristoylphosphatidylcholine can be deposited by vesicle adsorption. This system should allow the application of techniques such as the surface forces apparatus (SFA) technique to investigate interbilayer forces, membrane‐polymer interactions, and other dynamic membrane properties under near invivo conditions.

The x-ray surface forces apparatus for simultaneous x-ray diffraction and direct normal and lateral force measurements

We describe the experimental setup and principles of operation of the second-generation x-ray surface forces apparatus that allows for the first time simultaneous x-ray scattering and direct force measurements.

Synthesis and characterization of a semiflexible liquid crystalline polyester with a broad nematic region

Abstract In this paper, we report on the synthesis and detailed characterization of a new semiflexible nematic liquid crystalline polyester which could serve as a ‘model’ polyester for a variety of physical and physico-chemical investigations. The polymer is a nematic liquid over a wide temperature range–from the glass transition temperature at ∼95°C to the isotropic transition at ∼240°C. We expect this polyester to be particularly useful for studying the effect of flow on the orientation of liquid crystalline polymers, as well as the production and removal of disclinations.

Complex fluids at interfaces

Abstract The modification of the surface properties of solids by complex fluid coatings is ubiquitous in our modern world. However, many fundamental questions about the solid/coating interface remain to be answered such as: (1) how does the surface quality vary with time, (2) what affects the adhesion between the surfaces. (3) how are the properties of thin complex fluid films influenced by the proximity of a solid interface, and (4) how may we modify these properties for particular functionality. In this paper, we report the results from two separate studies aimed at answering some of these questions. In each case, neutron reflectivity results obtained using the SPEAR reflectometer at the Manuel Lujan Jr. Neutron Scattering Center were used to probe the structure of the solid/complex fluid interface.