Papers

AVS2004 Session BI-TuA: Biomembranes on a Chip

Tuesday, November 16, 2004 1:40 PM in Room 210D

Tuesday Afternoon

Start	Invited?	Item
1:40 PM		BI-TuA-2 Binding and Aggregation of α-Synuclein on Supported Lipid Bilayers J.S. Hovis, E.A. Gamble, M.C. Hull, J.-C. Rochet (Purdue University) Interest in α-synuclein was initiated with the observation that two mutations in the α-synuclein gene are linked to an early onset form of Parkinson's disease. α-Synuclein was further implicated in Parkinson's disease when the protein was found to be the main component of Lewy body inclusions in the brains of Parkinson's disease patients. In solution α-synuclein is natively unstructured while in the Lewy bodies it is primarily β-sheet in character. It has been shown that α-synuclein readily binds lipid vesicles containing negatively charged lipids and that upon binding the protein adopts an α-helical conformation. Interestingly, the aggregation of α-synuclein into β-sheet fibrils appears to be enhanced in the presence of negatively charged lipids. Due to the small size of the vesicles used in the previous studies the growth of the aggregates could not be observed directly. To provide more insight into the necessary conditions for the aggregation of α-synuclein we have observed the binding of α-synuclein to supported lipid bilayers using epi-fluorescence microscopy and infrared spectroscopy. The extent of aggregation was observed to depend on time, salt concentration, protein concentration and lipid composition. Results will be presented highlighting the necessary conditions for aggregation and comparing the conditions needed for wild-type aggregation with those of two mutant proteins (A30P and A53T) which have been linked with early onset Parkinson's.
2:00 PM		BI-TuA-3 Bioanalytics in the Nanometer and Attoliter Range H. Vogel (Swiss Federal Institute of Technology Lausanne, Switzerland) Spatial compartmentalisation is a prerequisite for the creation of living matter. Without the existence of clearly defined borders, differentiation and diversity at the cellular level would not be possible. Most scientific disciplines that deal with dissolved molecules are concerned with the same problem of subdividing solutions in miniaturised autonomous units, either to increase the functional complexity of a system, reduce reagent consumption, monitor fast chemical kinetics or even to study single-molecules. I will report on our recent progress that allows the massively parallel isolation of attoliter- sized artificial and native, cell-derived vesicles and their self-assembled positioning with 100-nm precision in ordered arrays on surfaces. The broad application for investigating (bio)chemical reactions and cellular signaling processes in individual containers by electrical and optical techniques will be discussed. The biological processes which will be presented are mediated on and across cellular membranes via transmembrane receptors such as transport-, channel-proteins or G protein-coupled receptors to mention some important examples. Our novel approaches are important for the elucidation of the molecular basis of receptor function and signal transduction processes as well as for applications in the field of screening for novel therapeutic compounds.
2:40 PM		BI-TuA-5 Construction and Characterization of Planar Lipid Bilayers Supported on Conductive Thin Polymer Films: Toward Artificial Photosynthetic Supramolecular Devices L. Wang, T. McBee, S. Marikkar, C. Ge, N. Armstrong, S. Saavedra (The University of Arizona) We are developing a new type of biomimetic photosynthetic device based on a photoactive lipid bilayer supported on a planar optical waveguide electrode overcoated with indium-tin oxide (ITO). The bilayer serves as a host for incorporation of artificial photosynthetic centers, which are prepared by Moore, Gust, and Moore (Arizona State University). A water-swollen, conductive polymer cushion is used to couple the bilayer to the ITO surface. The polymer cushion is required to planarize the ITO support and render it compatible with vesicle fusion, as well as act as a transducer of light-generated proton flux across the lipid layer, so that changes in flux can be detected both electrochemically and spectroscopically. In this presentation, we will summarize recent progress in preparation and characterization of lipid bilayers deposited on conductive polymer films composed of poly(aniline) (PANI) and poly(acrylic acid) (PAA), which are deposited by layer-by-layer self-assembly on ITO. A variety of lipid compositions, polymer compositions, and assembly conditions have been compared. An array of methods has been used to characterize these assemblies, emphasizing the diffusive properties of the lipid components, the spectroscopic and electrochemical responses of the PANI/PAA film, and the barrier properties of the lipid layer. Different lipid systems exhibit different diffusive properties; these appear to be correlated with the degree to which the potentiometric response of the PANI/PAA is blocked by lipid bilayer deposition. Egg phosphatidylcholine/cholesterol appears to form a continuous, nearly pinhole free bilayer on 2(PAA/PANI)/ITO, which is attributed to the role of cholesterol as a stabilizer in supported lipid films.
3:20 PM		BI-TuA-7 Nanoscale Dissection of a T Cell Immunological Synapse J.T. Groves (University of California, Berkeley) Coordinated rearrangement of cell membrane receptors into large-scale patterns is emerging as a broadly significant theme of intercellular signal transduction. In an effort to help unravel the mechanisms governing protein organization at intercellular synapses and the role of this organization in signal transduction, we have dissected living T cell immunolgical synapses in a hybrid live cell - supported membrane configuration. Nanometer-scale patterns of fluid lipid membranes, displaying cell recognition and signaling molecules, have been constructed on solid substrates by a combination electron-beam and scanning-probe lithographic techniques, along with membrane self assembly. When doped with appropriate proteins, supported membranes mimic and antigen presenting cell and can form synapses with living T cells. The substrate nanostructures guide the mobility of membrane-linked proteins and, correspondingly, the motion of their cognate partner proteins within living cells. A critical feature of this strategy is that proteins displayed in the supported membrane exhibit diffusive mobility. This enables formation of functional synaptic structures with living cells by permitting the necessary protein rearrangements. The manner in which precisely defined geometrical restrictions frustrate or facilitate synapse formation and signaling in living cells can be used to elucidate the mechanisms and functional consequences of molecular patterns at intercellular synapses.