Cluster size regulates protein sorting in the immunological synapse.

During antigen recognition by T cells, signaling molecules on the T cell engage ligands on the antigen-presenting cell and organize into spatially distinctive patterns. These are collectively known as the immunological synapse (IS). Causal relationships between large-scale spatial organization and signal transduction have previously been established. Although it is known that receptor transport during IS formation is driven by actin polymerization, the mechanisms by which different proteins become spatially sorted remain unclear. These sorting processes contribute a facet of signal regulation; thus their elucidation is important for ultimately understanding signal transduction through the T cell receptor. Here we investigate protein cluster size as a sorting mechanism using the hybrid live T cell-supported membrane system. The clustering state of the co-stimulatory molecule lymphocyte function-associated antigen-1 (LFA-1) is modulated, either by direct antibody crosslinking or by crosslinking its intercellular adhesion molecule-1 ligand on the supported bilayer. In a mature IS, native LFA-1 generally localizes into a peripheral ring surrounding a central T cell receptor cluster. Higher degrees of LFA-1 clustering, induced by either method, result in progressively more central localization, with the most clustered species fully relocated to the central zone. These results demonstrate that cluster size directly influences protein spatial positioning in the T cell IS. We discuss a sorting mechanism, based on frictional coupling to the actin cytoskeleton, that is consistent with these observations and is, in principle, extendable to all cell surface proteins in the synapse.

Discrete arrays of liquid-crystal-supported proteolipid monolayers as phantom cell surfaces.

The phospholipid bilayers of living cell membranes exist almost universally in a liquid state. This enables motion and spatial reorganization of membrane components on multiple length scales, which is an essential feature of many biological processes. There is great interest in the development of molecularly defined interfaces between synthetic materials and living cells. To this end, there is a need for solid substrate materials that can be derivatized with fluid, membrane-like interfaces. Herein, we describe array fabrication of discrete liquid-crystal areas supporting phospholipid monolayer membranes, and characterize the interactions with several different membrane surface proteins [avidin series, cholera toxin, green fluorescent protein (GFP), intercellular adhesion molecule (ICAM) and major histocompatibility complex (MHC)]. Three different linkage strategies (biotin, nickel chelating lipids complexing with histidine, and the choleratoxin binding unit (CTB) associating with G(M1) are evaluated. Additionally, experiments with live immunological T cells forming active synapses at the interface exhibit the specific nature of the surface.

Quantitative fluorescence microscopy using supported lipid bilayer standards.

Routine quantitative analysis of biomolecule surface density by fluorescence microscopy has been limited by the difficulty of preparing appropriate calibration standards that relate measured fluorescence intensity to actual surface concentration. Supported lipid bilayers are planar fluid films of uniform density and composition which can incorporate a variety of lipidated fluorophores and work well as fluorescence standards. Here, we outline a straightforward strategy to calibrate digital micrographs of fluorescent surfaces such as planar cellular junctions for comparison to supported bilayer standards. It can be implemented with standard microscopy equipment. To illustrate the advantages of this approach, we quantify cell- and bilayer-side protein density patterns in a hybrid immunological synapse between a T-cell and a supported bilayer.

Electrical manipulation of supported lipid membranes by embedded electrodes.

Alkanethiol modified gold electrodes patterned over a silica surface provided a dual hydrophobic/hydrophilic surface suitable for phospholipid monolayer and bilayer formation over the alkylated gold and glass surfaces, respectively. The phospholipid monolayer and bilayer were connected, allowing free diffusion of lipids within both leaflets of the glass-supported bilayer over the alkanethiol/gold-to-glass interface. Application of large alternating current fields to these electrodes irreversibly switched the gold electrodes to diffusion barriers. Enclosure of the electrode devices within protein barriers revealed a resting state surface potential driven reorganization of the charged fluorescent probes. Application of lower magnitude direct current fields resulted in electrophoretic redistribution of the membrane probes and electro-osmotic reorganization of membrane associated proteins.

Kinetic control of histidine-tagged protein surface density on supported lipid bilayers.

Nickel-chelating lipids are general tools for anchoring polyhistidine-tagged proteins to supported lipid bilayers (SLBs), but controversy exists over the stability of the protein-lipid attachment. Here, we show that chelator lipids are suitable anchors for building stable, biologically active surfaces but that a simple Langmuirian model is insufficient to describe their behavior. Desorption kinetics from chelator lipids are governed by the valency of surface binding: monovalently bound proteins desorb within minutes (t1/2 approximately 6 min), whereas polyvalently bound species remain bound for hours (t1/2 approximately 12 h). Evolution between surface states is slow, so equilibrium is unlikely to be reached on experimental timescales. However, by tuning incubation conditions, the populations of each species can be kinetically controlled, providing a wide range of protein densities on SLBs with a single concentration of chelator lipid. We propose guidelines for the assembly of SLB surfaces functionalized with specific protein densities and demonstrate their utility in the formation of hybrid immunological synapses.

Reversing the action of noncompetitive inhibitors (MK-801 and cocaine) on a protein (nicotinic acetylcholine receptor)-mediated reaction.

The nicotinic acetylcholine receptor (nAChR) is one of five structurally related membrane proteins required for communication between approximately 10(12) cells of the mammalian nervous system. The receptor is inhibited by both therapeutic agents and abused drugs. Understanding the mechanism of noncompetitive allosteric inhibitors of the nicotinic acetylcholine receptor is a long-standing and intensely investigated problem. During the past two decades, many attempts have been made to find drugs that prevent cocaine inhibition, including the synthesis of hundreds of cocaine analogues and derivatives, so far without success. The use of newly developed transient kinetic techniques in investigations of the inhibition of the receptor by the anticonvulsant MK-801 [(+)-dizocilpine] and the abused drug cocaine led to an inhibition mechanism not previously proposed. This mechanism indicates the properties of compounds that would prevent allosteric inhibition of the receptor and how to test for such compounds. Here we present the first evidence that small organic compounds (cocaine derivatives) exist that prevent cocaine and MK-801 inhibition of this receptor. These compounds are RTI-4229-70, a previously synthesized cocaine derivative, and based on its structure four newly synthesized cocaine derivatives, RCS-III-143, RCS-III-140A, RCS-III-218, and RCS-III-202A. Because the nAChR desensitizes rapidly, to make the required measurements a cell-flow technique with a time resolution of 10 ms was used to equilibrate BCH(3) cells containing the fetal mouse muscle-type nAChR with carbamoylcholine. The resulting whole-cell current pertaining to the nondesensitized nAChR was determined. Inhibitors and compounds that alleviate inhibition were tested by their effect on the whole-cell current.