| Spatial reorganization of membrane proteins is emerging as a critical method by which cells modulate signaling. However, difficulty remains in the design of experimental systems to explore the dynamics of such reaction networks quantitatively. Silica-supported lipid bilayers (SLBs) are ideally suited to this task, providing well-controlled, biofunctional surfaces that maintain the hallmark two-dimensional fluidity of cellular membranes. We seek to expand the utility of SLBs as platforms for biological experimentation by developing tools for the straightforward incorporation, manipulation and measurement of biomolecules in fluid lipid layers.;Here we describe methods of forming and characterizing SLBs, and demonstrate the efficacy of nickel-chelating lipids for reversibly tethering polyhistidine-tagged proteins to bilayers in a fluid manner. With a combination of experimentation and kinetic modeling, we reveal the non-equilibrium behavior that governs adsorption in such systems, and establish guidelines for the efficient design of surfaces with known protein density.;We then employ surfaces functionalized with polyhistidine-tagged proteins to investigate protein sorting in the immunological synapse. After briefly outlining the historical development of model membrane systems as tools for studying T-cell activation, we form and characterize hybrid immunological synapses between live T-cells and protein-functionalized SLBs. With novel fluorescent fusion proteins we track the dynamics of synaptic assembly, and show that micron-scale sorting in the synapse arises from differential force coupling to the actin cytoskeleton. Artificially cross-linking proteins on the T-cell surface biases their distribution towards the center, suggesting that the radial position of proteins in the mature synapse is determined by their clustering state, and thus, the valency of their attachment to centripetal actin flow.;The tools developed here are widely applicable to problems in T-cell signaling and we propose several directions for future experimentation. |
