Spatiotemporal Organization of Signaling Proteins on the Cell Membrane Studied by Spatially Patterned Supported Lipid Bilayers

Membrane proteins in the cell are dynamically organized to process the information from the environment and can precisely regulate cell signaling and functions. The spatiotemporal dynamics of proteins can be investigated by means of fluorescence microscopy to elucidate the mechanism of cell signaling. In addition, a fluid lipid bilayer on a flat glass surface with two-dimensional mobility of lipid molecules, called supported lipid bilayer (SLB), has proved a powerful tool to investigate cell-cell interactions and reconstitute membrane protein functions in vitro.The T cell is activated by an antigen presenting cell (APC) when a T cell receptor (TCR) is engaged by a major histocompatibility complex (MHC) on the APC. To precisely regulate T cell activation, the T cell organizes membrane proteins and other signaling molecules into particular spatial organization. We applied spatial patterning technologies to the SLB to probe and modulate spatiotemporal dynamics of the T cell signaling, and investigated its mechanism by fluorescence microscopy. In Chapter 2, the SLB embedded with a regular array of gold nanoparticles (nanodot array) was used to probe T cell receptor (TCR) microclusters on the T cell membrane. This nanodot array probes membrane protein assemblies below the diffraction limit of light in living cells by a mechanical means, which complements super-resolution microscopy. In Chapter 3, the spatiotemporal dynamics of Linker for Activation of T cells (LAT), another important protein in T cell signaling, was investigated by localized stimulation to the T cell using a polymer-patterned SLB. This method effectively separated the sites of T cell activation far apart from each other, and elucidated the LAT dynamics upon T cell activation more clearly than ever.ESCRT proteins play an essential role in membrane budding and scission, and it is suggested that they use membrane curvatures to regulate their functions. In Chapter 4, we made a cover glass with nano-hollows of ~100 nm in depth and ~200 nm in diameter, and investigated the interaction between the SLB on the nano-curvature and ESCRT proteins. Highly selective accumulation of ESCRTs into the part of SLB with nano-curvature was observed, which indicated the ESCRTs sense the artificial nano-curvature just as they do in vivo. This experimental platform opens up possibilities for precise kinetic studies on ESCRTs in vitro