Immune cells are complex and dynamic systems. A key example, T-cell activation requires the formation of an immunological synapse between the T-cell and its target. Formation and signaling across this synapse involves the spatially and temporally coordinated assembly of transmembrane receptors, lipid rafts, soluble proteins and the local reorganization of the cytoskeleton. Even subtle defects in individual molecules can disrupt this process with dire consequences for immune system function. Modern optical microscopy, in particular in its combination with genetically- encoded, fluorescence-based reporters, has been a key experimental tool to follow the molecular processes at the immunological synapse. However, until now we have been limited to observing this process. It is our goal to develop molecular tools that allow us to manipulate the immunological synapse with the same spatial and temporal resolution, with which we can now observe it. Specifically we propose to design and test genetically-encoded, light-switched versions of the Wiskott-Aldrich Syndrome Protein (WASP), a key regulator of actin skeleton reorganization at the immunological synapse. Our design is based on coupling the conformational equilibria of WASP and the small bacterial photoreceptor PYP (Photoactive Yellow Protein) so that light activation alleviates WASP's auto-inhibition and thus stimulates actin polymerization via activation of the Arp2/3 complex. The Wiskott-Alrdrich syndrome protein is a key molecular player in target recognition by immune cells, cell migration during wound healing and dendrite growth during neuronal development and remodeling. Consequently malfunction of this protein leads to a series of debilitating diseases including, but not limited to the eponymous Wiskott-Aldrich syndrome. We are proposing to develop new research tools to better understand the function of this protein and to help develop cures for the diseases caused by its malfunction. ? ? ?
