New probes for optical switching of protein interactions and protein fluorescence. Cell protrusion is characterized by spatial and temporal fluctuations in the concentrations, interactions and activities of membrane receptors, signaling proteins and 2nd messengers that include PDGF receptor, Ca2+, PIP2/PIP3, CapG and the barbed end of the actin filament. These molecular events are coupled to the rapid polymerization of actin filaments at sites close to the activated receptor. Efforts to elucidate the mechanism of cell protrusion are made difficult, in part, because of the paucity of probes to map specific interactions at the barbed end, and the need for specialized techniques to rapidly map, manipulate and measure the interactions and activities of specific signaling molecules and their target proteins in live cells. We describe a technology/hypothesis-driven approach to meet the research objective that introduces new optical probes including optical switches and imaging techniques that will be used to establish how molecular signals generated during receptor-mediated signaling are coupled to the spatial and temporal regulation of the CapG-barbed end complex, actin polymerization and cell protrusion. The proposal has four specific aims:
Aim 1 : To design and characterize new reactive optical switches and their conjugates.
Aim 2 : To design, synthesize and characterize optical switches to reversibly modulate cell Ca2+.
Aim 3 : To understand how a change in cell Ca2+ is coupled to the regulation of free barbed ends.
Aim 4 : To establish the role of PIP2/PIP3- dependent uncapping of barbed-ends during cell protrusion. The feasibility of the proposed studies is demonstrated through key preliminary achievements and recent publications. We believe that the unique properties of the new optical switches and associated technologies introduced in this proposal will spur the development of new microscope techniques and advance mechanism-driven studies of complex and dynamic processes in cell and molecular physiology. ? ? ?
