Project Summary What spatial and temporal controls regulate subcellular macromolecular complexes at the cell surface (plasma membrane) and how such spatial and temporal controls impact various disease states, is not known. The long-term goal of this proposal is to find new targets for diagnostic and treatment approaches that address the subcellular changes that occur in macromolecular complexes at the plasma membrane in diseases ranging from cancer, ciliopathies such as polycystic kidney disease, and various neuropathies. The objective of this proposal is to assess the novel role of a key protein in tethering numerous factors to the plasma membrane to precisely control the location and timing of the formation of subcellular complexes involved in cell division, growth signaling and nerve signaling transduction. The proposal will use an innovative combination of techniques from biological, physical and biochemical sciences. These include recently- in-house-developed techniques using microfluidics and optogenetics to encapsulate cytoplasm in various sizes of our choosing in which the membrane composition, cell cycle state, and protein composition can all be precisely controlled both spatially and temporally. The proposed research is significant, because it will determine which proteins in these newly identified pathways should be therapeutic targets for which diseases and in which cell types. It is also significant because it will develop a platform that can be extended to other proteins to study their roles at the plasma membrane alone or in combination with other factors, opening new avenues for dissecting macromolecular complexes at the plasma membrane in various contexts. This work will develop foundational resources that will be used by other researchers. The results will have a positive impact immediately because they will establish a better understanding of various cancers, ciliopathies and neuropathies and lead to new diagnostic and therapeutic targets for these diseases, and long-term because they lay the groundwork to develop new techniques for dissection of a multitude of different complexes at the plasma membrane.