This project has two overall goals. One is to determine how phosphoiylated inositol phospholipids (also called phosphoinositides or PPI) are distributed in lipid bilayer membranes, how the headgroups are oriented with respect to the membrane surface, under what conditions they form domains within membranes, and how the packing of these lipids affects their interactions with proteins in the underlying solution or cytosol. The second related goal is to determine the structural changes in the cytoskeletal protein gelsolin induced by binding of specific inositol lipids that cause gelsolin to lose its ability to bind actin. Unlike other inositol lipid-binding proteins such as those with pleckstrin homology or FYVE domains where PPI-binding is thought to dock the proteins without altering their structure, gelsolin and other actin binding proteins contain a distinct site where inositol lipids induce a change in secondary structure that may act as an allosteric regulatory switch to disrupt other regions of the protein. The significance of this work is that inositol lipids, or more usually the enzymes that form or degrade them, are implicated in many essential cellular functions including motility, apoptosis, and cell division. Some of the enzymes regulating PPI formation are directly implicated in a number of genetic diseases including Lowe's syndrome, or potentially function as tumor suppressors or regulators of apoptosis. PPIs are also the basis for drug design based on non-hydrolysable analogs that have potential to act as antimitogenic agents. Consequently there has been a great deal of work directed at measuring or manipulating the expression or function of proteins that produce or degrade inositol lipids or that are downstream targets of PPIs produced in response to signals generated at activated transmembrane receptors. Information about the lipids themselves: where they are located, which of the many potential downstream targets are bound to specific cellular lipids at any time, how lipid kinases are regulated by access to substrate, etc lags far behind in part because reagents and methods to localize and determine the concentrations of cellular lipids are less well developed than the methods of molecular biology. However recent studies of cell membrane strongly support the idea that lipids including PPIs are locally concentrated in specialized areas and new fluorescent PPI derivatives as well as improved methods to measure lipid membrane characteristics make it possible to complement what is known about PPI-binding proteins with molecular level information about the lipid ligands of these proteins. This project will employ a series of physical chemical methods on purified lipid systems together with biochemical studies of lipid-protein interactions, and fluorescence microscopy and genetic manipulation of cellular PPI levels to generate fundamental data concerning the properties of these unusual lipids, how they affect the function a specific actin binding protein (gelsolin) and how in a cellular context, altering PPI levels works to reorganize the cytoskeleton.
