The mechanisms which organize synaptic proteins at neurotransmitter release sites illustrate how a panoply of interacting molecules can orchestrate cell-cell communication. The knowledge of how synapses are assembled and how these molecular components are regulated is central to understanding synapse function and synapse plasticity. In recent years we have contributed to the understanding of synapse assembly and synapse plasticity by the genetic analysis of Discs-Large (DLG), a tumor suppressor and PDZ-containing protein that is essential for proper synapse assembly and function. We demonstrated that DLG is crucial for the in vivo clustering of two synaptic proteins- the Shaker K+ channel and the cell adhesion molecule Fasciclin II (Fasli). Moreover, we demonstrated that the clustering function of DLG can be dynamically regulated by neuronal activity, through Ca++/calmodulin- protein kinase II (CaMKII)-dependent phosphorylation of DLG. Although these studies have established a foundation for understanding the mechanisms of synapse development many elements responsible for this process remain to be discovered. We have now obtained evidence for two additional proteins, GUK-holder (GUKH) and SCRIBBLE (SCRIB), that are likely to function in the formation of a cytoskeletal scaffold that organizes protein complexes at the synapse. The present proposal employs Drosophila genetics to probe the mechanisms of action of these proteins at the synapse. Three sets of experiments are proposed. The first uses a genetic and molecular approach to understand the mechanism for the interactions between DLG, GUKH, and SCRIB as well as for how these interactions might be regulated. The second focuses on the functional analysis of GUKH and SCRIB, by analyzing mutations in the genes. Finally, the third approach is designed to identify synaptic SCRIB and GUKH binding partners that are likely to interface with the synaptic cytoskeleton. We expect that the proposed studies will substantially contribute to what is rapidly becoming a complex but coherent picture of how synapses are formed and are modified. Because many of these proteins are highly conserved across diverse phylogenies, our studies in Drosophila will also be relevant to vertebrates and mammals. An understanding of the factors involved in organizing a synapse will be essential to decipher the mechanisms underlying a number of neuropathologies, as well as to design strategies to repair nervous system damage after stroke, trauma, or disease.
