REGULATION OF THE NEUROMUSCULAR JUNCTION BY THE CYTOSKELETON-ASSOCIATED PROTEIN, ADDUCIN

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The neuromuscular junction (NMJ) includes the motoneuron terminal, the synaptic gap and post-synaptic muscle. My research program seeks to understand how the post-synaptic region of the NMJ is constructed and is modified by synaptic activity, using the fruit fly (Drosophila) as a model. The long-term goal of my research program is focused on understanding how the cytoskeleton of the post synapse regulates the morphology and function of the NMJ as it is a protein lattice upon which critical synaptic proteins such as scaffolding molecules and neurotransmitter receptors are integrated. We have found that the cytoskeletal protein adducin (Add; known as Hts in Drosophila: Add/Hts), an actin-spectrin binding protein, regulates synapse structure at the NMJ. In Drosophila, we found that post-synaptic Add/Hts over-expression causes NMJ synaptic overgrowth and the phosphorylation of the NMJ scaffolding molecule Discs large (Dlg), a membrane-associated guanylate kinase with homology to SAP-97. We have data that Add/Hts also translocates to the nucleus, likely from the synapse, suggesting that Add/Hts is a synapto-nuclear protein.******Our short-term objectives include: ***Aim 1: To examine Add/Hts distribution at the NMJ synapse in relation to other proteins including Dlg: We hypothesize that when Add/Hts is phosphorylated following synaptic activation it translocates away from the synapse to the cell nucleus where it alters nuclear export of transcripts for several kinases (e.g. Ca2+/calmodulin-dependent protein kinase II; CaMKII) which phosphorylate Dlg leading to a delocalization of Dlg at the synapse. These actions will de-stabilize' the synapse allowing growth or retraction to occur.***Aim 2: To determine the role of Add/Hts in the cell nucleus: We will identify molecular interactions between Add/Hts and other nuclear proteins and RNA species, such as CaMKII transcripts. ***Aim 3: To explore the role of Add/Hts as a regulator of export from the nucleus: We will determine the mechanism by which Add/Hts alters the levels of specific protein kinases in the synapse including CaMKII and partitioning defective-1/ microtubule affinity-regulating kinase, both of which phosphorylate Dlg, and whether Add/Hts actions in the cell nucleus assists in the export of transcripts for these protein kinases.******This work is original and important as our previous and preliminary data point to a novel view of how cytoskeletal-associated proteins modify synaptic function. We now think that signaling information in the synapse does not stay in the synapse, but is disseminated widely in the cell, including to the nucleus, likely by Add/Hts itself, as well as other molecules. The proposed experiments provide a relatively simple, tractable model of how the functions of Add/Hts can be determined in an intact animal and lead to an understanding of how it can regulate synaptic form and function at the NMJ.