Identifying the Mechanisms and Localization of Activity-Dependent CaMKII Synthesis - Project Summary At the Drosophila neuromuscular junction (NMJ), repetitive stimulation has been shown to induce various forms of protein synthesis-dependent plasticity, including growth of immature “ghost” boutons and potentiation of miniature excitatory junction potential (mEJP) frequency. There is a concurrent increase in calcium/calmodulin-dependent protein kinase II (CaMKII) protein abundance, which is required for plasticity. The molecular mechanisms of this increase in CaMKII are not well understood, and previous studies examining this effect did not differentiate between the presynaptic and postsynaptic sides of the NMJ. The Drosophila NMJ has a wide variety of genetic tools to separate the motoneuron and muscle, making it an ideal model for separating presynaptic and postsynaptic mechanisms. Preliminary data indicate that after spaced depolarization with a high potassium solution, CaMKII levels increase in both the presynaptic and postsynaptic sides of the NMJ. Additionally, this increase was blocked by application of cycloheximide, a translation inhibitor, indicating that CaMKII accumulation is dependent on protein synthesis and is unlikely to be due to protein translocation or transport. The goal of this research proposal is to identify the molecular mechanisms and localization of CaMKII synthesis on both sides of the synapse. In the first part of Aim 1, GFP reporter constructs will be used to determine the sequences in the CaMKII 3’UTR that are required for activity- dependent translation. In the second part of Aim 1, the trans-acting proteins that bind to the UTR will be identified using RNA affinity purification. The results from these experiments will elucidate the specific cis- and trans-acting factors that are required for activity-dependent CaMKII protein synthesis, and determine whether similar mechanisms act pre- and post-synaptically. In Aim 2, Flp-Frt recombination will be used to delete either the CaMKII catalytic domain or the 3’UTR and the effect on potentiation of mEJP frequency will be determined. The excision of the domains will be done exclusively in the presynaptic or postsynaptic compartment, so that the contribution of either the motoneuron or muscle can be clearly separated. In Aim 3, the subcellular localization of newly-synthesized CaMKII will be compared to that of pre-existing CaMKII. The function of this newly-synthesized CaMKII pool is unknown, and this experiment will determine whether the newly-synthesized CaMKII is targeted to a distinct subcellular nanodomain. Ultimately, this research plan will clarify how compartment-specific CaMKII synthesis may be contributing to long-term functional changes in the synapse and may identify new avenues of research into CaMKII-dependent plasticity. While conducting this research, in-depth training will be provided in a variety of crucial skills, including research design, statistical analysis, communication skills, super-resolution microscopy, and image analysis.
