Investigation of Nuclear Envelope Budding Molecular Mechanisms during Synaptogenesis

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The development of new connections between neuronal cells in the brain is a critical process which ultimately underlies our ability to learn and remember. This proposal focuses on a newly-discovered process called Nuclear Envelope Budding (NEB) that allows cells to make targeted changes to the structure of their connections with other cells. Three sets of experiments will be carried out, examining (1) molecular changes that underlie NEB, (2) the mechanical means through which NEB is accomplished, and (3) how NEB actually functions in newly-developing cell-to-cell connections. An exciting and novel aspect of this latter set of experiments is the use of live cell microscopy to document the development of new connections in real time as they form in a living organism. These studies will provide novel and critical insights into how the brain develops and into how our experiences can lead to changes in our neuronal circuitry.This proposal takes advantage of the well-established Drosophila neuromuscular junction (NMJ) model of synapse outgrowth and maturation to further our understanding of how RNA granules are formed and subsequently trafficked to synaptic sites during development. In particular, these studies will focus on the recently described process of Nuclear Envelope Budding (NEB), a nuclear export pathway by which RNA granules that contain postsynaptic transcripts are released from postsynaptic muscle nuclei. In Aim 1, we will investigate how Pin1, a highly conserved enzyme, functions to regulate NEB via nuclear lamina remodeling. In Aim 2, we will explore the role of a non-canonical Nuclear Pore Complex that we hypothesize loads mRNA transcripts into RNA granules forming at the periphery of the nucleus. Finally, in Aim 3, we will take advantage of a new tool to image trafficking dynamics of RNA granules in postsynaptic muscles at the time of synaptogenesis. This proposal utilizes high resolution imaging modalities including array tomography, immunogold EM and live cell imaging. These approaches, combined with the genetic tractability of the in vivo system, poise this study to make a significant contribution to our understanding of RNA granule biology and provide exciting new mechanistic insight into the bidirectional communication that occurs between the nucleus and the forming synapse during NMJ development.