Investigation of Nuclear Envelope Budding Molecular Mechanisms during Synaptogenesis

大脑中神经细胞之间新连接的发展是一个关键的过程，它最终奠定了我们学习和记忆的能力。这项提议的重点是一个新发现的过程，称为核包膜萌发(NEB)，允许细胞对其与其他细胞连接的结构进行有针对性的改变。将进行三组实验，检查(1)NEB基础上的分子变化，(2)NEB实现的机械手段，以及(3)NEB在新发展的细胞到细胞连接中的实际功能。后一组实验的一个令人兴奋和新颖的方面是使用活细胞显微镜来实时记录新连接在活生物体中形成的过程。这些研究将为大脑如何发育以及我们的经历如何导致神经元电路的变化提供新的和关键的见解。这一建议利用突触生长和成熟的果蝇神经肌肉接头(NMJ)模型来进一步理解RNA颗粒是如何在发育过程中形成并随后被输送到突触部位的。特别是，这些研究将集中在最近描述的核包膜萌发(NEB)过程，这是一种核输出途径，包含突触后转录产物的RNA颗粒从突触后肌肉核释放出来。在目标1中，我们将研究高度保守的PIN1酶是如何通过核膜重塑来调节NEB的。在目标2中，我们将探索非规范核孔复合体的作用，我们假设该复合体将mRNA转录加载到在核外围形成的RNA颗粒中。最后，在目标3中，我们将利用一个新的工具来显示突触发生时突触后肌肉中RNA颗粒的运输动力学。这项建议利用高分辨率成像方式，包括阵列断层扫描、免疫金EM和活细胞成像。这些方法，再加上体内系统的遗传可控性，使这项研究能够为我们理解RNA颗粒生物学做出重大贡献，并为NMJ发育过程中细胞核和形成突触之间的双向交流提供令人兴奋的新的机制见解。

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.