Investigation of Nuclear Envelope Budding Molecular Mechanisms during Synaptogenesis

突触发生过程中核膜出芽分子机制的研究

• 批准号: 1557975
• 负责人: Sean Speese
• 金额: $ 59.99万
• 依托单位: Oregon Health & Science University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1557975&HistoricalAwards=false
• 关键词: Investigation; Nuclear; Envelope; Budding; Molecular

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.

大脑中神经元细胞之间新连接的发展是一个关键过程，最终奠定了我们学习和记忆的能力。 这项提案的重点是一种新发现的称为核膜出芽（NEB）的过程，该过程允许细胞对其与其他细胞的连接结构进行有针对性的改变。将进行三组实验，检查（1）NEB背后的分子变化，（2）NEB完成的机械手段，以及（3）NEB在新发展的细胞与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记录新连接在活生物体中形成时的真实的发展。 这些研究将为大脑的发育和我们的经历如何导致神经元回路的变化提供新的和关键的见解。这一提议利用了成熟的果蝇神经肌肉接头（NMJ）突触生长和成熟模型，以进一步了解RNA颗粒如何形成，并随后在发育过程中运输到突触部位。 特别是，这些研究将集中在最近描述的核包膜出芽（NEB）的过程中，核出口途径，其中包含突触后转录的RNA颗粒从突触后肌肉细胞核释放。在目的1中，我们将研究如何Pin1，一个高度保守的酶，功能通过核纤层重塑调节NEB。 在目标2中，我们将探讨一个非典型的核孔复合物，我们假设负载mRNA转录到RNA颗粒形成在细胞核的周边的作用。 最后，在目标3中，我们将利用一种新的工具来成像突触发生时突触后肌肉中RNA颗粒的运输动力学。 该提案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，结合体内系统的遗传易处理性，使这项研究对我们理解RNA颗粒生物学做出了重大贡献，并为NMJ发育过程中细胞核和形成突触之间的双向通信提供了令人兴奋的新机制见解。