Exocytosis fuels plasma membrane expansion in developing neurons

胞吐作用促进发育中神经元的质膜扩张

• 批准号: 10402882
• 负责人: Stephanie Gupton
• 金额: $ 38.4万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10402882
• 关键词: Affect; Area; Axon; Biochemical; Biological; Cell Shape; Cell membrane; Cells; Clathrin; Complex; Computer Vision Systems; Computer software; Data; Detection; Development; Diffusion; Distal; Endocytosis; Event; Excision; Exocytosis; Extracellular Space; Frequencies; Growth Cones; Heterogeneity; Image; In Vitro; Lateral; Machine Learning; Mediating; Membrane; Methodology; Microfluidics; Modeling; Molecular; Morphogenesis; Morphology; Nervous system structure; Neurites; Neurons; PHluorin; PTK2 gene; Pathway interactions; Phosphorylation; Population; Protein Tyrosine Kinase; Proteins; Proteomics; Publishing; Regulation of Exocytosis; Resistance; Role; Running; Rupture; SNAP receptor; Side; Stretching; Surface; Synapses; Techniques; Tetanus; Time; Total Internal Reflection Fluorescent; Variant; Vesicle; axon guidance; cell behavior; in vivo; innovation; novel; physical model; response; spatiotemporal; synaptogenesis; vesicular SNARE proteins

Project Summary During development, neurons acquire a polarized, elongated, and complex morphology, which requires a significant expansion of plasma membrane surface area. Surface increases have been estimated to reach upward of 20% per day, which far exceeds concomitant neuronal volume increases. We previously demonstrated that SNARE-mediated exocytosis is required during neuritogenesis and axon branching, presumably to provide membrane material to the expanding plasma membrane, which can only stretch ~2-3% prior to rupture. Asymmetric exocytosis has also been implicated in the attractive axonal turning responses that are critical for axon guidance. Achieving proper neuronal morphogenesis and connectivity is central to the formation of a functional nervous system. Together these factors underscore the significance of regulated exocytosis in developing neurons, even prior to synaptogenesis. Over the last 40 years, a multitude of components involved in exocytosis have been identified, although this list is not exhaustive. Further, mechanisms that regulate the mode, progression, frequency, or spatiotemporal organization of vesicle fusion with the membrane, all of which are poised to modulate neuronal morphogenesis, have not been defined. To visualize exocytic events in developing neurons, we express a pH-sensitive variant of GFP (pHluorin) attached to the lumenal side of a v-SNARE, such as VAMP2 or VAMP7, to illuminate the occurrence of fusion pore opening between the acidic vesicular lumen and the neutral extracellular space. Analysis of such images has remained a time-intensive, non-automated bottleneck, delaying understanding of this fundamental cellular behavior. We developed a fully-automated computer- vision software for the detection and analysis of VAMP-pHluorin mediated exocytic events that will quantitatively reveal the spatial and temporal organization and regulation of exocytosis in developing neurons at a level of detail previously unattainable. We exploit this innovative methodology along with unbiased proteomics, microfluidics, biochemical and cell biological approaches to investigate the relationship between exocytosis and neuronal morphogenesis and identifying the molecular mechanisms that regulate exocytosis in developing neurons.

项目概要 在发育过程中，神经元获得极化、拉长且复杂的形态，这 需要显着扩大质膜表面积。表面增加有 据估计每天可达 20% 以上，远远超过伴随的神经元 音量增加。我们之前证明了 SNARE 介导的胞吐作用是必需的 在神经发生和轴突分支期间，可能为神经细胞提供膜材料 质膜膨胀，在破裂前只能拉伸约 2-3%。不对称 胞吐作用也与至关重要的有吸引力的轴突转向反应有关 用于轴突引导。实现适当的神经元形态发生和连接是神经元的核心 功能性神经系统的形成。这些因素共同强调了 在发育中的神经元中调节胞吐作用，甚至在突触发生之前。过去 40 多年来，许多参与胞吐作用的成分已被识别，尽管这个列表 并不详尽。此外，调节模式、进展、频率或 囊泡与膜融合的时空组织，所有这些都准备好 调节神经元形态发生，尚未定义。可视化胞吐事件 发育中的神经元，我们表达了附着在神经元上的 GFP (pHluorin) 的 pH 敏感变体 v-SNARE 的腔侧，例如 VAMP2 或 VAMP7，以阐明融合的发生 酸性囊泡腔和中性细胞外空间之间的孔开口。分析 此类图像的处理仍然是一个耗时的、非自动化的瓶颈，延迟了 了解这种基本的细胞行为。我们开发了全自动计算机 用于检测和分析 VAMP-pHluorin 介导的胞吐事件的视觉软件 将定量揭示胞吐作用的时空组织和调节 以以前无法达到的细节水平开发神经元。我们利用这一创新 方法论以及公正的蛋白质组学、微流体学、生物化学和细胞生物学 研究胞吐作用与神经元形态发生之间关系的方法 并确定调节发育神经元胞吐作用的分子机制。