Exocytosis fuels plasma membrane expansion in developing neurons

项目摘要 在发育过程中，神经元获得极化、拉长和复杂的形态，这 需要显著扩大质膜表面积。表面增加有 据估计达到每天20%以上，远远超过伴随的神经元 音量增加。我们以前证明了SNARE介导的胞吐作用是必需的。 在神经元发生和轴突分支过程中，可能是为了向 质膜膨大，破裂前只能拉伸~2-3%。不对称 胞吐作用也与吸引人的轴突转向反应有关，这是至关重要的。 用于轴突引导。实现适当的神经元形态发生和连接是 形成一个有功能的神经系统。这些因素加在一起，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。在过去40年中 多年来，已经确定了与胞吐作用有关的多种成分，尽管这份清单 并不是包罗万象。此外，调节模式、进程、频率或 囊泡与膜融合的时空组织，所有这些都有可能 调节神经元的形态发生，目前还没有定义。要将胞外事件可视化，请在 在发育神经元的过程中，我们表达一种对pH敏感的GFP变体(PHluorin)，该变体附着在 V型圈套的管腔侧，如VAMP2或VAMP7，以说明融合的发生 酸性泡状腔和中性细胞外间隙之间的孔洞。分析 这类图像的存储仍然是一个耗时的、非自动化的瓶颈，延迟了 对这种基本的细胞行为的理解。我们开发了一台全自动计算机- VAMP-pHluorin介导的胞外事件的检测和分析的视觉软件 将定量揭示胞吐作用的时空组织和调控。 以以前无法达到的细节水平发育神经元。我们利用这一创新 方法学以及无偏见的蛋白质组学、微流体学、生化和细胞生物学 胞吐作用与神经元形态发生关系的研究进展 并确定调节发育中神经元胞吐的分子机制。

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.