Exocytosis fuels plasma membrane expansion in developing neurons

项目摘要 在发育过程中，神经元获得极化的、细长的和复杂的形态， 需要质膜表面积的显著扩大。表面增加， 据估计，每天高达20%，远远超过伴随的神经元 音量增大。我们以前证明，SNARE介导的胞吐作用是必需的， 在神经突发生和轴突分支过程中，可能是为了给神经细胞提供膜材料。 膨胀的质膜，其在破裂之前只能拉伸~2-3%。不对称 胞吐作用也涉及到吸引性轴突转向反应， 轴突引导实现适当的神经元形态发生和连接是神经元发育的核心。 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。在过去40 多年来，参与胞吐作用的多种组分已经被鉴定，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 调节神经元形态发生，尚未定义。为了可视化细胞外事件， 在神经元发育过程中，我们表达了一种对pH敏感的GFP变体（pHluorin）， v-SNARE的内腔侧，如VAMP 2或VAMP 7，以阐明融合的发生 酸性囊泡腔和中性细胞外间隙之间的孔开口。分析 这类图像的处理仍然是一个时间密集型、非自动化的瓶颈， 理解这种基本的细胞行为。我们开发了一种全自动电脑- 用于检测和分析VAMP-pHluorin介导的胞吐事件的视觉软件， 将定量地揭示空间和时间的组织和调控的胞吐作用， 神经元的发育达到了以前无法达到的细节水平。我们利用这一创新 沿着无偏蛋白质组学、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关系的方法 并确定调节发育中神经元胞吐作用的分子机制。

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.