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

基本信息

批准号：
10159320
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
项目类别：
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/10159320
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

项目摘要

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.

项目摘要在发育过程中，神经元获得了两极分化，伸长且复杂的形态，该形态是需要大量的质膜表面积扩展。表面增加具有估计每天达到20％以上，远远超过了伴随的神经元体积增加。我们以前证明需要旋转介导的胞吐作用在神经发生和轴突分支期间，大概是为了提供膜材料扩展的质膜，在破裂之前只能伸展约2-3％。不对称胞吐作用也与诱人的轴突转动反应有关用于轴突指导。实现适当的神经元形态发生和连通性是功能神经系统的形成。这些因素共同强调了甚至在突触发生之前，在发育中的神经元中调节胞吐作用。在过去的40个多年来，已经确定了众多参与胞吐作用的成分，尽管此列表并不详尽。此外，调节模式，进展，频率或囊泡融合与膜的时空组织，所有这些都准备调节神经元形态发生尚未定义。可视化外生事件发育神经元，我们表达了附着在 V-SNARE（例如VAMP2或VAMP7）的亮度侧，以阐明融合的发生酸性囊泡管道和中性细胞外空间之间的孔口。分析这样的图像仍然是耗时的，非自动化的瓶颈，延迟了了解这种基本的细胞行为。我们开发了一个完全自动的计算机 - 视觉软件，用于检测和分析鞋面 - 荧光素介导的外囊肿事件，这些事件将定量揭示空间和时间组织以及胞吞作用的调节以前无法实现的细节开发神经元。我们利用这一创新性方法论以及无偏的蛋白质组学，微流体，生化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中胞吐作用的分子机制。