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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.

项目总结