EXOCYTOSIS MODELING--KINETICS OF MEMBRANE AGGREGATION AND FUSION

胞吐作用建模——膜聚集和融合的动力学

• 批准号: 3968988
• 负责人: S J MORRIS
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3968988
• 关键词: calcium; cell membrane; chemical reaction; exocytosis; fluorescent dye /probe; liposomes; membrane fusion; membrane lipids; neurotransmitters; phospholipids; pinocytosis; spectrometry; stop flow technique; synaptic vesicles

Neurotransmitter and neuromodulator release takes place by exocytosis; the influx of calcium into the cell or nerve terminal triggers the fusion of the storage granule with the cell plasma membrane. The membrane fusion events can be modeled by studying the fusion of artifical or biological membranes with each other. A new multichannel, computer controlled stopped-flow rapid mixing spectrometer has been constructed to study the kinetics of these reactions. Our previous stopped-flow mixing studies have shown that the kinetics of both aggregation and fusion of small vesicular structures (artifical lipid vesicles, neurotransmitter storage granules, etc.) follow second order kinetics with fusion being aggregation rate limited. Our original stopped-flow assay for membrane fusion, based on resonance energy transfer between fluorescent phospholipids was subject to artifacts arising from the interactions of NBD-labelled phosphatidylethanolamine with the fusion catalysts such as Ca2+ interacting directly with the NBD probe and changing its quantum yield. We have developed a new assay, based on changes in the fluorescence of pyrene-labelled phosphatidylcholine. This assay examines the ratio of pyrene excimer/monomer fluorescence as a function of time after mixing. This ratio is independent of calcium concentration and is linearly dependent upon the distance between pyrene fluorophores, thus solving these problems and additionally allowing easy calculation of expected amplitude changes as the vesicles fuse together. This assay has been applied to the Mg-promoted fusion of artifical vesicles as well as protein-catalyzed fusion.

神经递质和神经调质的释放通过胞吐作用发生; 钙离子流入细胞或神经末梢， 储存颗粒与细胞质膜。 的膜融合 事件可以通过研究人工或生物的融合来建模 膜彼此。 一种新型多通道计算机控制 建立了一种停流快速混合光谱仪， 这些反应的动力学。 我们以前的停流混合研究 表明，小泡的聚集和融合的动力学 结构（人造脂质囊泡，神经递质储存颗粒， 等等）。符合二级动力学，融合为聚集速率 有限公司 我们最初的膜融合停流分析，基于共振 荧光磷脂之间的能量传递受到人为因素的影响 由NBD标记的磷脂酰乙醇胺与 融合催化剂如Ca 2+直接与NBD探针相互作用 并改变其量子产率。 我们开发了一种新的检测方法， 芘标记的磷脂酰胆碱荧光的变化。 这 测定法检测芘激基缔合物/单体荧光的比率， 混合后的时间函数。 这个比例与钙无关 浓度和线性依赖于芘之间的距离 荧光团，从而解决了这些问题，并另外允许容易的 计算当囊泡融合在一起时预期的幅度变化。 该方法已应用于人工囊泡的Mg促进融合 以及蛋白质催化的融合。