Dynamics of membrane tension and synaptic vesicle recycling

膜张力和突触小泡回收的动力学

• 批准号: 10594954
• 负责人: ERDEM KARATEKIN
• 金额: $ 42.11万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10594954
• 关键词: Adrenal Glands; Area; Behavior; Binding; Biological Assay; Bipolar Neuron; Cell membrane; Cell surface; Cells; Cellular biology; Charge; Chromaffin Cells; Coupled; Coupling; Cytoskeleton; Drops; Electric Capacitance; Electron Microscopy; Electrophysiology (science); Endocytosis; Endocytosis Inhibition; Exocytosis; F-Actin; Feedback; Fishes; Fluorescence Microscopy; Grant; Intervention; Knowledge; Maps; Measurement; Measures; Membrane; Membrane Fusion; Membrane Potentials; Methods; Modeling; Molecular; Nature; Nerve; Nervous System; Neuroendocrine Cell; Neurons; Neurotransmitters; Optics; Osmosis; Pattern; Presynaptic Terminals; Process; Property; Recovery; Recycling; Regulation; Reporting; Resistance; Resolution; Retrieval; Signal Transduction; Site; Slide; Speed; Structure; Synapses; Synaptic Membranes; Synaptic Transmission; Synaptic Vesicles; System; Testing; Thinness; Tube; Vesicle; Visualization; Work; Zebrafish; cell motility; confocal imaging; experimental study; laser tweezer; live cell imaging; neuronal cell body; optic tweezer; pharmacologic; postsynaptic; presynaptic; presynaptic neurons; response; retinal bipolar neuron; spatiotemporal; synaptic function; trafficking; ultra high resolution; viscoelasticity; voltage

Project Summary Information in the nervous system is relayed mostly at synapses, where neurotransmitter is released with great temporal precision from a presynaptic terminal via the fusion of membrane-bound synaptic vesicles (SVs) with the plasma membrane (PM), in a process called exocytosis. Components of these SVs are subsequently retrieved via endocytosis and recycled for reuse. This grant aims to understand the interplay between SV recycling and membrane tension gradients and associated membrane flows. In neurons and neuroendocrine cells, both exo- and endocytosis are influenced by osmotic forces, suggesting they are influenced by membrane tension, 𝜎𝜎. Conversely, membrane addition to the PM via exocytosis is expected to lower 𝜎𝜎, while endocytosis should restore it. In addition, 𝜎𝜎 has been suggested to be a possible signal for coupling exo- to endocytosis. Despite these key roles, there are no measurements of 𝜎𝜎 in synaptic terminals and how 𝜎𝜎 changes are related to exo-endocytosis is not known, mainly due to technical difficulties. The best method to probe 𝜎𝜎 is to pull a thin membrane tether from the PM using optical tweezers; the tether force reflects 𝜎𝜎. However, most terminals are small and tightly coupled to post-synaptic structures, making tether pulling impractical. We overcome this challenge using fish bipolar neurons which possess giant terminals, in a setup that combines optical tweezers with electrophysiology or photostimulation and with high- resolution fluorescence microscopy. We aim to 1) characterize PM flows at neuronal presynaptic terminals. After stimulation, membrane added at an exocytic site needs to flow (and the associated 𝜎𝜎 perturbation propagate) over the cell surface, then through the tether to produce a change in the measured tether force. We will characterize membrane flows. 2) Determine mechanisms of cell membrane flow regulation by the cytoskeleton. We found that F-actin is a major regulator of PM-cytoskeleton drag, but how it interacts with the PM at terminals and activity-dependent changes in its structure are not understood. We will characterize F- actin rearrangements upon stimulation at the optical and electron microscopy levels. 3) Establish the relationship between tension changes in response to stimulation, membrane flow, and exo-endocytosis coupling. We will confirm that 𝜎𝜎 changes we observed in preliminary experiments are due to exo-endocytosis and map the spatiotemporal relationship between exo- and endocyosis sites as a function of membrane flow to test the idea that exo-endocytosis coupling may depend on membrane flow. 4) Do electromechanical effect matter for exo- or endocytosis? We observed rapid voltage-induced tether force changes consistent with electromechanical effects. The relevance of these effects to exo-endocytosis is not known. We will characterize electromechanical effects and determine whether they may play a role during exo-endocytosis. Overall, these measurements will help generate a model of feedback between membrane trafficking and membrane flows.

项目摘要 神经系统中的信息主要通过突触传递，神经递质在突触中以极大的速度释放。 通过融合膜结合的突触囊泡（SV）与 质膜（PM），在一个过程中称为胞吐作用。这些SV的组件随后 通过内吞作用回收并回收再利用。这项资助旨在了解SV 再循环和膜张力梯度以及相关的膜流动。 在神经元和神经内分泌细胞中，胞吞和胞吞都受到渗透力的影响，这表明 它们受到膜张力的影响。𝜎𝜎相反，通过胞吐作用将膜添加到PM中， 预期会降低细胞凋亡，而内吞作用会恢复细胞凋亡。此外，细胞凋亡被认为是一种可能的 将胞吞作用与胞外作用结合信号。尽管有这些关键作用，但在突触中没有测量到突触后神经元的表达。 主要由于技术上的困难，尚不清楚末端的生物学特性以及细胞内分泌变化如何与胞吞作用相关。 最好的方法来探测微扰是拉一个薄膜系绳从PM使用光学镊子;系绳 力反映力。然而，大多数终末都很小，并且与突触后结构紧密耦合， 系绳拉动不切实际。我们利用鱼的双极神经元克服了这一挑战， 终端，在一个装置，结合光镊与电生理学或光刺激，并与高- 分辨率荧光显微镜。我们的目标是：1）表征神经元突触前末梢的PM流。 在刺激后，在胞吐位点处添加的膜需要流动（以及相关的微扰扰动）。 传播）在细胞表面上，然后通过系链以产生测量的系链力的变化。我们 将表征膜流动。2)确定细胞膜流动调节的机制， 细胞骨架我们发现，F-肌动蛋白是PM-细胞骨架阻力的主要调节因子，但它如何与细胞骨架相互作用， 终端PM及其结构中的活性依赖性变化尚不清楚。我们将描述F- 在光学和电子显微镜水平上的刺激后的肌动蛋白重排。3)建立 刺激引起的张力变化、膜流动和外吞作用之间的关系 偶合器.我们将证实，我们在初步实验中观察到的细胞内吞变化是由于外吞-内吞作用引起的 并绘制作为膜流函数的胞外和胞吞位点之间的时空关系， 测试外吞-内吞耦合可能依赖于膜流动的想法。4)做机电效应 是胞吞作用还是胞吞作用？我们观察到快速的电压诱导的系绳力变化， 机电效应这些效应与胞外-内吞作用的相关性尚不清楚。我们将描述 它们可以被用于检测细胞的机电效应，并确定它们是否可以在外吞作用期间发挥作用。 总的来说，这些测量将有助于产生膜运输和膜运输之间的反馈模型。 膜流动