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.

项目概要 神经系统中的信息主要在突触处传递，神经递质在突触处大量释放 通过膜结合突触小泡 (SV) 与突触前末端的融合实现时间精度 质膜 (PM)，这一过程称为胞吐作用。这些 SV 的组件随后被 通过内吞作用回收并回收再利用。这笔赠款旨在了解 SV 之间的相互作用 回收和膜张力梯度以及相关的膜流。 在神经元和神经内分泌细胞中，外吞作用和内吞作用均受到渗透力的影响，这表明 它们受到膜张力𝜎𝜎的影响。相反，通过胞吐作用将膜添加到 PM 中是 预计会降低𝜎𝜎，而内吞作用应该会恢复它。此外，𝜎𝜎 已被建议为可能的 用于耦合外吞作用和内吞作用的信号。尽管有这些关键作用，但在突触中还没有对𝜎𝜎进行测量 终端以及𝜎𝜎变化如何与外吞作用相关尚不清楚，主要是由于技术困难。 探测 𝜎𝜎 的最佳方法是使用光镊从 PM 上拉出薄膜系绳；系绳 力反映𝜎𝜎。然而，大多数终端都很小并且与突触后结构紧密耦合，使得 系绳拉动不切实际。我们利用鱼类双极神经元克服了这一挑战，这些神经元拥有巨大的 终端，在将光镊与电生理学或光刺激相结合的设置中，以及高 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。 刺激后，在胞吐位点添加的膜需要流动（以及相关的𝜎𝜎扰动） 传播）在细胞表面，然后通过系绳产生测量的系绳力的变化。我们 将表征膜流。 2) 确定细胞膜流量调节机制 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 PM 细胞骨架相互作用 终端的 PM 及其结构中与活动相关的变化尚不清楚。我们将表征 F- 在光学和电子显微镜水平上刺激后肌动蛋白重排。 3）建立 刺激、膜流和胞吐作用引起的张力变化之间的关系 耦合。我们将确认我们在初步实验中观察到的𝜎𝜎变化是由于外吞作用 并绘制外吞位点和内吞位点之间的时空关系作为膜流的函数 测试外吞-内吞耦合可能依赖于膜流的想法。 4）做机电效应 外吞作用或内吞作用重要吗？我们观察到电压引起的系绳力的快速变化与 机电效应。这些效应与胞吐作用的相关性尚不清楚。我们将表征 机电效应并确定它们是否在外吞作用过程中发挥作用。 总的来说，这些测量将有助于生成膜运输和膜运输之间的反馈模型 膜流动。