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: 9808543
• 负责人: ERDEM KARATEKIN
• 金额: $ 46.06万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-01 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9808543
• 关键词: Area; Artificial Membranes; Biological; Bipolar Neuron; Calcium; Caliber; Cell membrane; Cell surface; Cells; Cellular Structures; Cellular biology; Charge; Coupled; Couples; Coupling; Cytoskeleton; Drops; Electric Capacitance; Electrophysiology (science); Endocytosis; Endocytosis Inhibition; Excision; Exocytosis; F-Actin; Feedback; Fluorescence Microscopy; Goldfish; Grant; Image; Intervention; Knowledge; Label; Length; Measurement; Measures; Mechanics; Membrane; Membrane Fusion; Membrane Potentials; Methods; Micromanipulation; Modeling; Molecular; Nature; Nerve; Nervous system structure; Neuroendocrine Cell; Neurons; Neurotransmitters; Pharmacology; Physiologic pulse; Physiology; Presynaptic Terminals; Process; Property; Recovery; Recycling; Regulation; Reporting; Resistance; Resolution; Retrieval; Signal Transduction; Site; Structure; Surface; Swelling; Synapses; Synaptic Membranes; Synaptic Transmission; Synaptic Vesicles; System; Testing; Thinness; Tube; Vesicle; base; cell motility; cell type; confocal imaging; experimental study; laser tweezer; membrane model; presynaptic; presynaptic neurons; response; restoration; retinal bipolar neuron; synaptic function; trafficking; 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 on to a post-synaptic cell via the fusion of membrane bound synaptic vesicles (SVs) with the cell membrane, in a process called exocytosis. The 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 exocytosis and endocytosis are influenced by osmotic swelling or shrinking, suggesting they are influenced by membrane tension, 𝜎. Conversely, membrane addition to the presynaptic terminal via exocytosis is expected to lower 𝜎, while endocytosis should restore it. In addition, membrane tension has been suggested to be one of the possible signals for coupling exocytosis to endocytosis. However, despite these key roles, there are no measurements of membrane tension in synaptic terminals and how tension changes are related to exo-endocytosis is not known, mainly due to technical difficulties. The best method to probe 𝜎 is to pull a thin membrane tether from the cell surface using optical tweezers, manipulating a 1-3 μm diameter bead as a handle. The bead's displacement from the trap center provides the tether force, which reflects 𝜎. However, most terminals are small and are tightly coupled to post-synaptic structures, making tether pulling impractical. We overcome this challenge using goldfish bipolar cells which possess giant terminals, in a setup that combines optical tweezers with electrophysiology (to control stimulation and/or measure capacitance changes) and with high-resolution fluorescence microscopy (to label and identify sub- cellular structures and calcium imaging). We aim 1) to characterize the tether force response to electrical and mechanical perturbations that occur at a presynaptic terminal during activity. After stimulation, membrane added at an exocytic site needs to flow (and the associated tension perturbation propagate) over the terminal surface, then through the tether to produce a change in the measured tether force. We will characterize membrane flows in double-tether experiments and calibrate the tether response to step- changes in tether length. We will confirm that 𝜎 changes we observed in preliminary experiments (a drop ~1 s after stimulation, followed by recovery in ~10 s) are due to exo-endocytosis, and characterize rapid voltage- induced tether force changes. These will enable a quantitative understanding of measured 𝜎 changes associated with stimulation. Next, we will 2) characterize how membrane tension is regulated at a presynaptic nerve terminal. Combining pharmacological interventions with live imaging and 𝜎 measurements, we will test the hypothesis that F-actin is a major regulator of 𝜎 at the nerve terminal. We will manipulate 𝜎 and calcium independently to dissect calcium and 𝜎 requirements for SV turnover. These measurements will help generate a model of feedback between membrane trafficking and 𝜎 at the nerve terminal.

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 通过膜结合的融合从突触前末端到突触后细胞的时间精度 突触小泡 (SV) 与细胞膜的相互作用，这一过程称为胞吐作用。这些 SV 的组件是 随后通过内吞作用回收并回收再利用。这笔赠款旨在了解相互作用 SV 回收和膜张力梯度以及相关膜流之间的关系。 在神经元和神经内分泌细胞中，胞吐作用和内吞作用均受到渗透膨胀或渗透膨胀的影响。 收缩，表明它们受到膜张力𝜎的影响。相反，膜添加到 突触前末梢通过胞吐作用预计会降低 𝜎，而内吞作用则应恢复它。此外， 膜张力被认为是将胞吐作用与内吞作用耦合的可能信号之一。 然而，尽管有这些关键作用，但还没有测量突触末梢和突触末端的膜张力。 张力变化与胞吐作用之间的关系尚不清楚，主要是由于技术困难。最好的 探测𝜎的方法是使用光镊从细胞表面拉出薄膜系绳，操纵 直径为 1-3 μm 的珠子作为手柄。珠子从陷阱中心的位移提供了系绳力， 这反映了𝜎。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。我们利用金鱼双极细胞克服了这一挑战，该细胞拥有巨大的 终端，在将光镊与电生理学相结合的设置中（以控制刺激和/或 测量电容变化）并使用高分辨率荧光显微镜（标记和识别亚 细胞结构和钙成像）。我们的目标 1) 表征系绳力响应 活动期间突触前末梢发生的电和机械扰动。 刺激后，在胞吐位点添加的膜需要流动（以及相关的张力扰动 传播）在终端表面上，然后通过系绳产生测量的系绳力的变化。 我们将在双系绳实验中表征膜流，并将系绳响应校准为阶跃- 系绳长度的变化。我们将确认我们在初步实验中观察到的 𝜎 变化（下降约 1 秒） 刺激后，随后在〜10秒内恢复）是由于外吞作用，并表征快速电压- 引起系绳力的变化。这些将使人们能够定量地了解相关的测量𝜎变化 有刺激。接下来，我们将 2）描述突触前膜张力的调节方式 神经末梢。将药物干预与实时成像和𝜎测量相结合，我们将 检验 F-肌动蛋白是神经末梢 𝜎 的主要调节因子的假设。我们将操纵 𝜎 和 钙独立剖析钙和 SV 周转的需求。这些测量将有助于 生成神经末梢膜运输和 𝜎 之间的反馈模型。