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.

项目摘要 神经系统中的信息主要是在突触中传达的，在突触中，神经递质发行了很棒 临时精度从突触前末端到后突触细胞，通过膜结合的融合 与细胞膜的突触蔬菜（SVS）在称为胞吐作用的过程中。这些SV的组成部分是 随后通过内吞作用检索并回收以重复使用。该赠款旨在了解相互作用 在SV回收和膜张力梯度和相关的膜流之间。 在神经元和神经内分泌细胞中，胞吐作用和内吞作用都受到渗透壳的影响或 收缩，表明它们受膜张力的影响，𝜎。相反，膜添加 突触前末端通过胞吐作用预计将降低，而内吞作用应恢复。此外， 膜张力已被认为是偶联胞吞作用与内吞作用的可能信号之一。 但是，执行这些关键角色，在突触终端中没有膜张力的测量 张力变化如何与外胞吞作用有关，这主要是由于技术困难。最好的 探测方法是使用光学镊子从细胞表面拉动薄膜系绳，操纵 直径为1-3μm作为手柄。珠子从陷阱中心的位移提供了束缚力， 反映𝜎。但是，大多数终端都很小，并且与后突触结构紧密耦合，使 系绳拉动不切实际。我们使用具有巨大的金鱼双极细胞克服了这一挑战 终端，在将光学镊子与电生理学结合的设置中（控制刺激和/或 测量电容的变化）和高分辨率的荧光显微镜（以标记并鉴定亚 细胞结构和钙成像）。我们的目的1）表征绑扎力响应 活性过程中突触前末端发生的电和机械扰动。 刺激后，在外旋转部位添加的膜需要流动（以及相关的张力扰动 在末端表面上繁殖），然后通过系绳在测得的系带力中产生变化。 我们将在双螺旋实验中表征膜流，并校准序列的响应 系绳长度的变化。我们将确认我们在初步实验中观察到的变化（降低约1 s 刺激后，其次是在〜10 s中恢复的是由于外吞作用，并且表征了快速的电压 - 诱导的系带力变化。这些将使对测得的变化有定量理解 刺激。接下来，我们将2）表征如何在突触前调节膜张力 神经末端。将药物干预与实时成像和𝜎测量相结合，我们将 检验F-肌动蛋白是神经末端的主要调节剂的假设。我们将操纵𝜎 独立钙以剖析钙和SV周转的需求。这些测量将有所帮助 在神经末端产生膜运输和𝜎之间的反馈模型。