Dynamics of membrane tension and synaptic vesicle recycling

项目摘要 神经系统中的信息主要通过突触传递，神经递质在突触中大量释放。 膜结合突触小泡(SVS)融合突触前终末的时间精度 质膜(PM)，在一种称为胞吐作用的过程中。这些SV的组件随后被 通过内吞作用回收并循环再利用。这笔赠款旨在了解SV之间的相互作用 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，外吞和内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中是 预计会降低𝜎𝜎，而内吞作用应该会恢复它。此外，𝜎𝜎被认为是一种可能的 连接外吞和内吞的信号。尽管有这些关键作用，但突触中的𝜎𝜎还没有测量到 主要是由于技术上的困难，目前尚不清楚终末和𝜎𝜎变化与外内吞作用的关系。 探测𝜎𝜎的最好方法是使用光钳从PM中拉出一根薄膜绳索； 武力反映了𝜎𝜎。然而，大多数终末都很小，与突触后结构紧密相连，使 系绳不切实际。我们用鱼的双极神经元克服了这一挑战，这种神经元拥有巨大的 终端，其设置将光学镊子与电生理或光刺激相结合，并且具有高 分辨率荧光显微镜。我们的目标是1)表征神经元突触前终末的PM流。 刺激后，在胞外部位添加的膜需要流动(以及相关的𝜎𝜎扰动 在细胞表面传播)，然后通过系绳产生测得的系缆力的变化。我们 将表征膜流动。2)确定细胞膜流量调节的机制。 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 PM在终端和其结构中依赖于活动的变化不被理解。我们将描述F- 在光学和电子显微镜水平上，肌动蛋白在刺激后发生重排。3)建立 刺激引起的张力变化、膜流量和外吞作用之间的关系 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引起的 并将胞外区和胞内区之间的时空关系作为膜流量的函数映射到 测试外吞作用偶联可能依赖于膜流动的观点。4)做机电效果 物质是外吞还是内吞？我们观察到电压诱导的锚固力的快速变化与 机电效应。这些效应与胞外吞噬作用的相关性尚不清楚。我们将描述 机电效应，并确定它们是否可能在胞外吞噬过程中发挥作用。 总体而言，这些测量将有助于生成膜贩运和膜传输之间的反馈模型 膜流动。

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.