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。 通过膜结合融合从突触前终末到突触后细胞的时间精度 突触小泡(SVS)与细胞膜发生胞吐作用。这些SVS的组件包括 随后通过内吞作用被取回并循环再利用。这笔赠款的目的是为了了解 SV循环与膜张力梯度和相关膜流量之间的关系。 在神经元和神经内分泌细胞中，胞吐和内吞都受到渗透性肿胀或 收缩，表明它们受到膜张力的影响，𝜎。相反，将膜添加到 突触前末端通过胞吞作用有望降低𝜎，而内吞作用应使其恢复。此外, 膜张力被认为是胞吐和胞内结合的可能信号之一。 然而，尽管有这些关键作用，但没有测量突触终末和突触的膜张力 张力变化与外吞作用的关系尚不清楚，主要是由于技术上的困难。最好的 探测𝜎的方法是用光学镊子从细胞表面拉出一条薄膜系绳，操纵 直径1-3μm的珠子作为手柄。珠子从陷阱中心的位移提供了系缆力， 这反映了𝜎。然而，大多数终末都很小，并且与突触后结构紧密相连，从而使 系绳不切实际。我们用拥有巨型细胞的金鱼双极细胞克服了这一挑战 终端，其设置将光学镊子与电生理相结合(以控制刺激和/或 测量电容变化)和高分辨率荧光显微镜(以标记和识别亚 细胞结构和钙成像)。我们的目标是1)表征系缆力响应 在活动期间发生在突触前终末的电和机械扰动。 刺激后，在胞外部位添加的膜需要流动(以及相关的张力扰动 在终端表面上传播)，然后穿过系绳以在测量的系缆力中产生变化。 我们将在双系绳实验中表征膜流动，并校准系绳对阶跃的响应。 系绳长度的变化。我们将确认我们在初步实验中观察到的𝜎变化(下降~1 S 刺激后，随后恢复在~10 S)是由于外吞作用，并表现为快速电压- 诱导系缆力变化。这些将使我们能够定量地了解测量到的相关𝜎变化 带着刺激。接下来，我们将描述膜张力是如何在突触前调节的 神经末梢。将药物干预与实时成像和𝜎测量相结合，我们将 测试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𝜎需求的SV周转。这些测量将会有所帮助 在神经末梢建立膜转运和𝜎之间的反馈模型。

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.