Membrane Bending Machinery for Synaptic Vesicle Endocytosis

描述（由申请人提供）：大脑活动在很大程度上是由神经递质分泌驱动的，神经递质分泌来源于一小部分循环突触囊泡（SVs）。胞吐后，突触囊泡的膜和蛋白质成分被纳入质膜，必须通过内吞作用恢复，以维持突触的持续功能。SV内吞作用的细微变化可导致严重的脑功能缺陷。该项目的长期目标是确定控制SV内吞作用的分子机制。在这里，我们将研究嗜内蛋白，一种SV内吞作用所需的保守蛋白。在初步研究中，我们发现嗜内蛋白通过弯曲膜促进内吞作用，而其作为分子支架的功能并不需要内吞作用。我们发现神经末梢的大多数嗜内啡肽与sv结合，而不是与内吞位点结合。我们进一步发现，嗜内啡肽脱离SVs的速率受胞吐作用的调节。基于这些初步结果，我们提出了三个目标。在Aim 1中，我们开发了亲内肽的膜结合、寡聚和膜弯曲的实时检测。我们将使用这些实验来确定膜弯曲的速度是否足够快，以解释嗜内噬蛋白在内吞作用中的作用。在Aim 2中，我们将确定嗜内啡肽如何靶向SVs。我们假设无活性的亲内蛋白单体与SV蛋白rab3结合，从而将亲内蛋白招募到SV池中。我们将使用生化、遗传和成像方法来检验这个想法。我们还将确定磷脂酰肌醇4,5-二磷酸（PIP2）是否刺激内吞位点的亲内蛋白寡聚。在Aim 3中，我们将确定Synaptojanin的PIP2磷酸酶活性如何与内吞作用耦合。具体来说，我们将定义允许Synaptojanin感知由嗜内啡肽产生的膜曲率的分子机制。这些研究将为SV内吞作用的调节机制和BAR结构域蛋白的一般功能提供重要的新见解。

DESCRIPTION (provided by applicant): Brain activity is driven in large part by neurotransmitter secretion, which is derived from a small pool of recycling synaptic vesicles (SVs). Following exocytosis, membrane and protein components of synaptic vesicles are incorporated into the plasma membrane and must be retrieved by endocytosis to maintain continued synaptic function. Subtle changes in SV endocytosis can lead to severe defects in brain function. The long-term goal of this project is to determine the molecular mechanisms governing SV endocytosis. Here, we will investigate Endophilin, a conserved protein required for SV endocytosis. In preliminary studies, we showed that Endophilin promotes endocytosis by bending membranes whereas its function as a molecular scaffold is not required for endocytosis. We showed that the majority of Endophilin at nerve terminals is bound to SVs, not at endocytic sites. We further showed that the rate of Endophilin unbinding from SVs is regulated by exocytosis. Based on these preliminary results, we propose three Aims. In Aim 1, we develop real-time assays for Endophilin's membrane association, oligomerization, and membrane bending. We will use these assay to determine if the rate of membrane bending is sufficiently fast to account for Endophilin's function in endocytosis. In Aim 2, we will determine how Endophilin is targeted to SVs. We hypothesize that inactive Endophilin monomers bind to the SV protein RAB-3 and thereby recruit Endophilin to the SV pool. We will use biochemical, genetic, and imaging approaches to test this idea. We will also determine if phosphatidylinositol 4,5-bisphosphate (PIP2) stimulates Endophilin oligomerization at endocytic sites. In Aim 3, we will determine how Synaptojanin's PIP2 phosphatase activity is coupled to endocytosis. Specifically, we will define the molecular mechanisms that allow Synaptojanin to sense membrane curvature generated by Endophilin. These studies will provide significant new insights into the mechanisms regulating SV endocytosis and how BAR domain proteins function generally.