Membrane Bending Machinery for Synaptic Vesicle Endocytosis

摘要： 大脑活动在很大程度上是由神经递质分泌驱动的，神经递质来自于 突触小囊泡回收池(SVS)。胞吐后，膜和蛋白质 突触小泡的成分被结合到质膜中，并且必须 通过内吞作用恢复，以维持持续的突触功能。SV的微妙变化 内吞作用会导致严重的脑功能缺陷。这个项目的长期目标是 确定控制SV内吞作用的分子机制。在这里，我们将调查 内亲素，一种保守的蛋白质，是SV内吞作用所必需的。在初步研究中，我们发现 内嗜素通过弯曲细胞膜来促进内吞作用，而它的功能是 内吞作用不需要分子支架。我们发现大多数内亲和素在 神经末梢与SVS结合，而不是在内吞部位。我们进一步表明， 内毒素从SVS上解离受胞吐作用的调节。根据这些初步结果， 我们提出了三个目标。在目标1中，我们建立了内亲和素细胞膜的实时检测方法 缔合、齐聚和膜弯曲。我们将使用这些化验来确定 膜弯曲的速度足够快，足以解释内亲和素在 内吞作用。在目标2中，我们将确定内亲和素是如何针对SVS的。我们假设 非活性亲内素单体与SV蛋白RAB-3结合，从而募集亲内素 送到SV泳池。我们将使用生化、遗传和成像方法来测试这一想法。我们 还将确定磷脂酰肌醇4，5-二磷酸(PIP2)是否刺激内亲和素 内吞胞位的寡聚作用。在目标3中，我们将确定突触素的PIP2 磷酸酶的活性与内吞作用有关。具体地说，我们将定义分子 允许突触素感知内亲和素产生的膜弯曲的机制。 这些研究将为调节SV的机制提供重要的新见解 内吞作用和BAR结构域蛋白的一般功能。

Abstract: 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.