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由神经递质分泌驱动，神经递质分泌来源于一小部分再循环突触囊泡（SV）。胞吐作用后，突触囊泡的膜和蛋白质成分被整合到质膜中，并且必须通过胞吞作用被回收以维持持续的突触功能。SV内吞作用的细微变化可导致脑功能的严重缺陷。本项目的长期目标是确定SV内吞作用的分子机制。在这里，我们将研究内啡肽，SV内吞作用所需的保守蛋白质。在初步研究中，我们发现，内嗜蛋白通过弯曲膜促进内吞作用，而内吞作用不需要其作为分子支架的功能。我们发现，在神经末梢的大多数内嗜蛋白是结合到SV，而不是在内吞网站。我们进一步表明，从SV的内嗜蛋白解结合的速率是由胞吐调节。根据这些初步结果，我们提出了三个目标。在目标1中，我们开发了用于内啡肽的膜缔合、寡聚化和膜弯曲的实时测定。我们将使用这些测定来确定膜弯曲的速率是否足够快以解释内吞作用中的内嗜蛋白的功能。在目标2中，我们将确定Endophilin如何靶向SV。我们假设，无活性的亲内皮素单体与SV蛋白RAB-3结合，从而将亲内皮素募集到SV库中。我们将使用生物化学，遗传学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（PIP 2）是否刺激内吞位点的内嗜蛋白寡聚化。在目标3中，我们将确定突触蛋白的PIP 2磷酸酶活性是如何与内吞作用相结合的。具体来说，我们将定义的分子机制，使突触的感觉产生的内啡肽膜曲率。这些研究将提供重要的新的见解调节SV内吞作用的机制，以及如何BAR域蛋白的功能一般。

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.