Diversity Supplement (Monica Quinones-Frias): Roles of Recycling Endosomes in Neuronal Extracellular Vesicle Cargo Traffic

项目摘要 这项提议的目的是了解细胞膜运输机制如何控制细胞内的蛋白质。 包装和释放细胞外囊泡（EV）货物从突触在体内。电动汽车很小 由包括神经元在内的多种细胞类型释放的膜结合囊泡，携带对 信号和疾病。然而，我们对电动汽车货物运输的空间和 在神经元的极化和复杂形态内进行时间调节。我们已经开发了工具， 跟踪和操纵体内果蝇突触前末梢的EV交通，并发现 通过质膜循环内体途径的货物决定了它们是否被局部分选 用于在EV中包装和释放，而不是通过逆行运输从突触中耗尽。回收 内体主要是在非神经元细胞中研究的，对它们的寿命知之甚少， 功能，或动态突触前终端。我们知道内体的再循环在 信号传导、神经元形态发生、EV交通和突触传递。理解和 对这些重要过程进行治疗性干预将需要对这些机制有更深入的了解。 神经元再循环内体功能的重要性。在本研究中，我们将阐明突触EV的机制， 货物和再循环内体运输。为了实现这些目标，我们将使用果蝇遗传学， 生物化学、高分辨率显微镜和活细胞成像。1)我们将确定函数， 动力学和调节不同类型的突触再循环内体。为此，我们将制定 新的工具和方法来定义和控制功能不同的回收区室在突触。 使用这些工具，我们将测试膜交通机械如何分类的新机制假设 突触回收舱的货物。2)我们将确定电动汽车货运量如何取决于不同的 突触内吞作用模式：网格蛋白介导的内吞作用，在低神经元活性下起作用 以及在强烈的神经元活动期间起作用的活动依赖性大量内吞作用。这些 实验将确定EV的命运是否由不同的内化模式决定，如何回收 内体有助于这些功能，并提供了新的机制来连接活性，内体运输， EV释放鉴于突触膜运输机制的保守性，我们的发现和 这些工具将在神经系统功能的许多方面为电动汽车交通的新见解奠定基础， 包括人类神经系统疾病。

PROJECT SUMMARY The goal of this proposal is to understand how cellular membrane trafficking machinery controls the packaging and release of extracellular vesicle (EV) cargoes from synapses in vivo. EVs are small membrane-bound vesicles released by numerous cell types including neurons, carrying cargoes critical for signaling and disease. However, we understand very little about how EV cargo traffic is spatially and temporally regulated within the polarized and complex morphology of neurons. We have developed tools to track and manipulate EV traffic at Drosophila presynaptic terminals in vivo, and discovered that flux of cargoes through a plasma membrane-recycling endosome route determines whether they are locally sorted for packaging and release in EVs, rather than depleted from synapses by retrograde transport. Recycling endosomes have primarily been studied in non-neuronal cells, and very little is known about their lifetime, functions, or dynamics at presynaptic terminals. We do know that recycling endosomes play critical roles in signaling, neuronal morphogenesis, EV traffic, and synaptic transmission. Understanding and therapeutically intervening in these important processes will require a deeper knowledge of the mechanisms of neuronal recycling endosome function. In this proposal, we will elucidate the mechanisms of synaptic EV cargo and recycling endosome traffic in vivo. To achieve these goals, we will use Drosophila genetics, biochemistry, high-resolution microscopy, and live cell imaging. 1) We will determine the functions, dynamics, and regulation of different types of synaptic recycling endosomes. To this end, we will develop new tools and approaches to define and control functionally distinct recycling compartments at synapses. Using these tools, we will test novel mechanistic hypotheses for how membrane traffic machinery sorts cargoes at synaptic recycling compartments. 2) We will determine how EV cargo traffic depends on distinct modes of synaptic endocytosis: clathrin-mediated endocytosis, which operates under low neuronal activity and activity-dependent bulk endocytosis, which operates during intense neuronal activity. These experiments will ascertain if EV fate is determined by different modes of internalization, how recycling endosomes contribute to these functions, and provide new mechanisms to link activity, endosomal traffic, and EV release. Given the conserved nature of synaptic membrane trafficking machinery, our findings and tools will lay the foundation for new insights into EV traffic in many aspects of nervous system function, including in human neurological disease.