The Role of ER Architecture in Axonal Presynaptic Physiology

我这个项目的主要目的是研究内质网在轴突和突触前终末中的生理作用，例如钙处理，如何受到内质网结构特征(如小管连续性和直径)的影响。我将利用ER-Shaping-Protein突变体来操纵ER的结构，以了解狭窄的小管直径是否是ER管腔扩散的限制因素，了解三向连接的丢失如何影响ER连续性和突触前终末的ER水平，并评估atlastin的丢失以及随之而来的ER结构变化如何影响轴突和突触前终末ER功能的不同方面，如局部钙信号和管腔隧道。我将以三龄果蝇幼虫为模式生物进行解剖，以进入幼虫的中枢神经系统，并促进显微镜和相关技术。在我的项目中占很大一部分的一项技术是FRAP，它使研究选定的分子/蛋白质的动力学成为可能。在评估管径对ER管腔扩散的影响时，这一点尤为重要。我的项目的另一个主要组成部分将是使用钙成像技术(钙成像显微镜)来评估失去atlastin对钙功能的影响。从设备/技术的角度来看，该项目可以分为三种方式：使用FRAP/光激活来观察连续性，使用STED/传统的共聚焦显微镜来观察ER的组织和水平，以及钙成像来研究ER的钙动力学。

The main aim of my project is to investigate how are the physiological roles of the ER, for example calcium handling, affected by ER architectural features such as tubule continuity and diameter, in axons and presynaptic terminals. I will utilise ER-shaping-protein mutants to manipulate ER architecture in order to understand if narrow tubule diameter is a limiting factor in ER lumen diffusion, find out how ER continuity and ER levels at presynaptic terminals are affected by the loss of three-way junctions and assess how the loss of atlastin, and the ensuing ER architectural changes, affect different aspects of ER function, such as local calcium signalling and lumenal tunnelling, in axons and presynaptic terminals.Using third instar Drosophila larvae as a model organism I will perform dissections to gain access to the larva's central nervous system and facilitate microscopy and associated techniques. One technique which forms a large part of my project is FRAP which enables for the dynamics of a chosen molecule/protein to be investigated. This is of particular importance when assessing the effect of tubule diameter on ER lumen diffusion. Another major component of my project will be the use of calcium imaging techniques (calcium imaging microscope) to assess the effect that loss of atlastin has on calcium function.From an equipment/technical point of view, the project could be broken down in three ways; using FRAP/photoactivation to look at continuity, STED/traditional confocal microscopy to look at ER organisation and levels and calcium imaging to investigate ER calcium dynamics.