The Role of ER Architecture in Axonal Presynaptic Physiology

点击翻译按钮获取中文摘要

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.