Organisation and Roles of Axonal Endoplasmic Reticulum

动物和人类的运动依赖于神经细胞沿着被称为轴突的狭窄突起携带信号的能力，在人类中，轴突可以从细胞中心延伸一米，在长颈鹿或鲸鱼中甚至可以延伸几米。如果说细胞体有剑桥大学的演讲厅那么大，那么轴突就像一条走廊，从细胞体向外延伸，最远可达爱丁堡或巴黎，实现物质的运输和交流。维持长轴突的结构和功能需要大量的工程技术。这一点的重要性反映在当它出错时发生的问题上——诸如轴突退化、瘫痪或缺乏感觉的疾病。其中一些优先影响长轴突或离细胞体最远的轴突末端，这表明距离细胞体最远的轴突部分最容易受到通信或运输障碍的影响。沿轴突的交流如何维持其形式和功能？轴突具有许多以膜为界的内部细胞器，这些细胞器的运输和组织有助于工程和通讯，以维持更长的轴突。它们中的一些沿着被称为微管的轨道运输，并沿着轴突来回携带物质和信号。轴突内的另一个膜结合结构是被称为光滑内质网（ER）的小管，它们沿着轴突纵向运行。由于它们的长度和连续性，以及随后在神经元内长距离传导信号的潜力，它们被比作“神经元中的神经元”。然而，形成它们的机制，它们在轴突中的功能，以及它们的形式和功能之间的关系，人们知之甚少。通过确定轴突退化的遗传原因，可以揭示维持长轴突功能和完整性的机制。为了支持内质网在这方面的作用，以较长脊髓运动轴突变性为特征的遗传性痉挛性截瘫（HSP）通常是由参与内质网膜建模的蛋白质突变引起的。这些蛋白质插入膜的一面，使其弯曲，其中一些还有其他作用，如将小管融合成一个网络，或切断微管（细胞器运输的轨迹）。在酵母中，去除这两组蛋白质甚至会删除几乎所有的管状内质网。我们的目标是了解控制轴突内质网结构的机制，以及这对轴突功能的重要性。考虑到容易产生缺乏特定蛋白质或表达改变形式的突变和转基因果蝇，以及研究神经细胞（包括轴突）的试剂和方法的可用性，我们使用果蝇。果蝇的一些HSP基因突变甚至具有与人类HSP相似的表型：幼虫的前部（由较短的轴突控制）可以正常移动，但后部（由较长的轴突控制）不能。我们还发现果蝇热休克蛋白对非神经元细胞内质网的形成和较长轴突正常数量的内质网很重要——这是热休克蛋白基因影响较长轴突内质网的第一个直接证据，也意味着轴突内质网对轴突存活很重要。因此，我们将使用高分辨率电子显微镜检查轴突中管状内质网的详细组织。我们将开发在活体动物、单轴突和高分辨率电子显微镜下可视化内质网的方法，并将利用这些方法详细评估一系列蛋白质在轴突内质网的存在、程度、形态和网络组织中的作用。我们还将评估轴突内质网通过释放重要的信号分子钙离子对轴突信号传导的贡献程度，以及这种信号传导如何依赖于由热休克蛋白和相关蛋白决定的内质网形式。从长远来看，我们的方法可以阐明内质网在轴突损伤和神经元远程信号传导中的作用。

Animal and human movement depends on the ability of nerve cells to carry signals along narrow projections known as axons, which in humans can extend as much as a metre from the centre of the cell, the cell body - and even several metres in giraffes or whales. If the cell body has the size of a lecture theatre in Cambridge, the axon is like a corridor reaching out of it as far as Edinburgh or Paris, allowing transport of materials and communication. Maintaining the structure and function of long axons requires a lot of engineering. The importance of this is reflected in problems that occur when it goes wrong - diseases with effects such as axon degeneration, paralysis, or lack of sensation. Some of these preferentially affect long axons, or the ends of axons furthest from the cell body - suggesting impairment in communication or transport, that the parts of axons furthest from the cell body are most susceptible to. How does communication along axons maintain their form and function? Axons have many internal organelles that are bounded by membranes, whose transport and organisation contributes to the engineering and communication to maintain longer axons. Some of them are transported along tracks called microtubules, and carry materials and signals forward and back along axons. Another membrane-bound structure within axons are tubules known as smooth endoplasmic reticulum (ER), which run lengthwise along the axon. Due to their length and continuity, and the ensuing potential to conduct signals for long distances within neurons, they have been likened to a "neuron within a neuron". However, the mechanisms that form them, their function in axons, and the relationship between their form and function, are poorly understood. Mechanisms to maintain function and integrity of long axons can be revealed by indentifying the genetic causes of axon degeneration. In support of a role for ER in this, the disease Hereditary Spastic Paraplegia (HSP), characterised by degeneration of longer spinal motor axons, is often caused by mutations in proteins with roles in modeling ER membrane. These proteins insert in one face of the membrane, thus curving it, and some have additional roles such as fusing tubules into a network, or severing microtubules (tracks along which organelles are transported). In yeast, removing two groups of these proteins even deletes nearly all tubular ER. We aim to understand the mechanisms that govern the architecture of ER in axons, and the importance of this for axon function. We use the fruitfly Drosophila, given the ease of generating mutant and transgenic flies that lack particular proteins or express altered forms of them, and the availability of reagents and methods to study nerve cells, including axons. Some mutations in Drosophila HSP genes even have similar phenotypes to human HSP: larvae whose anterior (controlled by shorter axons) moves normally, but whose posterior (controlled by longer axons) cannot. We have also shown that a Drosophila HSP protein is important for shaping ER in non-neuronal cells, and for normal amounts of ER in longer axons - the first direct evidence that HSP genes affect ER in longer axons, and implying that axonal ER is important for axon survival. We will therefore examine the detailed organisation of tubular ER in axons, using high resolution electron microscopy. We will develop ways to visualise ER in live animals, in single axons, and in high resolution electron microscopy, and will use these to assess in detail the roles of a range of proteins on the presence, extent, morphology and network organisation of axonal ER. We will also assess how far axonal ER contributes to signaling in axons by release of an important signaling molecule, calcium ions, and how this signaling depends on ER form as determined by HSP and related proteins. In the longer term, our approach can illuminate roles of ER in axonal injury and long-range signaling in neurons.