Building a continuous and dynamic but neglected cell compartment: 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沿着传递信号的能力，在人类中，轴突可以从细胞中心（细胞体）延伸一米。维持长轴突以确保良好的沟通需要大量的工程。这反映在当它出错时发生的问题上--如轴突变性、瘫痪或缺乏感觉。其中一些，如遗传性痉挛性截瘫（HSP），它会导致下半身的选择性麻痹，优先影响离细胞体最远的轴突，因此可能会影响长轴突最容易受到影响的通信或运输等过程。一种可能易受影响轴突的疾病影响的结构是被称为平滑内质网（ER）的小管，其纵向穿过轴突，彼此融合和分裂以形成网络。由于它们的长度和连续性，以及它们携带长距离信号的潜力，它们被称为“神经元中的神经元”。为了支持这一网络的重要作用，HSP通常是由有助于ER模型的蛋白质突变引起的，通过插入ER膜的一面并使其弯曲。轴突ER是一个未被探索的隔室，在科学文献中几乎没有出现2- 30年。直到最近，我们才开发出可视化工具，并发现其中的缺陷;通过这种方式，我们发现，使用果蝇突变体去除一些HSP膜弯曲蛋白，会导致运动轴突中ER网络的中度破坏，即使我们去除了最知名的弯曲诱导蛋白，网络仍然基本上完好无损，大多数轴突仍然拥有至少一个小管。我们假设存在多种机制来实现至少一个小管的连续网络，并避免过多的小管。管必须成形;微管的生长必须需要在正确的位置合成新的膜;由于未附着的内质网微管在轴突上上下移动，因此必须有运输它们的机制，并可能感觉到哪里需要它们。为了检测对轴突ER网络的形成至关重要的机制，并帮助其响应细胞的需求，我们将使用果蝇遗传学。果蝇神经元的功能与我们非常相似。它们的生命周期短，易于大量处理，这使得它们易于遗传学，通过去除或改变基因并研究其后果，从而了解受影响的过程是如何工作的。首先，由于我们假设有额外的HSP基因对轴突ER的形成很重要，我们将制造缺乏这些基因的果蝇，并测试轴突ER是否受到影响，以及如何受到影响。然而，仅仅去除已知的基因并不能确定必须存在的新机制。大规模随机诱变和缺陷筛选是一种行之有效的方法，已经为胚胎发育或生物钟等生物过程带来了几项诺贝尔奖。因此，第二，我们将产生足够的随机突变，以破坏基因组某一部分中的大多数基因。在通过昆虫表皮可见的轴突中，用荧光标记物可视化ER足够快，可以筛选新的突变果蝇的轴突ER缺陷，从而找到大多数参与其组织的基因。一旦我们建立了稳定的突变株系并确认了它们的表型，我们将使用全基因组测序来鉴定几个新突变株中受影响的基因。这些信息将告诉我们每个突变体中哪些蛋白质受到影响，并帮助我们预测其作用。最后，我们将通过深入分析受影响的蛋白质在正常轴突中的行为以及ER在最有希望的突变体中的行为来测试其中的一些预测。通过深入研究具有新表型的一些基因，我们将逐步建立轴突ER如何形成，调节和响应需求的图片。

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 far as a metre from the centre of the cell, the cell body. Maintaining long axons to ensure good communication requires a lot of engineering. This is reflected in problems that occur when it goes wrong - conditions such as axon degeneration, paralysis, or lack of sensation. Some of these, like Hereditary Spastic Paraplegia (HSP), which causes selective paralysis of the lower body, preferentially affect the axons furthest from the cell body, and therefore may affect processes like communication or transport that long axons are most vulnerable to. One structure that may be vulnerable to diseases that affect axons are tubules known as smooth endoplasmic reticulum (ER), which run lengthwise through the axon, fusing and splitting from each other to form a network. Due to their length and continuity, and their potential to carry signals for long distances, they have been termed a "neuron within a neuron". In support of an important role for this network, HSP is often caused by mutations in proteins that help model ER, by inserting in one face of the ER membrane, and curving it. Axonal ER is an underexplored compartment that barely featured in the scientific literature for 2-3 decades. It is only recently that we have developed tools to visualize it and see defects in it; in this way, we have found that removing some HSP membrane-curving proteins, using fruitfly mutants, causes moderate disruption of the ER network in motor axons.Even when we remove the best known curvature-inducing proteins, the network is still substantially intact, with most axons still possessing at least one tubule. We hypothesise the existence of multiple mechanisms to achieve a continuous network of at least one tubule, and avoid too many tubules. Tubules must be shaped; tubule growth must require new membrane synthesis at the right location; since unattached ER tubules move up and down axons, there must be mechanisms to transport them, and possibly sense where they are needed. To detect mechanisms that are important to make the axonal ER network and help it respond to the needs of the cell, we will use fruitfly genetics. Fruitfly neurons function very similarly to our own. Their short life cycle, and ease of handling in large numbers, makes them amenable to genetics, by removing or altering genes and studying the consequences, and thus learning how the affected processes work. FIRST, since there are additional HSP genes that we hypothesise are important for shaping axonal ER, we will make flies that lack these genes, and test whether axonal ER is affected, and how. However, removing only known genes will not identify new mechanisms that must exist. Large-scale random mutagenesis and screening for defects is a proven approach for this, having led to several Nobel prizes for biological processes such as embryo development, or biological clocks. Therefore SECOND, we will generate enough random mutations to disrupt most genes in one part of the genome. Visualising ER with a fluorescent marker, in axons that are visible through the insect cuticle, is rapid enough to allow screening of new mutant flies for defects in axonal ER, and thus find most of the genes involved in its organisation. Once we have established stable mutant lines and confirmed their phenotypes, we will use whole-genome sequencing to identify the affected genes in several new mutants. This information will tell us what protein is affected in each mutant, and help us to predict its role. Finally, we will test some of these predictions by in depth analysis of how the affected proteins behave in normal axons, and how ER behaves in the mutants that appear most promising.By studying a few genes with new phenotypes in depth, we will gradually build up a picture of how axonal ER is formed, regulated and responds to needs.