Building a continuous and dynamic but neglected cell compartment: axonal endoplasmic reticulum

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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.