Hereditary spastic paraplegias (HSP) are a group of diseases showing degeneration of lower motor axons. A common cause of HSP is the mutation of proteins that shape the tubular endoplasmic reticulum (ER). The architecture of the ER tubular network that extends through axons is therefore likely to be integral to the maintenance and survival of axons. The continuity and ubiquity of the ER network throughout neurons has earned it the term ‘a neuron within a neuron’. The reasons for its striking continuity are not fully understood, but it could potentially make the ER a channel for long-distance signalling, independent of action potentials at the plasma membrane, or motor-based transport. ER tubules are also much narrower in axons than in most other locations. This presents a potential paradox, of why a cellular structure that is physically continuous over long distances shows constrained continuity in its lumen.

Narrow ER nanotubules could limit axial diffusion along the ER tubule lumen. I aimed to explore this model. I used Drosophila larval motor axons, in which I could use mutations in ER-shaping proteins to manipulate tubule properties and genetic tools to label and express sensors in individual motor axons in vivo. In FRAP (fluorescence recovery after photobleaching) experiments I found that wildtype ER tubules were permissive for recovery of a GFP-tagged luminal protein, despite the difficulty of visualizing the lumen by scanning EM (electron microscopy), implying that proteins can diffuse along this lumen. To test whether this lumen diameter is limiting for diffusion, I compared recovery in wildtype axonal ER tubules with that in tubules of a larger diameter, in a triple mutant lacking the ER-shaping proteins, Rtnl1, ReepA and ReepB. Luminal diffusion of GFP was significantly faster in the larger mutant ER tubules, with the time to half recovery in wider mutant axonal ER tubules approximately twice as fast as in wildtype. Therefore, the narrow ER nanotubule diameter in wildtype axons limits luminal protein diffusion. To begin to dissect the roles of the different classes of ER-shaping proteins in determining tubule diameter, I also compared the FRAP recovery rates of a single Rtnl1 mutant with wildtype, and found no difference, implying that loss of Rtnl1 alone did not increase the ability of axon ER tubules to support the rate of axial diffusion.

To better understand the parameters of molecular movement along the ER nanotubule lumen, I generated larvae that expressed ER lumen markers suitable for single-molecule imaging. On sparse chemical labelling of a luminal HaloTag protein, I found two classes of single-particle movement: slower and relatively bright particles that showed largely unidirectional movement, and faster less brightly labelled particles. The slower class showed no bias in the direction of luminal movement and also appeared to move faster than microtubule-based motors. The faster class are similar to the single-molecule movements previously described in non-neuronal ER tubules. One experimental constraint on this approach was an unfavourable ratio of signal to background labelling. Therefore, I initiated a neuronal cell culture model for single-particle imaging to mitigate the challenges of larval nerve preparation.

One physiologically important molecule that diffuses through the ER lumen is Ca²⁺, and constraints on this diffusion could have consequences for intracellular Ca²⁺ signalling. Ca²⁺ is much smaller than GFP or a HaloTag, and therefore its movement might be less constrained by small tubule diameter. I aimed to develop a strategy to test the effect of axonal ER tubule diameter on intraluminal Ca²⁺ diffusion. This involved making and testing two components: Drosophila stocks containing an ER-localised channelrhodopsin and stocks containing an axonal ER-localised Ca²⁺ sensor (HaloCaMP). I report on the development of these stocks.

In summary, I have shown that luminal protein diffusion is constrained in axon ER nanotubules. I have developed tools that can be useful for further understanding the nature of these constraints and their physiological consequences.