Two evolutionarily conserved HSP protein families (the reticulon and REEP families) play a critical role in ER shaping (Voeltz et al., 2006). However, removing them from Drosophila (fruit fly) did not delete all tubular ER in axons as predicted (Yalcin et al., 2017), implying that more proteins function in this process. We therefore performed a genetic screen to find more proteins, by testing for abnormal axonal ER in fly larvae lacking additional HSP proteins. We found roles of a few such HSP proteins on levels and continuity of axonal ER, and we studied one such protein, atlastin, along with reticulon and REEPs.
ER dynamics could be critical for its function. Fluorescent markers make it feasible to visualise axonal ER dynamics in live animals. We recorded axonal ER movement in larvae and found that it was surprisingly dynamic, with frequent forward and backward movement of tubules along axons (Fig 1). We also bleached ER fluorescence markers in normal axons, and in mutant axons lacking specific HSP proteins; in the former we found rapid recovery due to diffusion of fluorescent proteins from nearby ER. In HSP mutants, this recovery was impaired, implying that the ER network was disrupted in these mutants.
ER tubules are structures on the scale of nanometers, which cannot be explored by conventional light microscopy. In collaboration with Dr M Terasaki (University of Connecticut, USA), we performed electron microscopy (EM) on Drosophila larval axons and reconstructed the ER network at ultrastructural level. Our results show that in fly axons ER forms a continuous network with multiple branches and some dead ends, and ER tubules often show proximity to mitochondria or plasma membrane (Fig 2); however in axons lacking reticulon and REEP proteins, tubules were fewer, larger, with more gaps, consistent with reduced membrane curvature.
One clinical feature of HSP is progressive lower limb spasticity and weakness. Although third instar larvae of fruit flies are used for a wide range of analysis, they are not suited for progressive phenotype studies, since they progress immediately to pupation. Therefore, we developed a method to visualise axonal ER in fly adults which live for over 30 days. Fluorescently labelled ER proteins expressed in adult flies reveal labelled motor axons in live undissected adult legs. This now allows us to test HSP mutations for age-dependent or degenerative defects using this system; it also makes large mutant screens feasible since it does not require time-consuming dissection and immunostaining.
Exploitation and dissemination. Further understanding of the roles of HSP proteins might suggest targets or approaches for therapy of axon degeneration diseases, although further research on the physiological consequences of these ER defects is still required. For dissemination, we have published part of our work in a peer-reviewed journal, eLife (Yalcin et al., 2017), and are currently writing a second manuscript on our atlastin work. We have also presented the work at an annual departmental symposium in Cambridge and the European Drosophila Research Conference. Some images from this project were shown as part of a matching game in Cambridge Science Festival, an annual event which makes science accessible to public. We have also posted movies showing reconstructed axonal ER network, and talks that include the work, on the Departmental web page (see URL below).
Voeltz, Prinz, Shibata, Rist and Rapoport (2006). A class of membrane proteins shaping the tubular endoplasmic reticulum. Cell 124, 573-586.
Yalcin, Zhao, Stofanko, O'Sullivan, Kang, Roost, Thomas, Zaessinger, Blard, Patto,, et al. (2017). Modeling of axonal endoplasmic reticulum network by spastic paraplegia proteins. eLife 6:e23882.
