Axon ER comprises a mainly tubular network, shaped by membrane proteins including REEPs and reticulons. It is continuous over large distances, with unusually narrow nanotubules. Since mutations in ER-shaping proteins can lead to the axon degeneration disease, hereditary spastic paraplegia (HSP), the specialized geometry of axonal ER – its levels, continuity and narrowness – appears critical for axon function and survival. Our goal is to understand how this occurs, primarily by studying the physiological consequences of genetically altering this geometry in vivo in Drosophila larvae. We first tested whether ER geometry affected presynaptic calcium signaling. Loss of reticulon Rtnl1 leads to lowered amounts of tubular ER, but no obvious change in ER cisternae, nor in levels of resting ER calcium that resides mostly in larger cisternae. Despite the normal levels of resting calcium, calcium fluxes into cytosol, ER and mitochondria were significantly reduced, apparently because of lower levels of STIM due to loss of ER tubules. Given the importance of ER calcium in neuronal physiology, we speculate that the narrowness of axonal ER tubules might limit calcium diffusion along their length, with potential consequences for replenishment of locally depleted ER calcium stores. Triple mutant Rtnl1 ReepA ReepB larvae have ER tubules that are fewer and wider than wildtype. FRAP analysis of both genotypes, and of Rtnl1 mutants with normal diameter tubules, shows fastest recovery of GFP in triple mutants, but normal slow recovery in Rtnl1 mutants, suggesting that narrow diameter constrains protein diffusion. Single-particle tracking suggests that the slower GFP recovery is not due to constrained single molecule movement in narrow ER nanotubules. We are developing optogenetic tools to test whether calcium movement in wildtype tubules is also constrained like protein movement, and are exploring potential consequences for presynaptic plasticity.