Axonal ER Architecture in Health and Disease"Axonal ER Architecture in Health and Disease"Cahir O'Kane\, PhD
Professor of Genetics
University of Cambridge
Axon ER comprises a mainly tubular network\, shaped by memb rane proteins including REEPs and reticulons. It is continuous over large d istances\, with unusually narrow nanotubules. Since mutations in ER-shaping proteins can lead to the axon degeneration disease\, hereditary spastic pa raplegia (HSP)\, the specialized geometry of axonal ER - its levels\, conti nuity and narrowness - appears critical for axon function and survival. Our goal is to understand how this occurs\, primarily by studying the physiolo gical consequences of genetically altering this geometry in vivo in Dro sophila larvae. We first tested whether ER geometry affected presynapt ic calcium signaling. Loss of reticulon Rtnl1 leads to lowered amounts of t ubular ER\, but no obvious change in ER cisternae\, nor in levels of restin g ER calcium that resides mostly in larger cisternae. Despite the normal le vels of resting calcium\, calcium fluxes into cytosol\, ER and mitochondria were significantly reduced\, apparently because of lower levels of STIM du e to loss of ER tubules. Given the importance of ER calcium in neuronal phy siology\, we speculate that the narrowness of axonal ER tubules might limit calcium diffusion along their length\, with potential consequences for rep lenishment of locally depleted ER calcium stores. Triple mutant Rtnl1 R eepA ReepB larvae have ER tubules that are fewer and wider than wildty pe. FRAP analysis of both genotypes\, and of Rtnl1 mutants with no rmal diameter tubules\, shows fastest recovery of GFP in triple mutants\, b ut normal slow recovery in Rtnl1 mutants\, suggesting that narrow diameter constrains protein diffusion. Single-particle tracking suggests th at the slower GFP recovery is not due to constrained single molecule moveme nt in narrow ER nanotubules. We are developing optogenetic tools to test wh ether calcium movement in wildtype tubules is also constrained like protein movement\, and are exploring potential consequences for presynaptic plasti city.