Investigating the role of Synaptotagmin‐1 at ribbon synapses

Abstract

AbstractBackgroundPhotoreceptors possess a specialized form of proteinaceous electron‐dense projection: the synaptic ribbon that exhibits graded neurotransmitter release. The molecular mechanism underlying graded release is unknown. Data suggests that synaptic vesicles at ribbon synapses undergo homotypic compound fusion where vesicles may only travel a short distance to the neighboring vesicles and fuse with each other around the ribbons1. Here we demonstrate that Synaptotagmin‐1 (Syt1) functions in compound fusion as it promotes full formation of the SNARE complex fusion machinery in response to calcium influx2. A better understanding of synaptic ribbon communication proteins may lead to novel drug targets to aid in therapeutic designMethodSyt1 knock‐down (KD) was developed using a CRISPRi system in Danio rerio larvae (n=100) and incubated for 5 days. Flash‐and‐freeze electron microscopy was performed on the Syt1 KD line and controls (n=100) by stimulating and freezing the samples at time points 0, 3, 6, 9 and 15 milliseconds after exposure to light for 30 seconds. Samples were fixed in epoxy resin and viewed under TEM. Blind analysis using ImageJ was performed to identify the presence of any compound fusion.ResultMean compound fusion frequency was highest at the 6ms and 9ms time points in Danio rerio controls. At the 6ms timepoint, compound fusion was seen at the middle of the ribbon and at 9ms, compound fusion was seen closer to the base. Prevalence of compound fusion was significantly reduced at the 6ms and 9ms time points in the Syt1 KD compared to controls.ConclusionSyt1 is can mediate compound fusion on a millisecond timescale during exocytosis.1. Matthews G, Sterling P. Evidence That Vesicles Undergo Compound Fusion on the Synaptic Ribbon. J Neurosci. 2008;28(21):5403‐5411. doi:10.1523/JNEUROSCI.0935‐08.20082. Chicka MC, Hui E, Liu H, Chapman ER. Synaptotagmin arrests the SNARE complex before triggering fast, efficient membrane fusion in response to Ca2+. Nat Struct Mol Biol. 2008;15(8):827‐835. doi:10.1038/nsmb.1463