Investigating Synaptic Vesicle Tethering at the Rod Photoreceptor Ribbon Synapse

• Applicant: Dr. Kaspar Gierke
• Subject Area: Molecular Biology and Physiology of Neurons and Glial Cells
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 528821243

In the mammalian retina, the transduction of light stimuli into membrane potential changes is mediated by rod and cone photoreceptors. At their highly specialized ribbon synapses, membrane potential changes trigger the graded and continuous release of the neuro-transmitter glutamate from presynaptic active zones (AZs) for further processing by the postreceptoral retinal network. In order to sustain the high amount of neurotransmitter release, photoreceptor AZs are extended by a structural organelle and name-giving hallmark of these synapses, the synaptic ribbon (SR). SRs of photoreceptors are plate-shaped, proteinaceous organelles, which extend several hundred nanometers into the terminals’ cytoplasm. The surface of SRs is covered with hundreds of SVs, which are tethered uniformly at a distance of ~40 nm via. This huge reservoir of tethered SVs is thought to support the continuous release of glutamate over a wide stimulus range. The molecule(s) that tether SVs and guide them towards the AZ for subsequent exocytosis, however, have remained elusive. Recently, we have identified Piccolino – a splice variant of the AZ protein Piccolo – as a component of SRs. Results from preceding experiments suggest that loss of Piccolino disturbs the uniform alignment of SVs that surround SRs. Moreover, we demonstrate that Piccolino exhibits a topology at the SR, facing with its N-terminus towards the cytoplasm and with its C-terminus towards the SR. Intriguingly, the N-terminal domains additionally exhibit strong affinity to lipids – possibly a mechanism, by which Piccolino is able to interact with SVs. Based on these preliminary findings, we hypothesize that Piccolino could be (one of) the SV tether at photoreceptor SRs. In this project, we want to test this hypothesis with the following approaches: (i) direct visualization of SV tethers with super-resolution electron tomography in the presence and absence of Piccolino. (ii) examining the lipid binding properties of Piccolino as a potential molecular mechanism underlying SV tethering. (iii) we will investigate retinal function in the presence and absence of Piccolino with electroretinographic recordings.

• DFG Programme: Research Grants