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The Biochemistry of Visual Processing in the Retina: Ribbon synapses at bipolar cell terminals Kirigin Elstad O’Day & Roberts labs http://webvision.med.utah.edu Overview of the retina http://webvision.med.utah.edu Ribbon Synapses: What do they do? • Transmit sensory information • Retina – photoreceptor and bipolar cell synaptic terminals. • Cochlea – hair cells. • Synaptic vesicles attach to the ribbon via short filaments. • Ribbons maintain fast, prolonged neurotransmitter release. http://webvision.med.utah.edu http://webvision.med.utah.edu http://webvision.med.utah.edu http://webvision.med.utah.edu http://webvision.med.utah.edu Ribbon Synapses: What are they made of? • MANY different proteins, including: – synapsin, cadherin, synaptotagmin, synaptobrevin, synaptogyrin, syntaxin, clathrin, myo1c, and MORE! – RIBEYE, Nok • How do these proteins control neurotransmitter release at the synapse? Can we understand ribbon synapses by studying protein function? • We need to measure some feature of the synapse – typically done by making mutants and comparing mutants to wildtype. • Morphology, anatomy, microanatomy, biochemistry, physiology, electrophysiology. • The goal: find a way to make retinal slice recordings from zebrafish axon terminals. http://users.rcn.com Previous Research www.sanger.ac.uk/ • Goldfish • Giant danio • Why zebrafish? The Experiment • Goal: design a way to make successful electrophysiological recordings from zebrafish bipolar synaptic terminals The Setup The zebrafish retina Ganglion cell layer Inner plexiform layer Inner nuclear layer horizontal cells bipolar cells amacrine cells Outer plexiform layer Outer nuclear layer photoreceptors Photoreceptors http://webvision.med.utah.edu Future directions… • Is there a phenotype for the absence of RIBEYE? – Currently, Yvonne Bradford (Roberts lab) is working to create morphlinos to block the synthesis of RIBEYE. • What are morpholinos? – A short sequence of fake nucleotides that can bind to mRNA or pre-mRNA. – Inject into zebrafish egg. – Splice into mRNA knocks out the protein due to steric hindrance. – Problem: It doesn’t replicate, so as the cell divides, you continually cut the morpholino concentration. – Advantage: You don’t have to mess with DNA/genetics. Why RIBEYE? • RIBEYE exists only at ribbon synapses. • RIBEYE probably has catalytic activity that affects vesicle formation. • RIBEYE is composed of 2 domains: • Unique A domain specific to ribbons. • B domain identical with CtBP2, a lipid-chemistry protein. CtBP2 • CtBP2: catalytic activity converts lysophosphatidic acid phosphatidic acid. • CtBP2 functionally converts big vesicles into smaller ones. • Is RIBEYE involved in making vesicles whole again? Nok • Nok is a member of the membrane-associated guanylate kinase (MAGUK) family. •These proteins contain several protein-protein interaction domains and work as scaffolding factors that recruit signaling molecules to cell junctions and synaptic terminals. Acknowledgements… • Peter O’Day • Bill Roberts • Mark Rutherford • Rob Duncan • Jerry Gleason • SPUR Program www.yamanashi-med.ac.jp/ http://webvision.med.utah.edu