1. 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

2. Overview of the retina

6. http://webvision.med.utah.edu

7. 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.

8. http://webvision.med.utah.edu

11. 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?

13. 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

14. Goldfish Giant danio Why zebrafish? www.sanger.ac.uk/ Previous Research

15. The Experiment Goal: design a way to make successful electrophysiological recordings from zebrafish bipolar synaptic terminals

16. The Setup

17. The zebrafish retina Ganglion cell layer

21. Inner plexiform layer

32. Inner nuclear layer horizontal cells bipolar cells amacrine cells

38. Outer plexiform layer

42. Outer nuclear layer photoreceptors

43. Photoreceptors

57. http://webvision.med.utah.edu

58. 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.

59. 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.

60. 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?

61. 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.

62. Acknowledgements… Peter O’DayPeter O’Day Bill RobertsBill Roberts Mark RutherfordMark Rutherford Rob DuncanRob Duncan Jerry GleasonJerry Gleason SPUR ProgramSPUR Program www.yamanashi-med.ac.jp/

63. http://webvision.med.utah.edu