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Mechanisms of Vesicle Recycling

Mechanisms of Vesicle Recycling. Jackie Bonds Neus 586 March 12, 2012. Synaptic Vesicle Cycle. Richmond, 2007. SNARE Proteins. Synaptobrevin (v-SNARE) SNAP-25 (t-SNARE) Syntaxin (t-SNARE) Synaptotagmin - Ca 2+ sensor . Vesicle Fusion. SNARE complex and Ca 2+ sensor.

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Mechanisms of Vesicle Recycling

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  1. Mechanisms of Vesicle Recycling Jackie Bonds Neus 586 March 12, 2012

  2. Synaptic Vesicle Cycle Richmond, 2007

  3. SNARE Proteins • Synaptobrevin (v-SNARE) • SNAP-25 (t-SNARE) • Syntaxin (t-SNARE) • Synaptotagmin - Ca2+ sensor Vesicle Fusion

  4. SNARE complex and Ca2+ sensor

  5. Zippering Model for SNARE Catalyzed Membrane Fusion Trans-SNARE Complex Exerts an inward force Cis-SNARE Complex Südof and Rothman, 2009

  6. Botulinum toxin A…aka BoTox

  7. Two Types of Fusion • Full Fusion - the synaptic vesicle merges and becomes incorporated into the cell membrane. • Kiss and Run - the synaptic vesicle "kisses" the cellular membrane, opening a small pore for its neurotransmitter payload to be released through, then closes the pore and is recycled back into the cell

  8. Synaptojanin • After the vesicle pinches off, need to hydrolyze a protein known as PI(4,5)P2in order to get rid of the endocytic proteins still located on the vesicle • Synaptojanin-1 is recruited to the endocytic process at a later stage  It is the main phosphatase for PI(4,5)P2 in the brain

  9. Coupling between PI(4,5)P2 metabolism and synaptic vesicle recycling phosphatases PI(4,5)P2PI(4)P Kinases PI(4)PPI(4,5)P2

  10. Full fusion and kiss-and-run Harata et al, J Neurochem 2006

  11. How can we tell the difference? • There are techniques available that allow the investigator to determine what kind of fusion is occurring • Electrophysiology • Imaging • FM Dyes • SynaptopHlorin • Quantum Dots • Electron Microscopy

  12. Electrophysiology Südofet al, Cell 1994

  13. Imaging: FM dyes Gaffield & Betz, Nat Protoc 2009

  14. Imaging: SynaptopHluorin Miesenbock et al, Nature 1998 Sankaranarayanan et al, Biophys J 2000

  15. Imaging: Quantum Dots Zhang et al, Science 2009

  16. Electron microscopy Defects in synaptic vesicle endocytosis Hayashi et al, PNAS 2008

  17. Current Work…happening right down the hall! • Tomosynnegatively regulates fusogenic SNARE complex formation  presence of tomosyn creates a competitive SNARE • If tomosyn is KO or mutated, would expect to see: • Increase in docked vesicles • Increase in primed/fusion competent vesicles • Increase in vesicle release

  18. Tomosyn phosphorylation leads to decreased affinity for SNAREs and is PKA dependent

  19. Tomosyn Phosphorylation • Photo-activated Adenylyl Cyclase (PAC) with blue light increases the production of cAMP increased PKA activation  increased tomosynphosphorylation • In the presence/activation of PAC, what would you expect to see?

  20. Thank you! Any Questions? References: Special thanks to Drs. Janet Richmond, Dave Featherstone, and Liang Wei Gong

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