Mechanisms of Vesicle Recycling
Jackie BondsNeus 586
March 12, 2012
Synaptic Vesicle Cycle
Richmond, 2007
SNARE Proteins
• Synaptobrevin (v-SNARE)• SNAP-25 (t-SNARE) • Syntaxin (t-SNARE) • Synaptotagmin - Ca2+ sensor
Vesicle Fusion
SNARE complex and Ca2+ sensor
Trans-SNARE ComplexExerts an inward force
Cis-SNARE Complex
Südof and Rothman, 2009
Zippering Model for SNARE Catalyzed Membrane Fusion
Botulinum toxin A…aka BoTox
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
Synaptojanin
• After the vesicle pinches off, need to hydrolyze a protein known as PI(4,5)P2 in 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
Coupling between PI(4,5)P2 metabolism and synaptic vesicle recycling
PI(4)P PI(4,5)P2Kinases
PI(4,5)P2 PI(4)Pphosphatases
Full fusion and kiss-and-run
Harata et al, J Neurochem 2006
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
Südof et al, Cell 1994
Electrophysiology
Gaffield & Betz, Nat Protoc 2009
Imaging: FM dyes
Sankaranarayanan et al, Biophys J 2000
Imaging: SynaptopHluorin
Miesenbock et al, Nature 1998
Zhang et al, Science 2009
Imaging: Quantum Dots
Hayashi et al, PNAS 2008
Electron microscopy
Defects in synaptic vesicle endocytosis
Current Work…happening right down the hall!
• Tomosyn negatively 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
Tomosyn phosphorylation leads to decreased affinity for SNAREs and is PKA dependent
Tomosyn Phosphorylation
• Photo-activated Adenylyl Cyclase (PAC) with blue light increases the production of cAMP increased PKA activation increased tomosyn phosphorylation
• In the presence/activation of PAC, what would you expect to see?
Thank you! Any Questions?
References:
Special thanks to Drs. Janet Richmond, Dave Featherstone, and Liang Wei Gong