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