SNARE complex and Ca 2+ sensor Endocytic invagination 1.Bar-domain containing protein such as...
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SNARE complex and Ca2+ sensor
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Endocytic invagination1. Bar-domain containing protein such as
amphiphysin which inducing membrane curvature
2. Clathrin, which is recruited by AP-2, is critical in maintaining the curvature of endocytic vesicles
Clathrin-mediated endocytosis
Pinch-off: Dynamin
Vesicle uncoating:
Synaptojanin 1 that hydrolyzes PI(4,5)P2
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Clathrin-mediated endocytosis is a slow form of
endocytosis
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Full fusion and kiss-and-run
Harata et al, J Neurochem 2006
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Coupling between PI(4,5)P2 metabolism and synaptic vesicle recycling
PI(4)P PI(4,5)P2
KinasesPI(4,5)P2 PI(4)P
phosphatases
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Techniques for synaptic vesicle recycling
1. Electrophysiology
2. Imaging
3. Electron microscopy
• FM dyes• SynaptopHluorin• Quantum dots
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Geppert et al, Cell 1994
1. Electrophysiology: single stimulation
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1. Electrophysiology: a train of stimulations
Defects in synaptic vesicle endocytosis
Dickman et al, Cell 2005
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Autaptic hippocampal neuron
Bekkers & Stevens, PNAS 1991
1. Electrophysiology
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Distinct vesicle pools
Rettig & Neher, Science 2002
1. Electrophysiology
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Detection of RRP by hypertonic sucrose solution
Stevens & Sullivan, Neuron 1998
1. Electrophysiology
Hypotonic 0.5 M sucrose Hypotonic 0.5 M sucrose
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Rhee et al, Cell 2002
A reduction in RRP size in Munc-13 KO neuron
1. Electrophysiology
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Gaffield & Betz, Nat Protoc 2009
2. Imaging: FM dye
FM dyes
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Sankaranarayanan et al, Biophys J 2000
2. Imaging: SynaptopHluorin
SynaptopHluorin
Miesenbock et al, Nature 1998
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SynaptopHluorin reports synaptic vesicle exocytosis
Sankaranarayanan & Ryan, Nat Cell Biol 2000
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2. Imaging: SynaptopHluorin
Bafilomycin to separate exo/endocytosis
Mani et al, Neuron 2007
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2. Imaging: SynaptopHluorinTransgenetic mice expressing SynaptopHluorin
Li et al, PNAS 2005
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Full fusion and kiss-and-run
Harata et al, J Neurochem 2006
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Detection of full fusion and kiss-and-run by quantum dots
Zhang et al, Science 2009
2. Imaging: quantum dots
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Detection of full fusion and kiss-and-run by quantum dots
Zhang et al, Science 2009
2. Imaging: quantum dots
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3. Electron microscopy
(1) Endocytic vesicle biogenesis(2) Docked vesicles
Rettig & Neher, Science 2002
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Hayashi et al, PNAS 2008
3. Electron microscopyDefects in synaptic vesicle endocytosis
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Neuroendocrine chromaffin cellsNeuroendocrine chromaffin cells
Chromaffin granules: ~ 250 nm
Synaptic vesicles: 30 – 50 nm
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Adrenal gland
Chromaffin cells as the model systemChromaffin cells as the model systemfor vesicle cyclingfor vesicle cycling
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Carbon fiber+700 mV
Amperometry detects catecholamine release fromsingle vesicles by oxidization
Amperometrical current
Quantal sizeAmperometry
Chromaffin cellFusion pore
PM
Foot
Spike
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Whole cell capacitance technique
stimulation
Cap
acit
ance
(p
F)
60 fF
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Patch pipette Patc
h pi
pett
e
Detection in exocytosis and endocytosis of single vesicles by cell-attached capacitance technique
Conductance
Capacitance
Im
Exocytosis
Endocytosis
Kiss-and-run
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Detection the dynamics of fission-pore during endocytosis
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Cell-attached capacitance to detect single vesicle fusion
Conductance
Capacitance
Patch pipette
Patch pipette
Chromaffin cell
0.1 nS
1 fF
0.5 nS
10 ms
Conductance
Capacitance
Fusion poreconductance
Fusion pore
PM