SNARE complex and Ca2 sensor

1
(No Transcript)
2
SNARE complex and Ca2 sensor
3
Clathrin-mediated endocytosis
Endocytic invagination

1. 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

Pinch-off Dynamin
Vesicle uncoating Synaptojanin 1 that
hydrolyzes PI(4,5)P2
4
Clathrin-mediated endocytosis is a slow form of
endocytosis
5
Full fusion and kiss-and-run
Harata et al, J Neurochem 2006
6
Coupling between PI(4,5)P2 metabolism and
synaptic vesicle recycling
7
Techniques for synaptic vesicle recycling
8

1. Electrophysiology single stimulation

Geppert et al, Cell 1994
9

1. Electrophysiology a train of stimulations

Defects in synaptic vesicle endocytosis
Dickman et al, Cell 2005
10

1. Electrophysiology

Autaptic hippocampal neuron
Bekkers Stevens, PNAS 1991
11

1. Electrophysiology

Distinct vesicle pools
Rettig Neher, Science 2002
12

1. Electrophysiology

Detection of RRP by hypertonic sucrose solution
Hypotonic 0.5 M sucrose
Hypotonic 0.5 M sucrose
Stevens Sullivan, Neuron 1998
13

1. Electrophysiology

A reduction in RRP size in Munc-13 KO neuron
Rhee et al, Cell 2002
14
2. Imaging FM dye
FM dyes
Gaffield Betz, Nat Protoc 2009
15
(No Transcript)
16
2. Imaging SynaptopHluorin
SynaptopHluorin
Miesenbock et al, Nature 1998
Sankaranarayanan et al, Biophys J 2000
17
SynaptopHluorin reports synaptic vesicle
exocytosis
Sankaranarayanan Ryan, Nat Cell Biol 2000
18
2. Imaging SynaptopHluorin
Bafilomycin to separate exo/endocytosis
Mani et al, Neuron 2007
19
2. Imaging SynaptopHluorin
Transgenetic mice expressing SynaptopHluorin
Li et al, PNAS 2005
20
Full fusion and kiss-and-run
Harata et al, J Neurochem 2006
21
2. Imaging quantum dots
Detection of full fusion and kiss-and-run by
quantum dots
Zhang et al, Science 2009
22
2. Imaging quantum dots
Detection of full fusion and kiss-and-run by
quantum dots
Zhang et al, Science 2009
23
3. Electron microscopy

1. Endocytic vesicle biogenesis
2. Docked vesicles

Rettig Neher, Science 2002
24
3. Electron microscopy
Defects in synaptic vesicle endocytosis
Hayashi et al, PNAS 2008
25
Neuroendocrine chromaffin cells
Chromaffin granules 250 nm Synaptic vesicles
30 50 nm
26
Chromaffin cells as the model system for vesicle
cycling
27
Amperometry detects catecholamine release
from single vesicles by oxidization
Amperometry
Amperometrical current
Chromaffin cell
28
Whole cell capacitance technique
29
Detection in exocytosis and endocytosis of single
vesicles by cell-attached capacitance technique
Exocytosis
Endocytosis
Kiss-and-run
Im
30
Detection the dynamics of fission-pore during
endocytosis
31
Cell-attached capacitance to detect single
vesicle fusion