SNARE complex and Ca2+ sensor

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

Clathrin-mediated endocytosis is a slow form of

endocytosis

Full fusion and kiss-and-run

Harata et al, J Neurochem 2006

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

Techniques for synaptic vesicle recycling

1. Electrophysiology

2. Imaging

3. Electron microscopy

• FM dyes• SynaptopHluorin• Quantum dots

Geppert et al, Cell 1994

1. Electrophysiology: single stimulation

1. Electrophysiology: a train of stimulations

Defects in synaptic vesicle endocytosis

Dickman et al, Cell 2005

Autaptic hippocampal neuron

Bekkers & Stevens, PNAS 1991

1. Electrophysiology

Distinct vesicle pools

Rettig & Neher, Science 2002

1. Electrophysiology

Detection of RRP by hypertonic sucrose solution

Stevens & Sullivan, Neuron 1998

1. Electrophysiology

Hypotonic 0.5 M sucrose Hypotonic 0.5 M sucrose

Rhee et al, Cell 2002

A reduction in RRP size in Munc-13 KO neuron

1. Electrophysiology

Gaffield & Betz, Nat Protoc 2009

2. Imaging: FM dye

FM dyes

Sankaranarayanan et al, Biophys J 2000

2. Imaging: SynaptopHluorin

SynaptopHluorin

Miesenbock et al, Nature 1998

SynaptopHluorin reports synaptic vesicle exocytosis

Sankaranarayanan & Ryan, Nat Cell Biol 2000

2. Imaging: SynaptopHluorin

Bafilomycin to separate exo/endocytosis

Mani et al, Neuron 2007

2. Imaging: SynaptopHluorinTransgenetic mice expressing SynaptopHluorin

Li et al, PNAS 2005

Full fusion and kiss-and-run

Harata et al, J Neurochem 2006

Detection of full fusion and kiss-and-run by quantum dots

Zhang et al, Science 2009

2. Imaging: quantum dots

Detection of full fusion and kiss-and-run by quantum dots

Zhang et al, Science 2009

2. Imaging: quantum dots

3. Electron microscopy

(1) Endocytic vesicle biogenesis(2) Docked vesicles

Rettig & Neher, Science 2002

Hayashi et al, PNAS 2008

3. Electron microscopyDefects in synaptic vesicle endocytosis

Neuroendocrine chromaffin cellsNeuroendocrine chromaffin cells

Chromaffin granules: ~ 250 nm

Synaptic vesicles: 30 – 50 nm

Adrenal gland

Chromaffin cells as the model systemChromaffin cells as the model systemfor vesicle cyclingfor vesicle cycling

Carbon fiber+700 mV

Amperometry detects catecholamine release fromsingle vesicles by oxidization

Amperometrical current

Quantal sizeAmperometry

Chromaffin cellFusion pore

PM

Foot

Spike

Whole cell capacitance technique

stimulation

Cap

acit

ance

(p

F)

60 fF

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

Detection the dynamics of fission-pore during endocytosis

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