SNARE complex and Ca 2+ sensor

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 Clathrin-mediated endocytosis Pinch-off: Dynamin Vesicle uncoating: Synaptojanin 1 that hydrolyzes PI(4,5)P 2

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)P 2 metabolism and synaptic vesicle recycling PI(4)P PI(4,5)P 2 Kinases PI(4,5)P 2 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 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 Electrophysiology

Distinct vesicle pools Rettig & Neher, Science Electrophysiology

Detection of RRP by hypertonic sucrose solution Stevens & Sullivan, Neuron Electrophysiology 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 Imaging: FM dye FM dyes

Sankaranarayanan et al, Biophys J 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: SynaptopHluorin Transgenetic 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 Imaging: quantum dots

Detection of full fusion and kiss-and-run by quantum dots Zhang et al, Science Imaging: quantum dots

3. Electron microscopy (1) Endocytic vesicle biogenesis (2) Docked vesicles Rettig & Neher, Science 2002

Hayashi et al, PNAS Electron microscopy Defects in synaptic vesicle endocytosis

Neuroendocrine chromaffin cells Chromaffin granules: ~ 250 nm Synaptic vesicles: 30 – 50 nm

Adrenal gland Chromaffin cells as the model system for vesicle cycling Techniques: Capacitance measurements Amperometry Big vesicle size ( nm in diameter) Oxidizable catecholamine

Carbon fiber +700 mV Amperometry detects catecholamine release from single vesicles by oxidization Amperometrical current Quantal size Amperometry Chromaffin cell Fusion pore PM Foot Spike

Whole cell capacitance technique stimulation Capacitance (pF) 60 fF

Patch pipette 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 Chromaffin cell 0.1 nS 1 fF 0.5 nS 10 ms Conductance Capacitance Fusion pore conductance Fusion pore PM