2. SNARE complex and Ca 2+ sensor

3. 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

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)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

7. Techniques for synaptic vesicle recycling 1.Electrophysiology 2.Imaging 3.Electron microscopy FM dyes SynaptopHluorin Quantum dots

8. Geppert et al, Cell 1994 1.Electrophysiology: single stimulation

9. 1.Electrophysiology: a train of stimulations Defects in synaptic vesicle endocytosis Dickman et al, Cell 2005

10. Autaptic hippocampal neuron Bekkers & Stevens, PNAS 1991 1.Electrophysiology

11. Distinct vesicle pools Rettig & Neher, Science 2002 1.Electrophysiology

12. Detection of RRP by hypertonic sucrose solution Stevens & Sullivan, Neuron 1998 1.Electrophysiology Hypotonic 0.5 M sucrose

13. Rhee et al, Cell 2002 A reduction in RRP size in Munc-13 KO neuron 1.Electrophysiology

14. Gaffield & Betz, Nat Protoc 2009 2. Imaging: FM dye FM dyes

16. Sankaranarayanan et al, Biophys J 2000 2. Imaging: SynaptopHluorin SynaptopHluorin Miesenbock et al, Nature 1998

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. Detection of full fusion and kiss-and-run by quantum dots Zhang et al, Science 2009 2. Imaging: quantum dots

22. Detection of full fusion and kiss-and-run by quantum dots Zhang et al, Science 2009 2. Imaging: quantum dots

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

24. Hayashi et al, PNAS 2008 3. Electron microscopy Defects in synaptic vesicle endocytosis

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

26. Adrenal gland Chromaffin cells as the model system for vesicle cycling Techniques: Capacitance measurements Amperometry Big vesicle size (150-450 nm in diameter) Oxidizable catecholamine

27. 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

28. Whole cell capacitance technique stimulation Capacitance (pF) 60 fF

29. Patch pipette Detection in exocytosis and endocytosis of single vesicles by cell-attached capacitance technique Conductance Capacitance Im Exocytosis Endocytosis Kiss-and-run

30. Detection the dynamics of fission-pore during endocytosis

31. 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