1.Phosphoinositide metabolism in synaptic vesicle recycling 2.Techniques in synaptic vesicle recycling SNARE complex and Ca 2+ sensor Clathrin-mediated endocytosis Invagination Constriction Uncoating Fission Clathrin triskelion AP-2 (binds to clathrin an PIP 2 ) Clathrin-mediated endocytosis Invagination Constriction Uncoating Fission Peter et al, Science 2004 Slepnev & De Camilli, Nat Rev Neurosci 2000 Clathrin-mediated endocytosis Invagination Constriction Uncoating Fission Slepnev & De Camilli, Nat Rev Neurosci 2000 Full fusion and kiss-and-run Harata et al, J Neurochem 2006 1.Phosphoinositide metabolism in synaptic vesicle recycling 2.Techniques in synaptic vesicle recycling PI(4,5)P 2 concentrates on the plasma membrane PI(4,5)P 2 PI(3)P PI(3,4,5)P 3 PI(4)P PI(3,5)P 2 PI(4,5)P 2 Functions of PI(4,5)P 2 : Exocytosis and endocytosis Ion channel regulation Cell adhesion Signal transduction Actin polymerization/cell motility 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 Ca 2+ dependent interaction between synaptotagmin and PI(4,5)P 2 Schiavo et al, PNAS 1996 Ca 2+ Endocytosis.org PI(4)P PI(4,5)P 2 Kinases PI(4,5)P 2 PI(4)P phosphatases Coupling between PI(4,5)P 2 metabolism and synaptic vesicle recycling Type I PI(4)P 5-kinase: PIPK I PIPK I PIPK I PI(4)P PI(4,5)P 2 Kinases Exocytosis defect in PIPK I KO neuron Di Paolo et al, Nature 2004 PIPK I mEPSCs in cultured neurons outside cytosol PI(4,5)P 2 adaptors clathrin cargo PI(4,5)P 2 is critical for clathrin-mediated endocytosis Endocytic Adaptors Interacting w/ PIP2: AP-2; AP-180; Epsin; N-WASP; Dynamin PI(4,5)P 2 metabolism is coupled to synaptic vesicle recycling PI(4)P PI(4,5)P 2 Kinases PI(4,5)P 2 PI(4)P and PI Phosphatases Synaptojanin 1 is a main phosphatase for PI(4,5)P 2 metabolism in the brain Cremona et al, Cell 1999 Synaptojanin 1 WT KO Cremona et al Synaptojanin 1 is critical for clathrin-mediated endocytosis presynaptically. 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 Advantages Disadvantages 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 edicalschool/departments/physiology/faculty/Re gularFaculty/Pages/Betz.aspx 2. 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 2. Imaging: Synaptophysin-pHluorin Granseth et al, Neuron 2006 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