1. Phosphoinositide metabolism in synaptic vesicle recycling

2. Techniques in synaptic vesicle recycling

Docking

Priming Ca2+

Fusion

Vesicle reserve

Pre-

Vesicle pools

Morphologically, vesicles are classified as docked or undocked. Docked vesicles are further subdivided into primed and unprimed pools depending on whether they are competent to fuse when cells stimulated.

Munc-18

Munc-13

SNARE complex

SNARE complex and Ca2+ sensor

Invagination Constriction

UncoatingFission

Steps of clathrin-mediated endocytosis

• Invagination to create curvature.

• Fission to separate the vesicle.

• Uncoating to form a real vesicle.

Splenv & De Camilli, 2000

Endocytic invagination mediated by BAR-domain containing protein

Peter et al., 2003

Clathrin is critical to maintain the curvature of endocytic vesicle

Hinrichsen et al., 2006

Endocytic invagination• Bar-domain containing protein such as

amphiphysin is critical for invagination by inducing membrane curvature

• Clathrin, which is recruited by AP-2, is critical in maintaining the curvature of endocytic vesicles

Clathrin-mediated endocytosis

Dynamin is important for pinch-off of endocytic vesicle

Reconstruction of dynamin 1 knockout synapse from electron tomography data.

Hayashi et al., 2008

Synaptojanin 1 is critical for vesicle uncoating

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

1. Phosphoinositide metabolism in synaptic vesicle recycling

2. Techniques in synaptic vesicle recycling

PI(4,5)P2 concentrates on the plasma membrane

PI(4,5)PPI(4,5)P22

PI(3)PPI(3)PPI(3,4,5)PPI(3,4,5)P33

PI(4)PPI(4)PPI(3,5)PPI(3,5)P22

PI(4,5)P2

Functions of PI(4,5)P2:

•Exocytosis and endocytosis

•Ion channel regulation

•Cell adhesion

•Signal transduction

•Actin polymerization/cell motility

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

Ca2+ dependent interaction between synaptotagmin and PI(4,5)P2

Schiavo et al, PNAS 1996

Ca2+

Endocytosis.org

PI(4)P PI(4,5)P2

KinasesPI(4,5)P2 PI(4)P

phosphatases

Coupling between PI(4,5)P2 metabolism and synaptic vesicle recycling

Type I PI(4)P 5-kinase:PIPK IαPIPK IβPIPK Iγ

PI(4)P PI(4,5)P2Kinases

Exocytosis defect in PIPK Iγ KO neuron

Di Paolo et al, Nature 2004

PIPK Iγ

mEPSCs in cultured neurons

Invagination Constriction

UncoatingFission

Importance of lipids in endocytosis

• Invagination to create curvature.

• Fission to separate the vesicle.

• Uncoating to form a real vesicle.

Splenv & De Camilli, 2000

PI(4,5)P2 metabolism is coupled to synaptic vesicle recycling

PI(4)P PI(4,5)P2

KinasesPI(4,5)P2 PI(4)P and PI

Phosphatases

Synaptojanin 1 is a main phosphatase for PI(4,5)P2 metabolism in the brain

Cremona et al, Cell 1999

Synaptojanin 1 WT

KO

Cremona et al. 1999

Synaptojanin 1 is critical for clathrin-mediated endocytosis presynaptically.

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