Table of Contents
Endocytosis at Synapses
Small Synaptic Vesicle Cycle
Big Question
Endocytosis at Synaptic Terminals
Frog NMJ - 1 min stimulation at 10 Hz
Frog NMJ - 1 min stimulation at 10 Hz
Unstimulated Frog NMJ
Frog NMJ - 1 min stimulation at 10 Hz
Free coats and coated vesicles were only observed distant from Active zone
Early Evidence for Endocytosis
Heuser and Reese Model, 1973
Clathrin Mediated Endocytosis Current Working Model
Sequential stages of clathrin coated pits.
Large Reticulospinal Synapse in Lamprey Spinal Cord
Modern Methods to Investigate Recycling
Modern Methods to Study Recycling Amphipathic (impermeant) styryl dyes
FM143 at Frog Nerve Terminal
Method - Membrane Capacitance
Start Here Wednesday to remind where we were on Monday
Regulation of Endocytosis - Why?
Regulation of Endocytosis - How?
Calcium Dependence of Endocytosis
Calcium Dependence of Endocytosis
Calcium dependence of Endocytosis
Calcium dependence of Endocytosis
Recovery from Endocytosis
Clathrin Mediated Endocytosis
Clathrin Mediated Endocytosis
Role of Sorting Endosome
Clathrin Mediated Endocytosis is at least 1 pathway to new SV
Coat Components
Cycle of the Clathrin Coat
Cycle of the Clathrin Coat
Structure of Clathrin Coat
Coat Recruitment
AP2 - adaptor protein
Adaptor protein AP2
Acquisition of Curvature
Anti-Endophillin antibodies disrupt coat curvature
Regulation of Synaptic Vesicle size
Model for Regulation of Vesicle size
Fission - Dynamin
1973 - Drosophila Shibire (dynamin) mutant Fly muscle
Dynamin - GTPase activity - Fission
Antibodies - Immunogold to Dynamin
Dynamin Fission Models
Fission -
Uncoating
Uncoating -
Synaptojanin Knockout Mice
Recapture by synaptic vesicle cluster
Vesicle mixing during recycling at NMJ
Dynamic and Transient Accessory Factors
Eps15, Epsin, intersectin/DAP160, syndapin/pacsin
Calcium Regulation of Endocytosis
Role of Actin
Actin accumulates in vicinity of coated pits when endocytosis is inhibited (e.g. by anti-Endophillin)
"Kiss and Run"
When endocytosis is blocked, a small population of SVs remains
Kiss and Run at Synapses - Methods
Kiss and Run at Synapses - Stimulation Epochs
Same amount of glutamate released
Dye uptake differs depending on mode of stimulation even though same amount of glutamate released
Data - observation 1
Under AP's (red arrow) about 18% of vesicles are released without loss of dye, suggesting "Kiss and Run"
Three endocytic loops in SV cycle
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