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Synaptic vesicle endocytosis is critical for ensuring rapid local recycling of synaptic vesicles to enable ongoing neuronal activity. We utilized the sensitivity and robustness of the pHluorin assay of synaptic vesicle recycling to probe the control of endocytosis spatially and in response to varying stimulation conditions. We examined the role of the large GTPases dynamin 1 and dynamin 3 in synaptic vesicle endocytosis; which have been thought to be essential for the ssion of budding vesicles from the plasma membrane. Dynamin 1, 3 and 1/3 double knockouts indicated that there is redundant presynaptic function of the proteins. Loss of either protein alone results in a subtle phenotype; only in the double knockout is synaptic vesicle endocytosis strongly impaired. However, even lacking both dynamin 1 and dynamin 3 synaptic vesicles can still undergo many rounds of exocytosis and endocytosis. Individual nerve terminals from the same neuron often di er in neurotransmitter release characteristics. The extent to which endocytic retrieval of synaptic vesicle components di ers across nerve terminals from the same axon is unknown. We used pHluorin-based assays to undertake a large-scale analysis of endocytosis kinetics of individual boutons. Our data indicates that endocytosis kinetics are primarily set at a cell-wide level rather than at that of individual boutons. We observed a 4 fold range in cell wide time constants (from 5s to 20s) that was not dependent upon the type of neurotransmitter being utilized (excitatory/inhibitory), nor the history of activity of the neuron. In addition to cell to cell variation in endocytic kinetics, we also explored the modulation of endocytosis by the stimulation and/or Ca2+ in ux. We demonstrated two regimes of Ca2+ modulation, a Ca2+ dependent acceleration for small stimuli and a Ca2+ dependent slowing for larger stimuli. The acceleration is especially prominent at physiological temperature, accelerating the endocytic time constant by 50% over 25AP at 10Hz. The acceleration has a persistence time >20s suggesting an optimization of endocytosis for infrequent bursts of activity. Utilizing the dynamin 1/3 double knockout we showed that the acceleration depends on the successful dephosphorylation of dynamin at 2 serines previously identi ed as substrates for the Ca2+ dependent phosphatase, calcineurin. Removal of the F-Bar domain protein syndapin 1, a dynamin 1 binding partner that requires dynamin 1s dephosphorylation, also distortedthe acceleration. We examined the setpoint of synaptic vesicle endocytosis between boutons, between cells, and under di erent stimuli conditions; indicating that there is a lot of potential for modulation of the endocytic kinetics: a 4 fold range of cell wide modulation and a stimulus dependence that shows evidence for a clear optimal stimulus for minimizing the endocytosis time constant in a dynamin phosphorylation state dependent manner.


A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

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