Dynamin: Regulation of Vesicle Cycling and Resting Stability of Vesicles at Presynaptic Terminals

Compensatory synaptic vesicle endocytosis during kiss-and-run vesicle fusion is not fully understood. Specifically, it is unclear whether proteins involved in classical clathrin-mediated endocytosis (CME) are also required for vesicle retrieval following kiss-and-run vesicle fusion. Part of the impetus for the work presented in this thesis was to determine whether one such protein, dynamin, is responsible for vesicle retrieval during kiss-and-run fusion. Dynamin is a key protein in membrane fission during CME, providing an energy utilizing GTPase reaction to sever the vesicle from the plasma membrane. However, because vesicles may not fully collapse during kiss-and-run fusion it is unclear whether there exists an analogous role for dynamin in this process. Utilizing a 5-HT-mediated model of presynaptic inhibition favoring kiss-and-run vesicle fusion in a model synapse, the lamprey giant reticulospinal synapse, I present findings that demonstrate kiss-and-run fusion as a frequency-dependent, calcium-sensitive effect of 5-HT. Furthermore, I demonstrate that dynamin does not appear to be conclusively involved in compensatory endocytosis during kiss-and-run vesicle fusion. While addressing this question, I employed different means of pharmacologically inhibiting dynamin. These experiments revealed that inhibition of the action of dynamin, either by inactivating its GTPase activity, or by modifying its interaction with amphiphysin, depletes synaptic vesicle numbers at presynaptic active zones. This finding suggests that dynamin is important in maintaining vesicle pools in resting presynaptic terminals, but also provides new avenues for better elucidating potential roles of endocytic proteins in paradigms used to investigate kiss-and-run vesicle fusion.