Quantitative Analysis of Synaptic Vesicle Membrane Trafficking

by Katharina Johanna Seitz
Doctoral thesis
Date of Examination:2017-08-10
Date of issue:2018-08-02
Advisor:Prof. Dr. Silvio O. Rizzoli
Referee:Prof. Dr. Silvio O. Rizzoli
Referee:PD Dr. Halyna Shcherbata
Persistent Address: http://dx.doi.org/10.53846/goediss-6981

 

 

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Abstract

English

The fate of synaptic vesicles upon fusion to the plasma membrane is unclear. Two models have been proposed: vesicle proteins either stay clustered in patches, thus retaining the molecular identity of individual vesicles, or they disintegrate and intersperse with other surface-resident proteins. Although this problem has been investigated in numerous studies, no consensus has been reached as to which of the models is true. I developed a new method using single-domain antibodies and STED microscopy to selectively visualize newly exocytosed vesicle proteins, and determine changes in the sizes of vesicle protein clusters in the time of recovery after exocytosis. I applied the method to different proteins of interest in hippocampal neuronal cultures and Drosophila melanogaster neuromuscular junctions. Using this single-domain antibody-based vesicle tracking assay, I could show that synaptophysin-pHluorin and synaptobrevin-pHluorin remain clustered on the plasma membrane of synaptic boutons for at least the first minute after exocytosis. Patches of synaptobrevin-pHluorin that diffuse into axonal plasma membrane segments, however, disintegrate during the same time frame. This indicates that in addition to already proposed mechanisms for maintenance of vesicle integrity, there may exist an additional mechanism that is inherent to the environment of synaptic boutons, as opposed to the vesicles themselves. A supporting endocytic scaffold might aid in clustering synaptic vesicle proteins.
Keywords: neurobiology; synaptic vesicles; membrane trafficking
 
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