Visualization of the fate of synaptic vesicle proteins post fusion

• Applicant: Professor Dr. Jürgen Klingauf
• Subject Area: Molecular Biology and Physiology of Neurons and Glial Cells
• Term: from 2008 to 2011
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 54534290

During synaptic transmission small synaptic vesicles filled with neurotransmitter fuse with the plasma membrane to release their content. For maintaining synaptic transmission the exocytosed vesicle proteins have to be retrieved thereafter by compensatory endocytosis. What is the fate of synaptic vesicle proteins post fusion? Do they stay together in a raft-like structure, that can be retrieved efficiently in toto or do they disperse in the plasma membrane and have to be resorted and reclustered for retrieval? While we recently could show that synaptic vesicles exocytosed and retrieved by compensatory endocytosis are non-identical with respect to their protein complement, this does not necessarily imply dispersion of vesicle proteins after fusion. By optically recording single fusion events with high-resolution scanning microscopy we could show for four different transmembrane vesicle proteins fast diffusional dispersion (D = 0.2 µm2/s) post fusion. These experiments however relied on overexpression of the respective proteins, which may introduce artifacts. Thus we are aiming at generating a floxed synapto-TEV-pHluorin knock-in mouse by insertion of synaptobrevin-TEV-pHluorin into the original synaptobrevin locus. This way the endogenous proteins can be fully replaced by the fluorescently tagged version and the fusion process and post-fusion fate of the vesicle proteins can be studied at unprecedented resolution and detail.

• DFG Programme: Research Grants