Visualization of the fate of synaptic vesicle proteins post fusion
Final Report Abstract
The goal of the two-year project was to use gene targeting to replace an endogenous synaptic vesicle gene with a version expressing a transmembrane protein fused at the luminal part to the fluorescent and pH-dependent protein pHluorin (knock-in). This way we should have been able to replace all copies of the respective protein with fluorescently marked ones on synaptic vesicles, and therefore boost the signal-to-noise tremendously compared to current over-expression experiments, enabling robust ‘optical single vesicle recording’. As outlined below, our initial strategies failed as we always lost the pHluorin reporter cassette during vector integration in mouse embryonic stem (ES) cells. A likely explanation for this loss is an additional cross over event between the Neomycin flanking loxP site and the pHluorin cassette. Thus we are now following a different strategy. A second aim was to establish in our lab the preparation of giant retinal bipolar cell terminals from mice for imaging individual exocytosis events using TIRF microscopy. Bipolar cells form gigantic ribbon-type terminals that can be acutely isolated from mouse retina and placed face down onto glass coverslips, enabling TIRFM. This part of the project was successfull. We were able to develop a new class of fluorescent lipid-based probes, based on a pH-sensitive organic dye, cypHer, a derivative of the cyanine Cy5, coupled to phospholipids, as a promising complementary assay to genetically encoded fluorescent proteins like pHluorin fusion proteins for studying exo- and endocytosis as well as resorting and recycling of specific lipids parallel to protein retrieval.
Publications
- A readily retrievable pool of synaptic vesicles. Nat Neurosci. 14:833-9. (2011)
Hua Y, Sinha R, Thiel CS, Schmidt R, Hüve J, Martens H, Hell SW, Egner A, Klingauf J
- Amyloid precursor protein is trafficked and secreted via synaptic vesicles. PLoS One. 6:e18754 (2011)
Groemer TW, Thiel CS, Holt M, Riedel D, Hua Y, Hüve J, Wilhelm BG, Klingauf J
