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| Item Type: | Preprint |
|---|---|
| Title: | Endogenous tagging of Munc13-1 with a SNAP tag as a tool for monitoring synapse nanoarchitecture |
| Creators Name: | Kowald, M., Bachollet, S.P.J.T., Benseler, F., Steinecker, M., Kaushik, S., Soykan, T., Siqi, S., Birke, R., Ilic, D., Brose, N., Hörnberg, H., Lehmann, M., Rizzoli, S.O., Broichhagen, J. and Lipstein, N. |
| Abstract: | ABSTRACT: Synaptic function is governed by highly regulated protein machineries, whose abundance and spatial localization change continually. Studies to determine the dynamic changes in synaptic proteins nanoarchitecture typically rely on immunolabeling, or on the expression of fluorescent proteins. The former employs chemical fluorophores and signal amplification, but requires fixation. The latter disturbs the cells minimally, but uses suboptimal fluorophores. Self-labeling tags have been introduced to combine the advantages of these approaches, and here we introduce a knock-in mouse line where the essential synaptic protein Munc13-1 is endogenously fused to the self-labeling SNAP tag. We demonstrate efficient Munc13-1-SNAP labeling in fixed neurons and brain sections by various SNAP dyes, as well as by a novel bright and far-red compound, SBG-SiR-d12, which we introduce here. SBG-SiR-d12 is designed as a membrane impermeable dye, but we repurposed it to stain cytosolic Munc13-1-SNAP in permeabilized, fixed cells and tissue, where we find it outperforms other dyes as evaluated by both conventional and super-resolution microscopy. Finally, we show that Munc13-1-SNAP can also be monitored by live cell imaging. We conclude that the Munc13-1 SNAP mouse line is a useful tool for the analysis of Munc13-1 nanoarchitectural dynamics in synapses, with a potential for wide adoption. |
| Keywords: | Active Zone, Munc13-1, Unc13a, Synapse, SNAP Tag, SBG-SiR-d12 |
| Source: | bioRxiv |
| Publisher: | Cold Spring Harbor Laboratory Press |
| Article Number: | 2024.10.08.617143 |
| Date: | 10 October 2024 |
| Official Publication: | https://doi.org/10.1101/2024.10.08.617143 |
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