Endogenous SNAP-Tagging of Munc13-1 for Monitoring Synapse Nanoarchitecture

Authors:

Maria Kowald, Sylvestre P. J. T. Bachollet, Fritz Benseler, Maria Steinecker, Moritz Boll, Sofia Kaushik, Tolga Soykan, Siqi Sun, Ramona Birke, Dragana Ilic, Nils Brose, Hanna Hörnberg, Martin Lehmann, Silvio O. Rizzoli, Johannes Broichhagen, Noa Lipstein

Keywords:

Active zone; Munc13-1; Unc13a; Synapse; SNAP tag; SBG-SiR-d12

Abstract:

Synaptic function is governed by highly regulated protein machineries, whose abundance and spatial localization change continually. Studies to determine dynamic changes in synaptic protein 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 enables monitoring of proteins by live microscopy but uses suboptimal fluorophores. Self-labeling tags have been introduced to combine the advantages of these two approaches, and here we introduce a knock-in mouse line where the essential presynaptic protein Munc13–1 is endogenously fused to the self-labeling SNAP tag. We demonstrate efficient Munc13–1-SNAP labeling in fixed cultured neurons and in brain sections by various SNAP dyes, as well as by a novel far-red and cell impermeable compound, SBG-SiR-d12. We introduce and characterize SBG-SiR-d12 as a highly efficient dye for SNAP-tag labeling of extracellular epitopes and of intracellular proteins such as Munc13–1 in fixed and permeabilized tissue. Finally, we show that Munc13–1-SNAP can be labeled in living neurons and monitored through live-cell imaging using confocal and super resolution microscopy. We conclude that the Unc13a^SNAP mouse line is a useful tool for labeling the presynaptic compartment and for the analysis of presynaptic nanoarchitectural dynamics, with potential for wide adoption.

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