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

Area of Science:

• Neuroscience
• Cell Biology
• Molecular Biology

Background:

• Synaptic function relies on dynamic protein machineries.
• Existing methods for studying synaptic protein changes (immunolabeling, fluorescent proteins) have limitations.
• Self-labeling tags offer a way to combine advantages of fixation-based and live imaging techniques.

Purpose of the Study:

• To introduce a knock-in mouse line for endogenous labeling of the presynaptic protein Munc13-1 using the SNAP self-labeling tag.
• To characterize a novel far-red, cell-impermeable dye (SBG-SiR-d12) for SNAP-tag labeling.
• To demonstrate the utility of this system for live-cell imaging and analysis of synaptic nanoarchitecture.

Main Methods:

• Generation of a knock-in mouse line expressing Munc13-1 fused to the SNAP tag.
• Labeling of Munc13-1-SNAP in fixed and live neurons using various SNAP dyes, including SBG-SiR-d12.
• Live-cell imaging using confocal and super-resolution microscopy.

Main Results:

• Efficient labeling of endogenous Munc13-1-SNAP in fixed and live neurons.
• Characterization of SBG-SiR-d12 as an effective dye for extracellular and intracellular SNAP-tag labeling.
• Successful monitoring of Munc13-1 dynamics in living neurons via advanced microscopy techniques.

Conclusions:

• The Unc13a^SNAP mouse line is a valuable tool for presynaptic compartment analysis.
• This system facilitates the study of presynaptic nanoarchitectural dynamics.
• The developed labeling strategy has broad potential for neuroscience research.