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Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence
Published on: January 29, 2019
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Identifying Synaptic Proteins by In Vivo BioID from Mouse Brain
Akiyoshi Uezu1, Scott Soderling2
1The Department of Cell Biology, Duke University Medical School, Durham, NC, USA. akiyoshi.uezu@duke.edu.
Methods in Molecular Biology (Clifton, N.J.)
|May 25, 2019
Summary
Researchers developed in vivo BioID (iBioID) to study inhibitory synapses in the mouse brain. This new method enables purification and analysis of previously intractable inhibitory synaptic protein complexes.
Area of Science:
- Neurobiology
- Molecular Biology
- Biochemistry
Background:
- Central nervous system synapses are either excitatory or inhibitory.
- Excitatory postsynaptic protein complexes are well-characterized.
- Inhibitory postsynaptic densities have been biochemically intractable.
Purpose of the Study:
- To adapt the BioID proximity labeling method for in vivo analysis of inhibitory synaptic complexes.
- To develop a protocol for purifying biotinylated proteins from inhibitory synapses in the mouse brain.
Main Methods:
- Adaptation of the BioID technique for in vivo application (iBioID).
- Utilizing a biotin ligase mutant (BirA*) fused to synaptic bait proteins.
- Labeling and purification of biotinylated proteins from mouse brain tissue.
Main Results:
- Successful implementation of iBioID for targeting inhibitory synaptic complexes.
- Enabled the labeling and purification of proteins proximal to synaptic bait proteins in vivo.
- Demonstrated the feasibility of studying previously intractable inhibitory synaptic structures.
Conclusions:
- The iBioID method provides a powerful new tool for investigating inhibitory synapses.
- This technique can be applied to study other challenging substructures in vivo.
- Advances understanding of inhibitory synaptic neurobiology and protein complexes.
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