A DARPin-based molecular toolset to probe gephyrin and inhibitory synapse biology
- University of Zurich, Switzerland
Abstract
Neuroscience currently requires the use of antibodies to study synaptic proteins, where antibody binding is used as a correlate to define the presence, plasticity, and regulation of synapses. Gephyrin is an inhibitory synaptic scaffolding protein used to mark GABAergic and glycinergic postsynaptic sites. Despite the importance of gephyrin in modulating inhibitory transmission, its study is currently limited by the tractability of available reagents. Designed Ankyrin Repeat Proteins (DARPins) are a class of synthetic protein binder derived from diverse libraries by in vitro selection, and tested by high-throughput screening to produce specific binders. In order to generate a functionally diverse toolset for studying inhibitory synapses, we screened a DARPin library against gephyrin mutants representing both phosphorylated and dephosphorylated states. We validated the robust use of anti-gephyrin DARPin clones for morphological identification of gephyrin clusters in rat neuron culture and mouse brain tissue, discovering previously overlooked clusters. This DARPin-based toolset includes clones with heterogenous gephyrin binding modes that allowed for identification of the most extensive gephyrin interactome to date, and defined novel classes of putative interactors, creating a framework for understanding gephyrin's non-synaptic functions. This study demonstrates anti-gephyrin DARPins as a versatile platform for studying inhibitory synapses in an unprecedented manner.
Data availability
All relevant mass spectrometry data has been deposited to the ProteomeXchange Consortium via the PRIDE (http://www.ebi.ac.uk/pride) partner repository.Project Name: Gephyrin interactome from mouse brain lysates using anti-gephyrin antibody and anti-gephyrin DARPinsProject accession: PXD033641Project DOI: 10.6019/PXD033641
Article and author information
Author details
Shiva K Tyagarajan
Funding
Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (310030_192522 /1)
- Shiva K Tyagarajan
Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (310030_192689)
- Andreas Plückthun
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: The collection of embryonic and adult tissue was performed in accordance with the European Community Council Directives of November 24th 1986 (86/609/EEC). Tissue collection was performed under license ZH011/19 approved by the Cantonal Veterinary office of Zurich.
Copyright
© 2022, Campbell et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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A DARPin-based molecular toolset to probe gephyrin and inhibitory synapse biology
eLife 11:e80895.
https://doi.org/10.7554/eLife.80895
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