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.
Metrics
-
- 1,577
- views
-
- 238
- downloads
-
- 8
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
A DARPin-based molecular toolset to probe gephyrin and inhibitory synapse biology
eLife 11:e80895.
https://doi.org/10.7554/eLife.80895
Further reading
-
- Neuroscience
The increasing use of tissue clearing techniques underscores the urgent need for cost-effective and simplified deep imaging methods. While traditional inverted confocal microscopes excel in high-resolution imaging of tissue sections and cultured cells, they face limitations in deep imaging of cleared tissues due to refractive index mismatches between the immersion media of objectives and sample container. To overcome these challenges, the RIM-Deep was developed to significantly improve deep imaging capabilities without compromising the normal function of the confocal microscope. This system facilitates deep immunofluorescence imaging of the prefrontal cortex in cleared macaque tissue, extending imaging depth from 2 mm to 5 mm. Applied to an intact and cleared Thy1-EGFP mouse brain, the system allowed for clear axonal visualization at high imaging depth. Moreover, this advancement enables large-scale, deep 3D imaging of intact tissues. In principle, this concept can be extended to any imaging modality, including existing inverted wide-field, confocal, and two-photon microscopy. This would significantly upgrade traditional laboratory configurations and facilitate the study of connectomes in the brain and other tissues.
-
- Neuroscience
- Physics of Living Systems
Neurons generate and propagate electrical pulses called action potentials which annihilate on arrival at the axon terminal. We measure the extracellular electric field generated by propagating and annihilating action potentials and find that on annihilation, action potentials expel a local discharge. The discharge at the axon terminal generates an inhomogeneous electric field that immediately influences target neurons and thus provokes ephaptic coupling. Our measurements are quantitatively verified by a powerful analytical model which reveals excitation and inhibition in target neurons, depending on position and morphology of the source-target arrangement. Our model is in full agreement with experimental findings on ephaptic coupling at the well-studied Basket cell-Purkinje cell synapse. It is able to predict ephaptic coupling for any other synaptic geometry as illustrated by a few examples.
