A DARPin-based molecular toolset to probe gephyrin and inhibitory synapse biology
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Evaluation Summary:
The authors developed a set of synthetic proteins, Designed Ankyrin Repeat Proteins (DARPins), that bind gephyrin, the main scaffold protein at inhibitory postsynaptic sites, and characterize them extensively to study gephyrin cluster morphology and biochemistry. In several aspects, DARPins outperform traditional antibodies. This study is clear, well organized and well written, demonstrating that DARPins can be important tools synaptic, cellular and circuit neuroscience fields.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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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 nonsynaptic functions. This study demonstrates anti-gephyrin DARPins as a versatile platform for studying inhibitory synapses in an unprecedented manner.
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Evaluation Summary:
The authors developed a set of synthetic proteins, Designed Ankyrin Repeat Proteins (DARPins), that bind gephyrin, the main scaffold protein at inhibitory postsynaptic sites, and characterize them extensively to study gephyrin cluster morphology and biochemistry. In several aspects, DARPins outperform traditional antibodies. This study is clear, well organized and well written, demonstrating that DARPins can be important tools synaptic, cellular and circuit neuroscience fields.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The paper by Campell et al., describes the isolation and characterization of Designed Ankyrin Repeat Proteins (DARP) that recognize distinct forms of gephryin. Gephyrin is a key determinant for postsynaptic accumulation of both glycine and GABAAR at synapses and thereby determines the efficacy of fast synaptic inhibition. In addition to this gephyrin regulates the synthesis of molybedum-cofactor, an essential co-factor for a number of metabolic enzymes.
The authors create DARPs that recognize specific splice forms of gephyrin and versions that discriminate between phosphorylated and dephosphorylated forms of gephyrin. These new tools reveal the differential recruitment of gephyrin isoforms to axo-axonic and somatodendritic synapses. In addition to these new tools allow the efficient one-step purification of …
Reviewer #1 (Public Review):
The paper by Campell et al., describes the isolation and characterization of Designed Ankyrin Repeat Proteins (DARP) that recognize distinct forms of gephryin. Gephyrin is a key determinant for postsynaptic accumulation of both glycine and GABAAR at synapses and thereby determines the efficacy of fast synaptic inhibition. In addition to this gephyrin regulates the synthesis of molybedum-cofactor, an essential co-factor for a number of metabolic enzymes.
The authors create DARPs that recognize specific splice forms of gephyrin and versions that discriminate between phosphorylated and dephosphorylated forms of gephyrin. These new tools reveal the differential recruitment of gephyrin isoforms to axo-axonic and somatodendritic synapses. In addition to these new tools allow the efficient one-step purification of differing gephyrin isoforms and their respective binding partners.
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Reviewer #2 (Public Review):
In the manuscript, Campbell and coworkers have developed a set of synthetic proteins, Designed Ankyrin Repeat Proteins (DARPins), which are able to bind gephyrin, the main scaffold protein at inhibitory postsynaptic sites. The authors extensively characterize DARPins for the localization of gephyrin clusters and the study of the gephyrin clusters morphology. In addition, the Authors show that DARPins are a valid tool for biochemical studies. Through the manuscript the DARPins performances are compared with those of commercially available anti-gephyrin antibodies. The Authors demonstrate that, in several aspects, DARPins outperform antibodies.
This study is clear, well organized and well written. It provides compelling evidence that DARPins represent an important tool for the advancement of the synaptic, …
Reviewer #2 (Public Review):
In the manuscript, Campbell and coworkers have developed a set of synthetic proteins, Designed Ankyrin Repeat Proteins (DARPins), which are able to bind gephyrin, the main scaffold protein at inhibitory postsynaptic sites. The authors extensively characterize DARPins for the localization of gephyrin clusters and the study of the gephyrin clusters morphology. In addition, the Authors show that DARPins are a valid tool for biochemical studies. Through the manuscript the DARPins performances are compared with those of commercially available anti-gephyrin antibodies. The Authors demonstrate that, in several aspects, DARPins outperform antibodies.
This study is clear, well organized and well written. It provides compelling evidence that DARPins represent an important tool for the advancement of the synaptic, cellular and circuit neuroscience fields.
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