Institute of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland.
Functional Genomics Centre, University of Zurich, Zurich, Switzerland.
Elife. 2022 Oct 31;11:e80895. doi: 10.7554/eLife.80895.
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.
神经科学目前需要使用抗体来研究突触蛋白，其中抗体结合被用作相关性来定义突触的存在、可塑性和调节。Gephyrin 是一种抑制性突触支架蛋白，用于标记 GABA 能和甘氨酸能突触后位点。尽管 Gephyrin 在调节抑制性传递中很重要，但由于现有试剂的可处理性，其研究目前受到限制。设计的锚蛋白重复蛋白（DARPins）是一类通过体外选择从各种文库中衍生的合成蛋白结合物，并通过高通量筛选进行测试，以产生特异性结合物。为了生成用于研究抑制性突触的功能多样化工具集，我们针对代表磷酸化和去磷酸化状态的 Gephyrin 突变体筛选了 DARPin 文库。我们验证了抗 Gephyrin DARPin 克隆在大鼠神经元培养物和小鼠脑组织中对 Gephyrin 簇的形态鉴定中的强大用途，发现了以前被忽视的簇。该 DARPin 基工具集包括具有异质 Gephyrin 结合模式的克隆，这些克隆允许鉴定迄今为止最广泛的 Gephyrin 相互作用组，并定义了新的潜在相互作用物类，为理解 Gephyrin 的非突触功能创建了一个框架。这项研究表明，抗 Gephyrin DARPins 是一种用于以前所未有的方式研究抑制性突触的多功能平台。
