Division of Developmental Biology, Department of Biology, Faculty of Science, Institute of Biodynamics and Biocomplexity, Utrecht University, 3584 CH Utrecht, the Netherlands.
Genetics. 2021 Dec 10;219(4). doi: 10.1093/genetics/iyab163.
Interactions among proteins are fundamental for life and determining whether two particular proteins physically interact can be essential for fully understanding a protein's function. We present Caenorhabditis elegans light-induced coclustering (CeLINC), an optical binary protein-protein interaction assay to determine whether two proteins interact in vivo. Based on CRY2/CIB1 light-dependent oligomerization, CeLINC can rapidly and unambiguously identify protein-protein interactions between pairs of fluorescently tagged proteins. A fluorescently tagged bait protein is captured using a nanobody directed against the fluorescent protein (GFP or mCherry) and brought into artificial clusters within the cell. Colocalization of a fluorescently tagged prey protein in the cluster indicates a protein interaction. We tested the system with an array of positive and negative reference protein pairs. Assay performance was extremely robust with no false positives detected in the negative reference pairs. We then used the system to test for interactions among apical and basolateral polarity regulators. We confirmed interactions seen between PAR-6, PKC-3, and PAR-3, but observed no physical interactions among the basolateral Scribble module proteins LET-413, DLG-1, and LGL-1. We have generated a plasmid toolkit that allows use of custom promoters or CRY2 variants to promote flexibility of the system. The CeLINC assay is a powerful and rapid technique that can be widely applied in C. elegans due to the universal plasmids that can be used with existing fluorescently tagged strains without need for additional cloning or genetic modification of the genome.
蛋白质之间的相互作用是生命的基础，确定两个特定的蛋白质是否发生物理相互作用对于全面了解蛋白质的功能可能至关重要。我们提出了秀丽隐杆线虫光诱导共聚集（CeLINC），这是一种光学二元蛋白质-蛋白质相互作用测定法，用于确定两种蛋白质是否在体内相互作用。基于 CRY2/CIB1 光依赖性寡聚化，CeLINC 可以快速且明确地鉴定荧光标记蛋白之间的蛋白质-蛋白质相互作用。通过针对荧光蛋白（GFP 或 mCherry）的纳米抗体捕获荧光标记的诱饵蛋白，并将其带入细胞内的人工聚集体中。荧光标记的猎物蛋白在聚集体中的共定位表明存在蛋白质相互作用。我们用一系列阳性和阴性参考蛋白对系统进行了测试。该测定系统的性能非常稳健，在阴性参考对中未检测到假阳性。然后，我们使用该系统测试了顶端和基底极性调节剂之间的相互作用。我们确认了 PAR-6、PKC-3 和 PAR-3 之间的相互作用，但未观察到基底外侧 Scribble 模块蛋白 LET-413、DLG-1 和 LGL-1 之间的物理相互作用。我们已经生成了一个质粒工具包，允许使用自定义启动子或 CRY2 变体来提高系统的灵活性。CeLINC 测定法是一种强大而快速的技术，由于可以使用现有的荧光标记菌株的通用质粒，而无需对基因组进行额外的克隆或遗传修饰，因此可以在秀丽隐杆线虫中广泛应用。
