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bioRxiv · 10.1101/2021.06.01.446599

CeLINC, a fluorescence-based protein-protein interaction assay in C. elegans

Kroll, J. R.·Remmelzwaal, S.·Boxem, M.
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Abstract

Interactions among proteins are fundamental for life and determining whether two particular proteins physically interact can be essential for fully understanding a proteins function. We present C. elegans light-induced co-clustering (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. Co-localization 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. SummaryWe have developed a protein-protein interaction assay for C. elegans to investigate whether pairs of proteins interact in vivo. C. elegans light-induced co-clustering (CeLINC) is based on trapping a fluorescently-tagged bait protein into artificial clusters, and observing whether candidate interacting prey proteins co-cluster with the bait protein. CeLINC can be widely applied as a single set of universal plasmids can be used with existing strains expressing fluorescently-tagged proteins.

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Kroll, J. R., Remmelzwaal, S., Boxem, M.. 2021-06-02. CeLINC, a fluorescence-based protein-protein interaction assay in C. elegans. https://doi.org/10.1101/2021.06.01.446599

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