CeLINC, a fluorescence-based protein-protein interaction assay in Caenorhabditis elegans.
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...
| Publicado en: | Genetics Vol. 219; no. 4; pp. 1 - 15 |
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| Autores principales: | , , |
| Formato: | diagnostic images pictorial tables/charts Journal Article |
| Publicado: |
Oxford University Press / USA
Dec2021
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| Acceso en línea: | Ver este registro en EBSCOhost |
| fields | @attributes: recordID: 1 pdfLink: plink: https://search.ebscohost.com/login.aspx?direct=true&db=ccm&AN=154166079&site=ehost-live header: @attributes: shortDbName: ccm uiTerm: 154166079 longDbName: CINAHL Complete uiTag: AN controlInfo: bkinfo: dissinfo: jinfo: jid: 00166731 GNT jtl: Genetics issn: 00166731 maglogo: N pubinfo: dt: Dec2021 vid: 219 iid: 4 pid: 622 pub: Oxford University Press / USA artinfo: ui: 154166079 154166079 154166079 10.1093/genetics/iyab163 154166079 ppf: 1 ppct: 14 formats: tig: atl: CeLINC, a fluorescence-based protein-protein interaction assay in Caenorhabditis elegans. aug: au: Kroll, Jason R. Remmelzwaal, Sanne Boxem, Mike affil: Division of Developmental Biology, Department of Biology, Faculty of Science, Institute of Biodynamics and Biocomplexity, Utrecht University, 3584 CH Utrecht, the Netherlands sug: subj: Caenorhabditis Elegans Biochemical Phenomena ab: 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. pubtype: Academic Journal doctype: diagnostic images pictorial tables/charts Journal Article ougenre: Article language: English refInfo: holdings: @attributes: islocal: N |
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