Fig. 3: DDX-23 enhances the partitioning of MAB-10 proteins into nuclear foci.

a Representative confocal fluorescent images of 4 animals expressing n5909[MAB-10::GFP] (green channel) and n6092[tagRFP-T::ddx-23] (red channel), which fluorescently tag mab-10 and ddx-23, respectively, at their endogenous loci. Inset: Hypodermal nuclei. Scale bar, 40 μm. b Yeast two-hybrid spot assay showing the interaction of DDX-23 protein fused to the GAL4 activation domain (upper right spot) but not of a control with the GAL4 activation domain-only (“Empty vector”) protein with MAB-10 protein fused to the GAL4 DNA-binding domain in quadruple drop-out plates (-His/-Ade/-Trp/-Leu) containing the competitive inhibitor of histidine synthesis 3-AT. c Schematic representation of the split GFP (spGFP) approach for assessing protein-protein interactions in vivo. Animals expressed an N-terminal GFP fragment (spGFPN) fused to DDX-23 (purple) in combination with a C-terminal GFP fragment (spGFPC) fused to MAB-10 (blue). A physical interaction between the DDX-23::spGFPN and MAB-10::spGFPC proteins leads to the emission of green fluorescence (depicted as a green circle). d Representative confocal images (merged DIC and GFP channels) of spGFP experiments using DDX-23::spGFPN and MAB-10::spGFPC (or spGFPC by itself as a control). Images are representative of 8–10 animals per experimental condition. Scale bar, 20 μm. e Confocal fluorescence images of animals expressing a MAB-10 translational reporter (P_mab-10::mab-10::mCherry::mab-10 3’UTR; red channel), DDX-23::spGFPN and MAB-10::spGFPC (or empty spGFPC as a control; green channel). Image is representative of five animals per condition. Inset: seam cell. Dotted white circle, nuclear circumference; scale bar, 20 μm. f Representative images of in vitro assays testing heterotypic interactions of purified DDX-23-mNeonGreen and MAB-10-mScarlet I (right panel pairs). DDX-23-mNeonGreen can self-interact in the presence of mScarlet I alone (left panel pairs). MAB-10-mScarlet I can self-interact in the presence of mNeonGreen alone (middle panel pairs) but interaction is enhanced in the presence of DDX-23-mNeonGreen (right panel pairs). Images are representative of three independent experiments. Micrographs were color-inverted for better visualization. Scale bar, 200 μm. g Schematic view of control mNeonGreen protein alone, wild-type DDX-23-mNeonGreen, mutant DDX-23(I383F)-mNeonGreen, and a DDX-23 protein lacking its IDR (DDX-23(ΔIDR)-mNeonGreen) used for recombinant protein production. h Representative images of in vitro interaction assays of control mNeonGreen protein alone, wild-type DDX-23-mNeonGreen, mutant DDX-23(I383F)-mNeonGreen, and DDX-23(ΔIDR)-mNeonGreen, each with purified MAB-10-mScarlet I. Images are representative of three independent experiments. Micrographs were color-inverted for better visualization. Scale bar, 50 μm. i Quantification of the number of MAB-10 spots when mixed in heterotypic assays with an mNeonGreen alone control, wild-type DDX-23, mutant DDX-23[I383F] or DDX-23(ΔIDR) in three independent experiments. Error bars, mean value +/− SEM. ***P = 0.0004 (mNeonGreen vs. DDX-23(WT)-mNeonGreen), ***P = 0.0007 (DDX-23(WT)-mNeonGreen vs. DDX-23(ΔIDR)-mNeonGreen) and **P = 0.0024 (DDX-23(WT)-mNeonGreen vs. DDX-23[I383F]-mNeonGreen) (two-sided t tests). j Fraction of animals with discrete MAB-10 foci in 1-day-old wild-type, ddx-23(n5703), and ddx-23(n5705) adults. n = 30 independent animals were scored for each genotype. All strains contain the reporter n5909 [mab-10::gfp]. k DIC and confocal fluorescent images of hypodermal cells in 1-day-old wild-type, ddx-23(n5703) and ddx-23(n5705) adults expressing the reporter n5909 [mab-10::gfp]. Images are representative of 12–15 animals per genotype. Scale bar, 20 μm. l Corrected total fluorescent intensity of MAB-10::GFP in the hypodermal nuclei of 1-day-old wild-type (n = 15) and ddx-23(n5705) (n = 12) adults expressing the reporter n5909 [mab-10::gfp]. ns not significant, P = 0.2381 (two-sided t test). Source data for (i, j, l) are provided as a Source Data file.