Fig. 3: TRFs require each other for their loading to ciliary EVs.

a Flattened z-stack shows that disruption of trf-2 abrogates loading of TRF-1::mScarlet to PKD-2::GFP EVs. TRF-1::mScarlet stays in the cilium and does not reach the ciliary tip, as a representative fluorescent profiling shows (a’); fluorescence values are normalized to the average of minimum and maximum values for each cilium (n = 10 cilia). a” Scatter plot shows no correlation between PKD-2::GFP and TRF-1::mScarlet within shafts (blue) and tips (yellow) of cilia (n = 122 cilia) of the trf-2(tm5167) mutant. Pearson correlation coefficients from a two-sided test are reported alongside unadjusted p-values. For the full dataset that includes all rays, refer to Supplementary Fig. 6. Source Data are provided as SourceData_Main.xls and SourceData_SuppFig6.xls files. b Flattened z-stack shows that disruption of trf-1 abrogates ciliary localization of TRF-2::GFP, and thus, no TRF-2::GFP is loaded to PKD-2::mScarlet EVs. b’ Quantification of total fluorescence within the cilium shows that TRF-2::GFP ciliary levels in the trf-1 mutant are reduced tenfold compared to WT cilia, whereas PKD-2::mScarlet total levels remain unaffected. n = 73 cilia for WT, and 112 cilia for the trf-1(nr2014) mutant. Two-sided Wilcoxon Rank Sum test, p < 2.2e-16 for TRF-2::GFP, and p = 0.006035 for PKD-2::mScarlet. c Flattened z-stack through cell bodies of RnB neurons showing reduced cytoplasmic levels of TRF-2::GFP. c’ Quantification of cell body cytoplasmic levels of PKD-2::mScarlet and TRF-2::GFP as a ratio of average cytoplasmic fluorescence normalized by fluorescence within the nucleus on a single image plane, n = 96 cell bodies for WT, n = 147 cell bodies for the trf-1(nr2014) mutant. Two-sided Wilcoxon Rank Sum test, p < 2.2e-16 for TRF-2::GFP, and p = 0.4343 for PKD-2::mScarlet. The box-and-whiskers plots (b’ and c’) show the median values where the box covers two quartiles around the median, and the whiskers extend 1.5 quartiles from the median. Source Data are provided as SourceData_Main.xls file. d Homology of C. elegans TRFs to human TRAFs. e Working model of the molecular mechanism for loading TRFs to ciliary EVs. LOV-1 is required for loading both TRFs to ciliary EVs; in the lov-1 mutant, cilia produce EVs without TRFs. TRFs are required for loading each other to the ciliary EVs; in the trf-2 mutant, cilia produce EVs without TRF-1, whereas the trf-1 mutation abrogates TRF-2 ciliary localization. Additional supporting data for the model are presented in Supplementary Fig. 4, showing the colocalization of TRF-1::mScarlet and TRF-2::GFP, and Supplementary Fig. 5, showing that TRFs are not required for release of polycystins in ciliary EVs. Source Data are provided as SourceData_SuppFig4.xls and SourceData_SuppFig5.xls files.