Super-resolution imaging combined with intermolecular FRET reveals that JNK is localized at the transition fibers of MCCs and interacts with TF components. (A) A Xenopus epidermal MCC expressing RFP-Centrin stained for pJNK and acetylated tubulin. Scale bars, 5 µm. Top-down higher magnification view is shown. Scale bars, 1 µm. Side view (XZ projection) showing that pJNK is localized distally from the basal bodies. Fluorescence intensity profile along the white arrow. (B) (I) Representative Z-stack images of embryos injected with GFP-Chibby and stained for pJNK. (II) Images of embryos injected with GFP-IFT52 and stained for pJNK. (III) Images of embryos injected with GFP-CEP83 and stained for pJNK. (IV) Images of embryos injected with GFP-CEP123 and stained for pJNK. (V and III) Images of embryos injected with GFP-JNK and stained for CEP164. Scale bars, 5 µm. High-magnification top views and fluorescence intensity profiles are shown. Scale bars, 0.5 µm. Side views show XZ projection. (C) Quantification of the mean diameters of the rings of JNK (n = 39 BBs), Chibby (n = 63 BBs), IFT52 (n = 63 BBs from 4 MCCs), CEP83 (n = 57 BBs from 3 MCCs), CEP123 (n = 60 BBs from 4 MCCs), and CEP164 (n = 40 BBs from 3 MCCs) show that JNK’s ring has approximately the same diameter as IFT52, CEP83, CEP123, and CEP164. Bars represent mean ± SEM from two experiments. ***, P < 0.001, unpaired two-tailed t test. (D) Schematic diagram depicting JNK localization in relation to other markers. TZ, transition zone; BB, basal body; TF, transition fiber. (E) Multiciliated cell expressing mScarlet JNK (acceptor) and GFP-CEP123 (donor), before and after acceptor photobleaching. GFP intensity rises, showing that FRET is taking place between GFP and mScarlet and suggesting that JNK is interacting with CEP123. Data represent mean ± SEM (n = 5 MCCs from three embryos).