(A) Color-based time coding of a high-speed time-lapse movie of an MCC; Successive frames of the movie are color coded as indicated in the time key and overlaid; distinct colors in the overlay reveals ciliary movement. (B) Similar time-coding of an MCC after Heatr2-KD; the lack of color reflects the absence of ciliary movement between frames in the movie. (C) Membrane labeling with CAAX-RFP (pink) reveals normal cilia morphology in control MCCs (an projection of the confocal optical slices specifically through the apical surface is shown with; dashed lines indicate MCCs. (c’) Labelling with GFP-Dnai2 reveals normal localization to the motile axonemes shown in panel C. (D) Membrane labeling reveals normal morphology of motile cilia in MCCs after Heatr2 KD. (d’) GFP-Dnai2 is lost from the motile cilia shown in panel D. (E) Dnai2 is present in DynAPs visible in an en face projection through the cytoplasm of control MCCs (indicated by dashed lines). (F) Despite loss from motile axonemes, Dnai2 remains localized to DynAps in the cytoplasm of MCCs after Heatr2 knockdown. (G) Despite the presence of DynAPs in Heatr2-KD MCCs, the number of foci in these cells is significantly reduced relative to wild-type MCCs. p = 1.27 × 10 −10 by two-sample t-test (n = 25 wild-type cells, 26 Heatr2-KD cells across two experiments, three embryos each). (H, h’) FRAP reveals that Heatr2 knockdown significantly impairs the mobility of GFP-Ktu in DynAPs. Control vs. Heatr2-KD GFP-Ktu mobile fraction, p < 2.2 × 10 −16 by two-sample t-test (n = 25 vs. 31 observations, each in independent cells across three experiments, three embryos each). Scale bars 10 µm.