FIGURE 5:

Three-color Airyscan analysis of changes in IFT88 localization during ciliogenesis in cells subjected to amplibody-proExM. (A) Schematic representation of stages of the ciliogenesis process: (I) After serum starvation, PCVs positive for MyoVa (red) start to accumulate around and dock to the distal appendages of the MC (blue) beneath the plasma membrane; (II) PCVs are fused to form a large membrane structure, CV; (III) coupled with axonemal growth (formation of a ciliary shaft), PCVs are further docked to the CV, resulting in elongation of the CV to form the CS; and (IV) the CS membrane is finally fused with the plasma membrane, resulting in direct exposure of the ciliary membrane to the extracellular environment. During the ciliogenesis process, TFs and the TZ are formed at the ciliary base and together function as a gate that controls the entry and exit of soluble and ciliary membrane proteins. (B) Three-color Airyscan analysis, coupled with amplibody-proExM, of the localization of IFT88 (green), MyoVa (red), and CEP164 (blue) during ciliogenesis. RPE1 cells were cultured under serum-fed conditions (a) or under serum-starved conditions for 0.5 h (b–e) or 36 h (f), fixed, and incubated with antibodies against IFT88, MyoVa, and CEP164 before the amplibody-proExM treatment, and analyzed by Airyscan microscopy. Representative images of cells without PCVs (a), with PCVs (b), CV (c), or CS (d, early CS; e, late CS), or with mature cilia (f) are shown. Black arrows indicate the IFT88 signals at the axoneme tip and TFs (e and f), and the red arrow indicates IFT88 signals above the TFs (f). Scale bars, 1 µm. (C) Maximum-intensity projection images of IFT88 (green) and CEP164 (magenta) in cells cultured under serum-fed conditions (a) or serum-starved (S.S.) for 0.5 h (b) were created by rotating the image nine times at 40° intervals around the physical center. The set of nine images was then merged into one stack, and average projections were generated. (D) The axial distance of the distal edge of IFT88 signals from the distal end of CEP164-positive TFs in mature cilia. The yellow dashed line represents the position of CEP164. The axial distance (d) from IFT88 to CEP164 was measured (n = 40). (E) After serum starvation for 0.5 or 36 h, cells with IFT88 signals in the TZ were counted, and the percentages are shown as bar graphs. Values are means ± SEM from three independent experiments, and 100 cells were analyzed in each set of experiments. ****p < 0.0001 (unpaired t test).