Fig 2: PI(4,5)P2 localizes to a specific subdomain of the transition zone. (A) Ciliated hTERT-RPE1 cells were immunostained with PI(4,5)P2 (green), ARL13B (grayscale) and CEP164, TCTN1, MKS3, RPGRIP1L or AHI1 (red) antibodies and imaged by STED microscopy (confocal resolution image of the ARL13B stained axoneme is shown). Right panels show merged image at lower magnification. Arrows indicate transition zone PI(4,5)P2 signals, arrow heads indicate transition zone protein localization, bar indicates 1 µm. (B) Graph shows the lateral diameter between the highest intensity points of the PI(4,5)P2 or cilia protein marker puncta perpendicular to the plane of the axoneme. Bars represent mean ± SEM, n = 3 independent experiments, =30 cilia imaged per experiment and all cilia with two distinct PI(4,5)P2 or cilia marker protein puncta measured, statistical significance was determined using one-way ANOVA (p < 0.0001) followed by Tukey’s post hoc test ***p < 0.001, ****p < 0.0001. (C) Graph shows the axial distance between the highest intensity point of the PI(4,5)P2 signal and each cilia marker protein signal parallel to the plane of the axoneme. Bars represent mean ± SEM, n = 3 independent experiments, = 30 cilia imaged per experiment and all cilia with distinct PI(4,5)P2 or cilia marker protein puncta measured, statistical significance was determined using one-way ANOVA (p < 0.0001) followed by Tukey’s post hoc test, ****p < 0.0001. (D) Representative image showing the method used for lateral diameter and axial distance measurements, bar indicates 1 µm. (E) Ciliated hTERT-RPE1 cells were immunostained with PI(4,5)P2 (green), MKS3 (red) and ARL13B (grayscale) antibodies and imaged by STED microscopy (confocal resolution image of the ARL13B stained axoneme is shown). Right panel shows merged image at lower magnification. Arrow indicates ring shaped transition zone PI(4,5)P2 morphology, bar indicates 1 µm.

Fig 3: PI(3,4,5)P3 localizes to a specific subdomain of the transition zone. (A) Ciliated hTERT-RPE1 cells were immunostained with PI(3,4,5)P3 (green), ARL13B (grayscale) and TCTN1, MKS3, RPGRIP1L or AHI1 (red) antibodies and imaged by STED microscopy (confocal resolution image of the ARL13B stained axoneme is shown). Right panels show merged image at lower magnification. Arrows indicate transition zone PI(3,4,5)P3 signal, arrow heads indicate transition zone protein localization, bar indicates 1 µm. (B) Graph shows the lateral diameter between the highest intensity points of the PI(3,4,5)P3 or transition zone protein puncta perpendicular to the plane of the axoneme. Bars represent mean ± SEM, n = 3 independent experiments, 30 cilia imaged per experiment and all cilia with two distinct PI(3,4,5)P3 or transition zone protein puncta measured, statistical significance was determined using one-way ANOVA (p < 0.0001) followed by Tukey’s post hoc test, ****p < 0.0001. (C) Graph shows the axial distance between the highest intensity point of the PI(3,4,5)P3 signal and each transition zone protein signal parallel to the plane of the axoneme. Bars represent mean ± SEM, n = 3 independent experiments, 30 cilia imaged per experiment and all cilia with distinct PI(3,4,5)P3 or transition zone protein puncta measured, statistical significance was determined using one-way ANOVA (p < 0.0001) followed by Tukey’s post hoc test, **p < 0.01, ***p < 0.001, ****p < 0.0001. (D) Representative image showing the method used for the lateral diameter and axial distance measurements, bar indicates 1 µm. (E) Ciliated hTERT-RPE1 cells were immunostained with PI(3,4,5)P3 (green) and ARL13B (grayscale) antibodies and imaged by STED microscopy (confocal resolution image of the ARL13B stained axoneme is shown). Arrow indicates ring shaped transition zone PI(3,4,5)P2 morphology, bar indicates 1 µm.

Fig 4: Molecular architecture at the base of the primary cilium obtained by overlapping coordinate-defined superresolution images of TZ/TF proteins and an EM image.(a) A 7-color superresolution image obtained by merging multiple single-colored STED images illustrating the relative locations of important TZ/TF proteins. (b) A typical equally-magnified EM image of a primary cilium in an RPE-1 cell, where TFs are marked as blue dots, areas of microtubule doublets (MT) circled with red dashed lines, areas of the ciliary membrane (CM) covering the ciliary necklace (CN) circled with yellow dashed lines, and the distal end of the BB marked by a white dashed line. CP: ciliary pocket. (c) A merged image of (a,b) obtained by aligning the TFs in EM and CEP164 in STED. RPGRIP1L was close to the microtubule doublets; TMEM67 and TCTN2 were localized mostly toward the TZ membrane, while MKS1 was localized midway between the membrane and microtubules. CEP290, right above the BB, was localized at a different axial level from the other TZ proteins. Scale bar, 200 nm. (d) A localization model of TZ/TF proteins at the ciliary base pinpointing the positions of these proteins relative to each other and to known structural elements. Y-links are hypothesized to bridge across the level where RPGRIP1L and MKS1 are occupied, with dwelling transmembrane proteins such as TMEM67 and TCTN2 decorated as the ciliary necklace.

Fig 5: MT aster formation is required for Cep290 localization at CS and TZ assembly. a–c Percentage of cells with CS Cep290 after treatment with DMSO, 20 µM nocodazole (a), 1 µM taxol (b), or 10 µM ciliobrevin D (c) for 30 min. d RPE-1 cells transfected with control or FSD1 siRNA were subjected to nocodazole treatment for 2 h and released at indicated time points. Next, cells were fixed with 4% paraformaldehyde in PBS and stained with antibodies to a-tubulin to visualize microtubules (green) and Centrin2 to mark centrosomes (red). Percentage of cells with different microtubule regrowth statuses at indicated time points was quantified on the right panel. Scale bars, 5 µm (main image) and 1 µm (magnified region). e RPE-1 cells were transfected with indicated siRNA were subjected to nocodazole treatment for 2 h and released at indicated time points. Cells were then fixed and permeabilized with cold methanol and stained with antibodies to Cep290 (red), a-tubulin (green), and Centrin2 (purple). Percentage of cells with CS Cep290 after nocodazole release for 20 min was quantified on the right panel. Scale bars, 5 µm (main image) and 1 µm (magnified region). f Effects of FSD1, Ninein, or Kif3a depletion on the Cep290 localization at centriolar satellites in cycling RPE-1 cells. g, h Effects of FSD1, Ninein, or Kif3a depletion on the relative intensity of the transition zone components TMEM67 (g) and NPHP8 (h) at mother centrioles in quiescent cells. Data are presented as mean ± s.d. of three independent experiments. n number of cells. In all panels, statistical comparisons between two groups were carried out by two-tailed t-test. NS not significant, **P < 0.01, ***P < 0.001