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. 2021 Apr 17;296:100680. doi: 10.1016/j.jbc.2021.100680

Figure 3.

Figure 3

Targeting DIAPH3 to the ciliary base regulates cilia length and morphology.A–F, targeting DIAPH2 and DIAPH3 to the ciliary base via centrin or PACT causes elongation and bulb formation at the tip of the cilia. A, fusion constructs were made between GFP-centrin (CNTN1) and the indicated proteins (DIAPH2 and DIAPH3). Cells were transfected with GFP-CNTN1 (control), GFP-CNTN1-DIAPH2, or GFP-CNTN1-DIAPH3 and blotted for GFP to detect protein size and to ensure integrity of the fusion proteins. B, constructs were transfected into hTERT-RPE1 cells, which were then serum starved for 24 h to induce ciliogenesis, fixed, and stained for acetylated tubulin (cilia marker) and GFP (to detect transfected cells). Cilia are shown in boxes (left panels) and the boxed areas are magnified (right panels). Scale bars are 8 μm in the left panel and 1 μm in the right-side panels. Arrowheads point toward the bulbs at tips of cilia. C, quantification of bulbed versus nonbulbed cilia. Measurement of cilia length for GFP-CNTN1, GFP-CNTN1-DIAPH2, or GFP-CNTN1-DIAPH3 samples was presented in Box–Whisker graphs (as in Fig. 1C). D, targeting DIAPH3 to the ciliary base via PACT causes elongation and formation of bulbs at the tip of the cilia. Fusion constructs were made between GFP-PACT (centrosomal targeting domain) and DIAPH3. hTERT-RPE1 cells were transfected with GFP-PACT or GFP-PACT-DIAPH3 and serum starved for 24 h and fixed and stained for GFP and acetylated tubulin. Scale bars are 1 μm for each image. E–F, hTERT-RPE1 cells were transfected with CNTN1-GFP (control), GFP-CNTN1-DIAPH1, GFP-CNTN1-DIAPH3, GFP-PACT (control), GFP-PACT-DIAPH1, or GFP-PACT-DIAPH3. Cells were serum starved for 24 h to induce ciliogenesis. Quantification for ciliated cells with bulb (E) and length of bulbed versus nonbulbed cilia (F) was conducted. Error bars in E represent ± SD of three independent experiments; n = 50 cells each, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, ∗∗∗∗p < 0.001. Two-tailed t-test analysis was done to compare CNTN1-GFP control to the samples with GFP-CNTN1, while GFP-PACT control was compared with samples with GFP-PACT. Box and whisker plots (F) were performed as in Fig. 1C).