The G-domain of ARL13B contains a tubulin-interacting region. (A) Schematic representation of the main domains of the ARL13B protein (top) and GST-fused fragments of ARL13B (bottom) purified from E. coli. (B) GST pull-down from a solution of purified tubulin dimer. (C-F) GST pull-down from hTERT-RPE1 cell lysate. Bands corresponding to fusion proteins of the expected size are marked with red dots; degradation products run below.

ARL13B G-domain is dispensable for cilium localization but essential for cilium length regulation. (A) Domain structure of GFP-tagged wild-type and mutated ARL13B proteins expressed in mammalian cells as GFP fusions. (B-E) Cilia lengths in wild-type MEFs (B,D) and Arl13b^hnn MEFs (C,E) untransduced (B,C) or transduced with the indicated lentiviral expression vectors (D,E) were determined following immunofluorescence for ARL13B, acetylated tubulin and γ-tubulin in wild-type MEFs (B), IFT88, acetylated tubulin and γ-tubulin (C), and GFP, glutamylated tubulin and γ-tubulin (D,E). Nuclei were stained with DAPI. (F) Quantification of changes in cilia length distribution in wild-type and Arl13b^hnn MEFs expressing GFP-tagged ARL13B constructs compared to non-transduced controls. χ²-test, *P<0.0001 from non-transduced wild-type MEFs. n=number of cilia measured in two independent experiments. Scale bars: 5 µm.

The G-domain regulates ARL13B distribution along cilia. (A-J) Immunofluorescence of clonal Arl13b^hnn and wild-type MEFs untrasduced or expressing GFP-mARL13B, GFP-mARL13BΔGD (see Fig. S4A for expression levels of GFP-tagged variants of Arl13b). GFP, green, acetylated tubulin, red and γ-tubulin, white. (A,B) GFP-mARL13B and GFP-mARL13BΔGD uniformly distribute along cilia of wild-type MEFs. (C) Cilium of untransduced MEFs. (D) Ectopic GFP-mARL13B rescues the ciliary length defect and distributes uniformly along the ciliary membrane of Arl13b^hnn MEFs. (E,G) Ectopic GFP-mARL13BΔGD accumulates at the distal end (E) or at both ends (F) of cilia and in stump-like structures (G) of Arl13b^hnn MEFs but, it is dramatically reduced between cilia extremities (E and F). (K) Ultrastructural analysis by TEM of longitudinal sections of cilia of Arl13b^hnn MEFs. Clonal Arl13b^hnn MEFs transduced with GFP-mARL13BΔGD show accumulation of electron-dense deposit along the length of cilia (middle panel) and in stump-like structures (right panels). In contrast, clonal Arl13b^hnn MEFs transduced with GFP-mARL13B show no electron-dense accumulation along the cilia (left panel). Insets in K are gradient maps of the boxed areas. Note that axonemal microtubules (vertical yellow streaks) are visible in the cilium of Arl13b^hnn MEFs transduced with GFP-mARL13B, but not in the cilium of those transduced with GFP-mARL13BΔGD, which are mostly filled with electron-dense material (blue granules). (L) Color-coded examples of variable distribution of GFP-tagged ARL13B mutants along cilia of transduced Arl13b^hnn MEFs quantified in (M). Only the mGFP-ARL13BΔGD mutant lacking the G-domain aa sequence fails to distribute uniformly along the ciliary membrane of Arl13b^hnn MEFs. Wild-type and mutant ARLS13B carrying a single aa substitution affecting the GTPase active site uniformly distribute along the ciliary length. (M) Quantification of the distribution of GFP-tagged proteins in cilia of wild-type and Arl13b^hnn MEFs expressing GFP-tagged ARL13B constructs as indicated below the graph. Bars show percentage of cilia with even distribution of GFP-ARL13B proteins along the cilium (mean of two independent experiments, total number of cilia per experiment per construct was between 70 and 385, error bars represent standard error of the mean). In Arl13b^hnn MEFs, the difference between mARL13BdeltaGD and all other groups is significant (***P<0.001, one-way ANOVA with Tukey post-test for pairwise comparisons). Scale bars: 2 µm (J,L); 0.5 µm (K).

The ARL13B G-domain sequence but not its GTPase activity is required for normal distribution of transmembrane proteins along the ciliary membrane. (A-E) Immunofluorescence signal for SMO (red), GFP (green) and γ-tubulin (white) in wild-type and Arl13b^hnn MEFs untransduced (A and B) and Arl13b^hnn MEFs stably expressing GFP-tagged wild-type or mutant ARL13B (C-E). MEFs shown in (A-E) were treated with recombinant SHH. (F) Average of fluorescence intensity (arbitrary units)±standard deviation of Smo along cilium length. n=20 cilia were evaluated for each construct and pooled from three independent experiments. Only cilia longer than 2 µm were analyzed. Student's t-test analysis of the differences between Smo signal intensity from MEFs expressing wild-type and mutant ARL13B, the error bar represents the s.d. For ARL13B-ΔGD, P<0.0001 for the proximal 80% of the cilia. For ARL13B-T35N, not significant along the entire length of the cilium. (G-K) Immunofluorescence signal for mCherry (red), GFP (green) and γ-tubulin (white) in wild-type and Arl13b^hnn MEFs transduced with mCherry-SSTR3 lentivector (G-K) and in Arl13b^hnn MEFs stably expressing GFP-tagged wild-type or mutant ARL13B (I-K). (L) Average of fluorescence intensity of mCherry-SSTR3 as calculated in (F). Student's t-test analysis of the differences between mCherry-SSTR3 signal intensity from MEFs expressing wild-type and mutant ARL13B, the error bars represent the mean ±s.d. For ARL13B-ΔGD, P<0.001 for the proximal 60% of the cilia. For ARL13B-T35N, not significant along the entire length of the cilium. (M) Quantification of cilia with even distribution of mCherry-SSTR3 (in %). The distribution was considered uneven when the fluorescence signal in any region of the cilium decreased >50% as compared to the signal intensity of the remaining portion of the cilium. Dots show the results of three independent experiments, error bars show means ±s.d. Cell lines and GFP-tagged ARL13b constructs are indicated below the graph. Scale bars: 2 µm.