FIGURE 7:

ARL13B domains required for TULP3 interactions and ciliary localization. (A) Schematic representation of different ARL13B truncations used. n = 47–200 cilia counted. Arl13b^hnn MEFs had no HA-tagged INPP5E in cilia. ^#, the D1 fragment lacking a CCdomain (Supplemental Figure S7D) or ∆CC is not trafficked to cilia likely from indirect effects, such as formation of cellular aggregates upon loss of CC (Nozaki et al., 2017). (B) Arl13b^hnn cells stably expressing ^HAINPP5E along with the indicated C-LAP–tagged ARL13B fusions were serum starved for 48 h before fixing and immunostained for GFP, HA (INPP5E), and acetylated tubulin (AcTUB) and counterstained for DNA. Quantification in A, C, and D. (C) Violin plots of GFP ciliary fluorescence intensities from B. n > 30 cilia/condition. (D) Cilia lengths of cell lines from B. Only ciliated cells in the Arl13b^hnn background stably expressing HA-tagged INPP5E were counted. Arl13b^hnn cells are ∼20% ciliated (Larkins et al., 2011). n > 30 cilia/condition. (E) Model depicting TULP3’s role in trafficking ARL13B to cilia. ARL13B trafficking to cilia is primarily regulated by binding to TULP3 and TULP3’s binding to the IFT-A core complex. An N-terminus amphipathic helix that includes the palmitoylation site and precedes the GTPase domain mediates binding of ARL13B to TULP3 and directs trafficking to cilia even in the absence of palmitoylation and the RVxP sorting motif. The RVxP motif also provides an alternative mode of trafficking. TULP3 binding to short sequences in diverse cargoes is likely mediated by the tubby domain. Scale, 5 μm. *, p < 0.001 by Chi-square test. ****, p < 0.0001; ***, p < 0.001; **, p < 0.01; *, p < 0.05; ns, not significant (C–D). Adjusted p values are shown in C. Arrows and arrowheads indicate cilia positive and negative for the indicated proteins, respectively. See also Supplemental Figure S5.