Extended Data Figure 1: Arl13b–mCherry–GECO1.2 identifies primary cilia.

Arl13b–mCherry–GECO1.2 contains an improved genetically encoded calcium indicator³⁶ (GECI) with an apparent K_d of 450 nM (Extended Data Fig. 2), well suited to work within the reported range of ciliary Ca²⁺ concentrations ([Ca²⁺]_cilium)⁴⁰. The genomic integration site of the transgene is within a non-coding region of chromosome 1 (Extended Data Fig. 2) and transgenic animals maintained as homozygotes (Arl13b–mCherry–GECO1.2^tg) are viable, have average litter sizes, and do not show phenotypes consistent with cilia defects (for example, situs inversus, organ malformation; Extended Data Fig. 2). a, Frozen tissue section of P21 mouse kidney. GECO1.2 and mCherry are preferentially localized to cilia, identified by the cilia-specific marker, anti-acetylated tubulin antibody. b, c, Primary mIMCD cells isolated from kidneys of P14–P21 Arl13b–mCherry–GECO1.2 transgenic mice. Ciliary localization (arrow) of anti-polycystin 2 antibody in c. Scale bars, 10 μm. d, Two OHC hair bundles marked with the actin-binding peptide, phalloidin. One of the stereocilia bundles expresses Arl13b–mCherry–GECO1.2. Kinocilia on both OHCs marked with an antibody to acetylated tubulin, express Arl13b–mCherry–GECO1.2. Scale bar, 5 μm. e, Scanning electron micrograph of a primary mIMCD cell. Left, red dashed line outlines a single mIMCD cell; white circle indicates the primary cilium. Scale bar, 10 μm. Right, magnified image. No defects in cilia formation were evident following 3–4 days in culture. Scale bar, 500 nm. All images are representative of more than ten images taken of biological triplicates.