Figure 2—figure supplement 3.

Figure 2—figure supplement 3.

(A) bPAC activity measurements in HEK-TM cells. HEK293 cells express the CNGA2-TM ion channel, which opens upon cAMP binding and conducts Ca²⁺ (HEK-TM). bPAC-mCherry was co-expressed with the mNphp3(201)-tagged mCherry nanobody. Light-dependent activation of bPAC increases intracellular cAMP levels, leading to a Ca²⁺ influx, which was quantified using a fluorescent Ca²⁺ dye (GFP-certified FluoForte). bPAC activity was determined in the presence of mNphp3(201)-VHH_LaM-2 or mNphp3(201)-VHH_LaM-4 (fused to HA or eGFP). Co-expression with the ciliary protein Sstr3-eGFP was used as a negative control (ciliary localized protein, but not binding to bPAC or LAPD). bPAC activity was determined according to the maximum amplitude of the Ca²⁺ signal after light stimulation (465 nm light pulse, 1 s, 162 µW/cm²) compared to the ionomycin-evoked Ca²⁺ signal. 5 min before light stimulation, cells were treated with 25 µM of IBMX to inhibit phosphodiesterases and sustain a long-lasting increase in cAMP. NT: non-transfected cells. (B) LAPD activity measurements in HEK-TM cells. To measure LAPD activity, HEK-TM cells were pre-stimulated with 100 μM NKH477 to activate transmembrane adenylate cyclases (AC), thus increasing cAMP levels. Ca²⁺ influx was detected by a Ca²⁺ dye (Fluo4-AM). LAPD activity was determined in the presence of mNphp3(201)-VHH_LaM-2 or mNphp3(201)-VHH_LaM-4 (fused to HA or eGFP). Co-expression of the ciliary protein Sstr3-eGFP was used as a negative control (ciliary localized protein, but not binding to bPAC or LAPD). Fluo4-AM-loaded HEK-TM cells were incubated with 100 μM NKH477 during continuous 850 nm light illumination (0.5 µW/cm²). When reaching a steady-state, light was switched to 690 nm (0.5 µW/cm²) to stimulate LAPD activity. LAPD activity was determined as the maximal decrease compared to the maximal Ca²⁺ signal amplitude after NKH477 addition. Data are shown as individual data points (each data point represents and independent experiment and corresponds to the average of a duplicate or triplicate measurement) and mean ± S.D., p-values calculated using unpaired, two-sided Student's t-test compared to Sstr3-eGFP are indicated. All HEK-293 cells were non-ciliated.