Figure 6

(A) Cilia length of mIMCD-3 cells stimulated for 1 hr with 10 μM Forskolin (solvent: DMSO), normalized to the DMSO control. Data are shown as mean ± S.D., n = 3 with at least 40 cells per experiment. (B) Correlation of cilia length and eGFP fluorescence (a.u., average ciliary fluorescence of non-transfected control cells was subtracted) in the cilium in mIMCD-3 cells transiently expressing mNphp3(201)-VHHLaM-2-eGFP. Below 7.5 a.u., the cilia length is independent of the eGFP fluorescence (see inset, values are highlighted in green, slope not different from zero, correlation: p=0.07), whereas including values > 7.5 a.u., there is a linear correlation between the cilia length and the eGFP fluorescence in the cilium (slope different from zero, correlation: p<0.0001). (C) Length of cilia that show mNphp3(201)-VHH_LaM-2-eGFP localization and an eGFP fluorescence < 7.5 a.u., normalized to equally treated, non-transfected (NT) control cells. Data are shown as mean ± S.D., n = 7 with at least 18 cilia per experiment; p-values determined using unpaired, two-tailed Student’s t-test are indicated. (D) Correlation of cilia length and mCherry fluorescence in the cilium in mIMCD-3 cells transiently expressing mNphp3(201)-VHHLaM-2-eGFP and mCherry. Only cilia with an eGFP fluorescence below 7.5 a.u. were taken into account. There is no linear correlation between the mCherry fluorescence and cilia length (slope not different from zero, correlation: p=0.2). (E) mIMCD-3 cells (non-transfected, NT) kept in the dark (top) or stimulated with light (bottom, 1 hr, 465 nm, 38.8 µW/cm²) (F) mIMCD-3 bPAC-mCherry cells kept in the dark (left) or stimulated with light (right, 16 hr, 465 nm, 38.8 µW/cm²). (G) mIMCD-3 bPAC-mCherry transiently transfected with mNphp3(201)-VHH_LaM-2-eGFP kept in the dark (left) or stimulated with light (right, 1 hr, 465 nm, 38.8 µW/cm²). (H) Normalized cilia length after light stimulation (left 1 hr, right 16 hr; 465 nm, 38.8 µW/cm²) for mIMCD-3 bPAC-mCherry cells with or without transiently expressing mNphp3(201)-VHH_LaM-2-eGFP. Only cilia with an eGFP fluorescence < 7.5 a.u. were included and each data point was normalized to control cells. Data are shown as mean ± S.D., n = 3 with at least 25 cells per experiment; p-values determined using one-sample Student’s t-test compared to 100% are indicated. (I) mIMCD-3 LAPD-mCherry cells kept in the dark (left) or stimulated with light (right, 16 hr, 630 nm, 42.3 µW/cm²). (J) mIMCD-3 LAPD-mCherry transiently transfected with mNphp3(201)-VHH_LaM-2-eGFP kept in the dark (left) or stimulated with light (right, 16 hr, 630 nm, 42.3 µW/cm²). (K) Normalized cilia length after light stimulation (16 hr, 630 nm, 42.3 µW/cm²) for mIMCD-3 LAPD-mCherry with or without transiently expressing mNphp3(201)-VHH_LaM-2-eGFP. Only cilia with an eGFP fluorescence < 7.5 a.u. were included and each data point was normalized to control cells. Data are shown as mean ± S.D., n = 3–4 with at least 18 cells per experiment; p-values determined using one-sample Student’s t-test compared to 100% are indicated. Cells in E-G and I-J were stained with an Arl13B antibody (cyan) and DAPI (blue). All boxes indicate the magnified view below. Arrows indicate the direction and the length of the shift of the respective same-colored fluorescence channel. Scale bar for all images: 3 μm. (L) Spatial cAMP signaling controlling cilia length. Our data suggest a model, in which cAMP signaling in the cell body, stimulated by photoactivation of bPAC and an increase in cAMP levels, causes primary cilia shortening, whereas an increase of cAMP levels in the cilium results in primary cilia elongation. (M) Summary of the correlation between bPAC localization and photoactivation, cAMP levels, and cilia length.