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Genetically encoded calcium indicator illuminates calcium dynamics in primary cilia

Abstract

Visualization of signal transduction in live primary cilia constitutes a technical challenge owing to the organelle's submicrometer dimensions and close proximity to the cell body. Using a genetically encoded calcium indicator targeted to primary cilia, we visualized calcium signaling in cilia of mouse fibroblasts and kidney cells upon chemical or mechanical stimulation with high specificity, high sensitivity and wide dynamic range.

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Figure 1: 5HT6–G-GECO1.0 targets primary cilia and detects changes in ciliary Ca2+.
Figure 2: 5HT6–G-GECO1.0 detects ciliary Ca2+ influxes in response to ATP.
Figure 3: Laminar fluid flow induces dynamic calcium signals in primary cilia.

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Acknowledgements

We thank A. Seki and T. Meyer (Stanford University) for the 5HT6 construct, M. Fivaz (National University of Singapore) for the Lyn-YFP construct, A. Miyawaki (RIKEN) for the YC3.60 construct, L. Looger (Janelia Farm) for the GCaMP5G construct, G. Pazour (University of Massachusetts) for the GFP-CTS20 and GFP-CTS68 constructs, O. Griesbeck (Max Planck Institute) for the TNXXL construct, R. Reed (Johns Hopkins University) for mIMCD3 cells, and Y. Okubo, K. Kanemaru and H. Ishikawa for helpful comments on the manuscript. This study was supported in part by the US National Institutes of Health (NIH) (GM092930, DK065655 and DK090868 pilot funds provided by the Baltimore Polycystic Kidney Disease Research and Clinical Core Center) to T.I., and other grants to S.C., K.N., S.T., K.K. and T.K. from the Ministry of Education, Culture, Sports, Science and Technology of Japan and the Japan Society for the Promotion of Science. S.C.P. is supported by the Agency for Science, Technology and Research in Singapore.

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S.S., S.C.P., R.D., P.N.K. and T.I. generated constructs. K.K. developed the IA sequence under the supervision of T.K. The immunohistochemistry was performed by S.C., and K.N. and S.T. took the transmission electron microscopy images. S.S., S.C.P., R.D. and P.N.K. carried out cell biology experiments and microscopy under the supervision of T.I. S.C.P., S.S. and T.I. wrote the manuscript.

Corresponding author

Correspondence to Takanari Inoue.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–14 and Supplementary Table 1 (PDF 2286 kb)

5HT6–G-GECO1.0 can detect an increase in ciliary Ca2+ in NIH-3T3 cells.

The addition of 1 μM ionomycin causes a dramatic increase in fluorescence of 5HT6-G-GECO1.0 in cilia of NIH-3T3 cells. Scale bar is 5 μm. (AVI 10814 kb)

5HT6–G-GECO1.0 can detect ciliary [Ca2+] dynamics in response to ATP stimulation.

The addition of 10 μM ATP induces an increase in both cytosolic and ciliary Ca2+ in NIH-3T3 cells co-transfected with 5HT6-G-GECO1.0 and R-GECO1. The left image is cytosolic R-GECO1. The middle image is of the corresponding cell's cilia expressing 5HT6-G-GECO1.0. The right image is a merged image. The increase in cytosolic Ca2+ can be seen to precede the increase in ciliary Ca2+. Additionally, oscillations in cytosolic and ciliary Ca2+ can be observed in some cells. Scale bar is 10 μm. Video captured at 0.63 Hz. (AVI 116735 kb)

Ca2+ enters the base of the cilia and propagates to the tip after ATP stimulation.

Ca2+ can be seen entering the base of a cilia and traveling to the tip after the addition of 10 μM ATP in NIH-3T3 cells transfected with 5HT6-G-GECO1.0. Scale bar is 5 μm. Video was captured at 1.5 Hz. (AVI 27537 kb)

Dynamic Ca2+ signals detected in primary cilium bent with mechanical flow stimulation.

An upright-positioned primary cilium was subjected with laminar flow corresponding to 1dyne/cm2 shear stress approximately 2 minutes after the start of imaging. Flow was continued for 7.5 minutes before cilium was further imaged for another 4 minutes. Time-lapse imaging was performed at 15-second intervals. Z-projection of xy-planes are presented in this movie. The scale bar corresponds to 5 μm. The scale for the pseudo-colored GFP/ mCherry images has been included in Supplementary Figure 13c. (AVI 143475 kb)

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Su, S., Phua, S., DeRose, R. et al. Genetically encoded calcium indicator illuminates calcium dynamics in primary cilia. Nat Methods 10, 1105–1107 (2013). https://doi.org/10.1038/nmeth.2647

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