Fluorescence Microscopy Journal Club

Primary cilia are not calcium-responsive mechanosensors

by M. Delling, A. A. Indzhykulian, X. Liu, Y. Liu, T. Xie, D. P. Corey, and D. E. Clapham

Nature 2016, 531, 7596, pp. 656-660

In this paper, Delling et al question the widely held hypothesis that primary cilia act as Calcium responsive mechanosensors (CaRMS). This hypothesis has been used to explain a large range of biological observations, like left-right axis determination in embryonic development or polycystic kidney disease. The authors developed a transgenic mouse, Arl13b–mCherry–GECO1.2, expressing a ratiometric genetically encoded calcium indicator confined to the primary cilia and were able to demonstrate the complete lack of mechanically induced calcium increases in primary cilia upon mechanical stimulation. They found no CaRMS in osteocyte-like cells, mouse embryonic fibroblasts, or, indeed, any primary cilia examined. They found that in all cases where the calcium concentration in cilia increased, the calcium rise was initiated at other sites in the cell and diffused from the cytoplasm into the cilium.

They explain the previous hypothesis by out of focus data, motion, and light path artifacts in combination with low acquisition rates where calcium originating from the cytoplasm can diffuse into the cilium in less than 200 ms and be mistaken as originating from within the cilium. Temporal resolution is critical. The authors used the Opterra swept-field confocal in combination with a fast camera in order to reach supra video rate acquisitions in 2 colours, and performed the acquisitions in slit mode in order to both increase light delivery to the camera and avoid excessive photobleaching.

These findings have a significant implication on established models based on an incorrect hypothesis and encourage researchers to reinvestigate other mechanisms for regulation of ciliary ion channels.