Methods for Measuring Ciliary Calcium Dynamics: Potential Role for the Ciliary Inversin Compartment

Abstract

The primary cilium is a cellular organelle at the nexus of many human disease syndromes that include polycystic kidney disease (PKD), embryonic developmental defects and retinal degeneration. While the whole organism phenotypes of these “ciliopathy” syndromes are well understood, the cellular and molecular mechanisms remain unclear. The primary cilium has been implicated in a number of cellular signaling pathways, including Sonic Hedgehog, second messengers such as calcium and cyclic AMP, and fluid flow sensing (Berbari, Connor, Haycraft, & Yoder, 2009; Gerdes, Davis, & Katsanis, 2009; Mick et al., 2015; Nachury, 2014; Praetorius & Spring, 2001). However, outside of the Hedgehog signaling pathway few cellular phenotypes have been identified partly due to a lack of available assays for cilium function. In this study I sought to develop an assay to measure calcium ion (Ca2+) dynamics in the primary cilium of wild-type (WT) cells and use that assay to determine alterations in cells in which the primary cilium protein inversin was deleted by CRISPR/Cas9 gene editing. Ca2+ kinetics were measured by live cell time lapse fluorescence microscopy of the biosensor G-GECO1, that changes fluorescence intensity upon binding of Ca2+, fused to the Ca2+–insensitive fluorescent protein mCherry and the primary cilium-localized receptor 5HT6 (Su et al., 2013). I used three different methods to generate an abrupt increase in the ciliary calcium concentration: two-photon ablation of the cilium tip with a focused 800nm pulsed laser beam, ATP-mediated calcium release, and illumination of the caged calcium chelator NP-EGTA in a small area of the cell with a focused 405nm laser beam. The most robust method used NP-EGTA un-caging with the beam positioned in the cytoplasm near the cilium base. Using this method, I found that cells lacking inversin by CRISPR/Cas9 gene editing had longer peak time and emptying time of calcium ions from the primary cilium. This result provides the first cellular phenotype linking a ciliopathy gene and Ca2+ signaling. With this knowledge, detailed molecular pathways can be explored and potential therapeutic interventions developed.