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.

Figure 1

5HT6-G-GECO1.0 targets primary cilia and detects changes in ciliary Ca²⁺. (a) Schematic of 5HT₆-G-GECO1.0. G-GECO1.0 contains M13 (a skeletal muscle light-chain kinase), a circularly permuted GFP (cpGFP), and Calmodulin (CaM). (b) A primary cilium from a NIH-3T3 cell expressing 5HT₆-G-GECO1.0 stained with antibody against acetylated α-tubulin. Bar represents 3 μm. (c) Time-lapse imaging of a NIH-3T3 primary cilium expressing 5HT₆-G-GECO1.0 treated with ionomycin (Iono). AFU stands for arbitrary fluorescence unit. Bar represents 5 μm.

Figure 2

5HT₆-G-GECO1.0 detects ciliary Ca²⁺ influxes in response to ATP. (a,b) Fluorescence microscopy images of NIH-3T3 cell expressed with the indicated sensors showing response to ATP (a) or DMSO (b). Bar represents 5 μm. Time lapse images here were captured at 0.067 Hz. (c) High speed time lapse imaging (0.63Hz) reveals oscillations in cytosolic and ciliary Ca²⁺ levels in response to 10 μM ATP in NIH-3T3 cells expressing indicated sensors. Bar indicates 10 μm. AFU, arbitrary fluorescence unit.

Figure 3

Laminar fluid flow induces dynamic calcium signals in primary cilia (a) Fluorescence intensity of GFP divided by that of mCherry is indicated before and after flow administration. 5HT₆-mCherry-G-GECO1.0 activity is shown in blue (18 primary cilia from 7 independent experiments) and 5HT₆-mCherry-GFP (control) is in black (9 primary cilia from 3 independent experiments). Fluorescence signals have been normalized against baseline fluorescence before flow induction. Region highlighted in light blue indicates time points with flow. Error bars represent SEM. (b) Comparison of relative GFP intensities between 5HT₆-mCherry-G-GECO1.0 and 5HT6-mCherry-GFP before and after one minute flow induction. In each case, the GFP intensity without flow has been normalized to a value of 1. Statistical analysis: 5HT₆-mCherry-GFP −flow vs +flow (P = 0.9545); 5HT₆-mCherry-G-GECO1.0 −flow vs +flow (P = 0.000153); 5HT₆-mCherry-G-GECO1.0 +flow vs 5HT₆-mCherry-GFP +flow (P = 0.000128).