The β-cell primary cilium is an autonomous Ca²⁺ compartment for paracrine GABA signalling

Abstract

The primary cilium is an organelle present in most adult mammalian cells and is thought of as an antenna for detection of a variety of signals. Here we use intact mouse pancreatic islets of Langerhans to investigate signalling properties of the primary cilium in β-cells. Using cilia-targeted Ca2+ indicators we find that the resting Ca2+ concentration in the cilium is lower than that of the cytosol, and we uncover a Ca2+ extrusion mechanism in the cilium that effectively insulates the cilium from changes in cytosolic Ca2+. Stimuli that give rise to pronounced cytosolic Ca2+ concentration increases, such as glucose- and depolarization-induced Ca2+ influx, and mobilization of Ca2+ from the ER, was accompanied by minor increases in cilia Ca2+ concentrations that were spatially restricted to a small compartment at the base. Conversely, we observe pronounced Ca2+ concentration changes in the primary cilia of islet β-cells that do not propagate into the cytosol and show that paracrine GABA signalling via cilia-localized GABA- B1-receptors is responsible for this Ca2+ signalling. Finally, we demonstrate that the cilia response to GABA involves ligand-dependent transport of GABA-B1 receptors into the cilium.

Fluorescence imaging of beta cell primary cilia

“…cADDis sensor response appears not to be affected by tagging of the 5HT6 receptor, as multiple ciliary proteins including Arl13b and SSTR3 have been targeted in this fashion without changing the sensor response (54). Similarly, cilia-targeted calcium probes such as 5HT6-GECO (Addgene 47499) (55, 59) can be used for the study of ciliary calcium dynamics while serving as a surrogate label for primary cilia. If greater ciliary specificity still is desired, one can introduce transient transgene expression of fluorescent-tagged proteins such as the ciliary GPCRs somatostatin receptor 3 (SSTR3) and 5-hydroxytryptamine receptor (5HT6), or ADPribosylation factor-like protein 13 B (Arl13b), a small GTPase enriched in the cilia membrane (60-62), using transfection or viral transduction approaches.…”