124-OR: Repetitive Ca2+ Waves Emanate from a Stable Leader Cell in Mouse Islets

Pancreatic beta cells form a highly connected network within the islet, with transcriptomic and functional heterogeneity existing between individual cells. A sub-population of leader cells has been proposed to act as functional hubs, controlling islet responses to glucose and other secretagogues. Using high speed confocal Ca²⁺ imaging, we aimed here to examine (a) the role of these cells in wave propagation and (b) their stability over time.

Methods: Islets were isolated from C57BL6 male mice expressing the recombinant Ca²⁺ probe GCaMP6f selectively in the beta cell (Ins1Cre:GCaMP6^f/f). Islets were then infected with an adenovirus expressing photo-activatable- (PA) mCherry. Ca²⁺ imaging was performed across a single islet plane in modified KREBS-Ringer medium at 11mM glucose using a Zeiss LSM 810 laser-scanning microscope and data acquisition at 5 Hz. Cells were photopainted by excitation of PA-mCherry at 405nm.

Results: Leader cells were identified as those exhibiting the first detectable increase in Ca²⁺ signal during subsequent Ca²⁺ waves. The great majority of islets (85%, n=26, three independent experiments) displayed repetitive Ca²⁺ waves (1.10±0.26 min^-1), and 82.9±5.0% of waves propagated directly from an identifiable leader cell. There was an average of 2.5±0.4 leader cells per islet with 62.9±0.70% of waves emanating from a “principal” leader cell. Ca²⁺ imaging experiments performed 16 h after the original session revealed that 37±0.08% (n=4) of waves emanated from the same, now photopainted, leader cell.

Conclusions: These data suggest that (a) a complex, non-binary, heterogeneity exists between leader and follower beta cells, (b) leader behaviour is an intrinsic phenotype of a sub-population of cells with a half-life in this role of ∼12 h.