Molecular phenotyping of single pancreatic islet leader beta cells by "Flash-Seq"

AIMS: Spatially-organized increases in cytosolic Ca2+ within pancreatic beta cells in the pancreatic islet underlie the stimulation of insulin secretion by high glucose. Recent data have revealed the existence of subpopulations of beta cells including "leaders" which initiate Ca2+ waves. Whether leader cells possess unique molecular features, or localisation, is unknown. MAIN METHODS: High speed confocal Ca2+ imaging was used to identify leader cells and connectivity analysis, running under MATLAB and Python, to identify highly connected "hub" cells. To explore transcriptomic differences between beta cell sub-groups, individual leaders or followers were labelled by photo-activation of the cryptic fluorescent protein PA-mCherry and subjected to single cell RNA sequencing ("Flash-Seq"). KEY FINDINGS: Distinct Ca2+ wave types were identified in individual islets, with leader cells present in 73 % (28 of 38 islets imaged). Scale-free, power law-adherent behaviour was also observed in 29 % of islets, though "hub" cells in these islets did not overlap with leaders. Transcripts differentially expressed (295; padj < 0.05) between leader and follower cells included genes involved in cilium biogenesis and transcriptional regulation. Providing some support for these findings, ADCY6 immunoreactivity tended to be higher in leader than follower cells, whereas cilia number and length tended to be lower in the former. Finally, leader cells were located significantly closer to delta, but not alpha, cells in Euclidian space than were follower cells. SIGNIFICANCE: The existence of both a discrete transcriptome and unique localisation implies a role for these features in defining the specialized function of leaders. These data also raise the possibility that localised signalling between delta and leader cells contributes to the initiation and propagation of islet Ca2+ waves

State-of-the-art microscopy to understand islets of Langerhans:what to expect next?

The discovery of Langerhans and microscopic description of islets in the pancreas were crucial steps in the discovery of insulin. Over the past 150 years, many discoveries in islet biology and type 1 diabetes have been made using powerful microscopic techniques. In the past decade, combination of new probes, animal and tissue models, application of new biosensors and automation of light and electron microscopic methods and other (sub)cellular imaging modalities have proven their potential in understanding the beta cell under (patho)physiological conditions. The imaging evolution, from fluorescent jellyfish to real-time intravital functional imaging, the revolution in automation and data handling and the increased resolving power of analytical imaging techniques are now converging. Here, we review innovative approaches that address islet biology from new angles by studying cells and molecules at high spatiotemporal resolution and in live models. Broad implementation of these cellular imaging techniques will shed new light on cause/consequence of (mal)function in islets of Langerhans in the years to come