Spatiotemporal optical microscopy of vesicle-mediated hormone secretion in pancreatic cells

Insulin and glucagon, produced by pancreatic β and α cells respectively, play complementary roles in maintaining glucose homeostasis. The alteration of this homeostasis could lead to pathologies, one above all the Diabetes. While the dynamics of insulin secretory granules (ISGs) have been extensively studied, those of glucagon secretory granules (GSGs) remain less explored. In this thesis, after evaluating different labelling strategies, I analysed the motility and acidification of both granule types, providing the first classification of GSG dynamics under stimulation and perturbation. Labelling approaches such as genetically encoded reporters (C-pep-EGFP for ISGs, NPY-EGFP for GSGs) and the zinc probe ZIGIR were tested with image-derived mean square displacement (iMSD) analysis. ZIGIR was selected to perform dynamical studies based on Single-particle tracking (SPT), which revealed that GSGs mainly exhibit diffusive motion (~60%), with smaller immobile (~35%) and directed (~5%) fractions, mirroring ISGs behaviour in upkeeping conditions. Cytoskeletal perturbations confirmed the role of microtubules in active transport; interestingly, actin disruption increased ISGs motility but reduced GSGs dynamics. Secretory stimulation (1 mM glucose for GSGs; 16.7 mM for ISGs) enhanced both diffusive and directed motion. These findings indicate that, α and β cells exhibit similar granule dynamics yet rely on different cytoskeletal regulation. Even though ISGs are a well-studied granules, a calibrated measure of their pH is still missing. To investigate granule maturation, I employed the pH-sensitive E1GFP probe inserted within the C-peptide of proinsulin and performed calibrated pH measurements using Phasor-FLIM. ISGs exhibited a mean pH of ~5.8, with greater acidity near the membrane also during first phase of glucose-induced secretion. Preliminary measurements on NPY-E1GFP-labelled GSGs revealed a similar pH profile (~5.8). Two-photon confocal approach revealed heterogeneity in granule pH greater than expected; combining it with orbital tracking microscopy I measured pH fluctuations at millisecond time scale (with values ranging from 1.9 to 8.3), suggesting complex acidification dynamics requiring further validation. In conclusion, this thesis integrates advanced imaging and quantitative analysis to characterize the motion and acidification of pancreatic hormone granules, offering new insights into their regulation and potential implications for diabetes research.