Two Distinct Populations of Insulin Granules That Have Unique Properties

Purified secretory granules can be reconstituted into a planar supported membrane fusion assay. The addition of Munc18 and complexin to syntaxin-1/SNAP25 containing membranes allow granules to bind but not fuse in the absence of calcium, while calcium readily triggers fusion in the presence of PI(4,5)P2 (Kreutzberger et al. Sci Adv. 2017 and Kreutzberger et al. Nat. Comm. 2019). Combination of this assay with single cell imaging of secretion has revealed two sub-populations of insulin granules. One population of granules has an average diameter of ∼180 nm, contains synaptotagmin-7, fuses with a high sensitivity to calcium, and exhibits a slow mode of secretion. The other population of granules have an average diameter of ∼208 nm, contains synaptotagmin-9, fuses with a lower sensitivity to calcium, and has a faster mode of secretion. These sub-populations have distinct lipid compositions and were determined to form downstream of different lipid translocases of the ABC transporter family. Immuno-depletion of purified insulin granules identified proteins that are present in both populations as well as proteins that are enriched in only one or the other sub-population.