Intragranular targeting of syncollin, but not a syncollinGFP chimera, inhibits regulated insulin exocytosis in pancreatic β-cells

Abstract

Several proteins play a role in the mechanism of insulin exocytosis. However, these 'exocytotic proteins' have yet to account for the regulated aspect of insulin exocytosis, and other factors are involved. In pancreatic exocrine cells, the intralumenal zymogen granule protein, syncollin, is required for efficient regulated exocytosis, but it is not known whether intragranular peptides similarly influence regulated insulin exocytosis. Here, this issue has been addressed using expression of syncollin and a syncollingreen fluorescent protein (syncollinGFP) chimera in rat islet -cells as experimental tools. Syncollin is not normally expressed in -cells but adenoviral-mediated expression of both syncollin and syncollinGFP indicated that these were specifically targeted to the lumen of -granules. Syncollin expression in isolated rat islets had no effect on basal insulin secretion but significantly inhibited regulated insulin secretion stimulated by glucose (16·7 mM), glucagon-like peptide-1 (GLP-1) (10 nM) and glyburide (5µM). Consistent with specific localization of syncollin to -granules, constitutive secretion was unchanged by syncollin expression in rat islets. Syncollin-mediated inhibition of insulin secretion was not due to inadequate insulin production. Moreover, secretagogue-induced increases in cytosolic intracellular Ca 2+, which is a prerequisite for triggering insulin exocytosis, were unaffected in syncollin-expressing islets. Therefore, syncollin was most likely acting downstream of secondary signals at the level of insulin exocytosis. Thus, syncollin expression in -cells has highlighted the importance of intralumenal -granule peptide factors playing a role in the control of insulin exocytosis. In contrast to syncollin, syncollinGFP had no effect on insulin secretion, underlining its usefulness as a 'fluorescent tag' to track -granule transport and exocytosis in real time.

Exocyst Sec5 Regulates Exocytosis of Newcomer Insulin Granules Underlying Biphasic Insulin Secretion

“…Syncollin, a secretory granule protein originally found in pancreatic acinar cells, could be tagged with a pH-sensitive GFP variant, pHlourin, that fluoresces when exposed to the extracellular alkaline pH upon exocytosis of the secretory granule [35]; and which could be used as a surrogate granule cargo to tag insulin granules to assess exocytosis [38]. Control and Sec5 KD INS-1 cells were infected with adenovirus encoding syncollin-pHluorin and fusion events were indicated by abrupt brightening of pHluorin fluorescence followed by a cloud-like diffusion pattern indicating dispersion of the syncollin cargo into the cell exterior.…”

Direct interaction between Rab3D and the polymeric immunoglobulin receptor and trafficking through regulated secretory vesicles in lacrimal gland acinar cells

“…Syncollin is a protein that was originally characterized to be tightly associated with the luminal surface of zymogen granules in pancreatic acinar cells (2). Syncollin-GFP fusion protein has been used to label large protein-enriched secretory vesicles in a variety of cell types (23,24). Previous studies of syncollin-GFP in rabbit LGACs have shown that acini transduced with Ad encoding for syncollin-GFP (Ad-syncollin-GFP) expressed syncollin-GFP in large mature secretory vesicles underneath the apical plasma membrane in unstimulated lacrimal acini (27,28).…”

Neurexin-1α Contributes to Insulin-containing Secretory Granule Docking

“…SEAP is a recombinant cargo protein commonly used to assess constitutive secretion (42,50). We found that NRXN1 knockdown did not affect SEAP secretion at high glucose (Fig.…”