Insulin secretion by ‘kiss-and-run’ exocytosis in clonal pancreatic islet β-cells

Exocytotic release of neuropeptides and hormones is generally believed to involve the complete merger of the secretory vesicle with the plasma membrane. However, recent data have suggested that ‘kissand-run’ mechanisms may also play a role. To analyse secretory events in neuroendocrine β-cells, we imaged chimaeric reporters targeted to either the vesicle membrane [chimaeras of synaptobrevin-2 and pH-sensitive green fluorescent protein (synapto · pHluorin) or of phogrin (phosphatase on the granule of insulinoma) and enhanced green fluorescent protein (EGFP) (phogrin · EGFP)] or the lumen [neuropeptide Y (NPY) · pH-insensitive yellow fluorescent protein (Venus)] by evanescent wave microscopy. Unexpectedly, the frequency of NPY · Venus release events was only 17–27% of that of vesicle fusion reported with synapto · pHluorin, but not phogrin · EGFP, indicating that exocytosis of cargo peptides that is likely to require complete collapse of the vesicle into the plasma membrane is relatively rare. However, both the frequency and the kinetics of NPY · Venus release were modulated by stimulus strength or by overexpression of synaptotagmin IV, demonstrating the plasticity of ‘kiss-and-run’ fusion. Introduction The release of neurotransmitters and peptide hormones from cells involves the exocytotic fusion of secretory vesicles with the plasma membrane [1]. In the classical model of exocytosis, this involves complete fusion of the vesicular and plasma membranes, followed later by the retrieval of membrane at a different site [2]. Questioning this model, clathrinmediated endocytosis appears to be too slow to explain the observed rapid retrieval of membrane. Moreover, recruitment of a key regulator of endocytosis, dynamin, is observed on only ≈60% of vesicles retreating from the plasma membrane [3]. Based on these and other observations, we and others [3,4] have recently proposed that regulated exocytosis may occur in part by ‘kiss-and-run’ mechanisms, in which vesicles fuse transiently with the plasma membrane and release their contents through a partially opened fusion pore. In an effort to quantify the population of insulin release that occurs by kiss-and-run, we used three fusion constructs, between: (1) synaptobrevin-2 [vesicle-associated membrane protein 2 (VAMP2)] and pHluorin, a pH-sensitive green fluorescent protein (GFP) (synapto ·pHluorin) [5]; (2) the vesicle cargo neuropeptide Y (NPY) and Venus, a pHinsensitive GFP variant (NPY ·Venus) {Venus is derived from enhanced GFP (EGFP), but is much less affected by pH and has improved maturation kinetics [6]}; and (3) the membrane protein phogrin (phosphatase on the granule of insulinoma)