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Sci Rep. 2019 Apr 1;9(1):5403. doi: 10.1038/s41598-019-41891-x.

A novel optical tracer for VMAT2 applied to live cell measurements of vesicle maturation in cultured human β-cells.

Author information

1
Department of Chemistry and Biochemistry, California State University, Fullerton, California, USA.
2
Division of Experimental Therapeutics, Department of Medicine, Columbia University Medical Centre, New York, New York, USA.
3
Alberta Diabetes Institute, Ray Rajotte Surgical-Medical Research Institute, Department of Surgery, University of Alberta, Edmonton, AB, T6G 2E1, Canada.
4
Division of Endocrinology, Department of Medicine and Naomi Berrie Diabetes Center, Columbia University Medical Centre, New York, New York, USA.
5
Division of Endocrinology, Department of Medicine and Naomi Berrie Diabetes Center, Columbia University Medical Centre, New York, New York, USA. peh1@cumc.columbia.edu.

Abstract

The islet β-cells integrate external signals to modulate insulin secretion to better regulate blood glucose levels during periods of changing metabolic demand. The vesicular monoamine transporter type 2 (VMAT2), an important regulator of CNS neurotransmission, has an analogous role in the endocrine pancreas as a key control point of insulin secretion, with additional roles in regulating β-cell differentiation and proliferation. Here we report on the synthesis and biological characterisation of a fluorescent ligand for VMAT2 suitable for live cell imaging. Staining for VMAT2 and dopamine in live β-cell cultures show colocalisation in specific vesicles and reveal a heterogeneous population with respect to cell size, shape, vesicle number, size, and contents. Staining for VMAT2 and zinc ion, as a surrogate for insulin, reveals a wide range of vesicle sizes. Immunohistochemistry shows larger β-cell vesicles enriched for proinsulin, whereas smaller vesicles predominantly contain the processed mature insulin. In β-cell cultures obtained from nondiabetic donors, incubation at non-stimulatory glucose concentrations promotes a shift in vesicle diameter towards the more mature insulin vesicles at the expense of the larger immature insulin secretory vesicle population. We anticipate that this probe will be a useful reagent to identify living β-cells within complex mixtures for further manipulation and characterisation.

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