ArticleThe Biochemical journal2002
Monitoring of exocytosis and endocytosis of insulin secretory granules in the pancreatic beta-cell line MIN6 using pH-sensitive green fluorescent protein (pHluorin) and confocal laser microscopy.
Article in The Biochemical journal, 2002. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 32 papers.
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32 citing papers in PubMed, 73 citations in OpenAlex.
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- The PI(4)P phosphatase Sac2 controls insulin granule docking and release.The Journal of cell biology · 2019Article
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- Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets.Journal of visualized experiments : JoVE · 2017Article
- A Ratiometric Sensor for Imaging Insulin Secretion in Single β Cells.Cell chemical biology · 2017Article
- Exploring cells with targeted biosensors.The Journal of general physiology · 2017Review
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- The secretory deficit in islets from db/db mice is mainly due to a loss of responding beta cells.Diabetologia · 2014Article
- Illumination of the spatial order of intracellular pH by genetically encoded pH-sensitive sensors.Sensors (Basel, Switzerland) · 2013Review
- Glucose principally regulates insulin secretion in mouse islets by controlling the numbers of granule fusion events per cell.Diabetologia · 2013Article
- Imaging beta-cell mass and function in situ and in vivo.Journal of molecular medicine (Berlin, Germany) · 2013Review
- cAMP-mediated stabilization of fusion pores in cultured rat pituitary lactotrophs.The Journal of neuroscience : the official journal of the Society for Neuroscience · 2013Article
- investigating acid production by Streptococcus mutans with a surface-displayed pH-sensitive green fluorescent protein.PloS one · 2013Article
- Evolving insights regarding mechanisms for the inhibition of insulin release by norepinephrine and heterotrimeric G proteins.American journal of physiology. Cell physiology · 2012Review
- Coupling of metabolic, second messenger pathways and insulin granule dynamics in pancreatic beta-cells: a computational analysis.Progress in biophysics and molecular biology · 2011Review
- Hormonal inhibition of endocytosis: novel roles for noradrenaline and G protein G(z).The Journal of physiology · 2010Article
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Authors and funding
6 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
The dynamics of exocytosis/endocytosis of insulin secretory granules in pancreatic beta-cells remains to be clarified. In the present study, we visualized and analysed the motion of insulin secretory granules in MIN6 cells using pH-sensitive green fluorescent protein (pHluorin) fused to either insulin or the vesicle membrane protein, phogrin. In order to monitor insulin exocytosis, pHluorin, which is brightly fluorescent at approximately pH 7.4, but not at approximately pH 5.0, was attached to the C-terminus of insulin. To monitor the motion of insulin secretory granules throughout exocytosis/endocytosis, pHluorin was inserted between the third and fourth amino acids after the identified signal-peptide cleavage site of rat phogrin cDNA. Using this method of cDNA construction, pHluorin was located in the vesicle lumen, which may enable discrimination of the unfused acidic secretory granules from the fused neutralized ones. In MIN6 cells expressing insulin-pHluorin, time-lapse confocal laser scanning microscopy (5 or 10 s intervals) revealed the appearance of fluorescent spots by depolarization after stimulation with 50 mM KCl and 22 mM glucose. The number of these spots in the image at the indicated times was counted and found to be consistent with the results of insulin release measured by RIA during the time course. In MIN6 cells expressing phogrin-pHluorin, data showed that fluorescent spots appeared following high KCl stimulation and remained stationary for a while, moved on the plasma membrane and then disappeared. Thus we demonstrate the visualized motion of insulin granule exocytosis/endocytosis using the pH-sensitive marker, pHluorin.
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