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. 1998 Jul 1;333(Pt 1):193–199. doi: 10.1042/bj3330193

Secretory-granule dynamics visualized in vivo with a phogrin-green fluorescent protein chimaera.

A E Pouli 1, E Emmanouilidou 1, C Zhao 1, C Wasmeier 1, J C Hutton 1, G A Rutter 1
PMCID: PMC1219572  PMID: 9639579

Abstract

To image the behaviour in real time of single secretory granules in neuroendocrine cells we have expressed cDNA encoding a fusion construct between the dense-core secretory-granule-membrane glycoprotein, phogrin (phosphatase on the granule of insulinoma cells), and enhanced green fluorescent protein (EGFP). Expressed in INS-1 beta-cells and pheochromocytoma PC12 cells, the chimaera was localized efficiently (up to 95%) to dense-core secretory granules (diameter 200-1000 nm), identified by co-immunolocalization with anti-(pro-)insulin antibodies in INS-1 cells and dopamine beta-hydroxylase in PC12 cells. Using laser-scanning confocal microscopy and digital image analysis, we have used this chimaera to monitor the effects of secretagogues on the dynamics of secretory granules in single living cells. In unstimulated INS-1 beta-cells, granule movement was confined to oscillatory movement (dithering) with period of oscillation 5-10 s and mean displacement <1 microm. Both elevated glucose concentrations (30 mM), and depolarization of the plasma membrane with K+, provoked large (5-10 microm) saltatory excursions of granules across the cell, which were never observed in cells maintained at low glucose concentration. By contrast, long excursions of granules occurred in PC12 cells without stimulation, and occurred predominantly from the cell body towards the cell periphery and neurite extensions. Purinergic-receptor activation with ATP provoked granule movement towards the membrane of PC12 cells, resulting in the transfer of fluorescence to the plasma membrane consistent with fusion of the granule and diffusion of the chimaera in the plasma membrane. These results illustrate the potential use of phogrin-EGFP chimeras in the study of secretory-granule dynamics, the regulation of granule-cytoskeletal interactions and the trafficking of a granule-specific transmembrane protein during the cycle of exocytosis and endocytosis.

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Selected References

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  1. Asfari M., Janjic D., Meda P., Li G., Halban P. A., Wollheim C. B. Establishment of 2-mercaptoethanol-dependent differentiated insulin-secreting cell lines. Endocrinology. 1992 Jan;130(1):167–178. doi: 10.1210/endo.130.1.1370150. [DOI] [PubMed] [Google Scholar]
  2. Ashcroft F. M., Kakei M. ATP-sensitive K+ channels in rat pancreatic beta-cells: modulation by ATP and Mg2+ ions. J Physiol. 1989 Sep;416:349–367. doi: 10.1113/jphysiol.1989.sp017765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Augustine G. J., Neher E. Calcium requirements for secretion in bovine chromaffin cells. J Physiol. 1992 May;450:247–271. doi: 10.1113/jphysiol.1992.sp019126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bonner-Weir S. Morphological evidence for pancreatic polarity of beta-cell within islets of Langerhans. Diabetes. 1988 May;37(5):616–621. doi: 10.2337/diab.37.5.616. [DOI] [PubMed] [Google Scholar]
  5. Greene L. A., Tischler A. S. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci U S A. 1976 Jul;73(7):2424–2428. doi: 10.1073/pnas.73.7.2424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Heim R., Cubitt A. B., Tsien R. Y. Improved green fluorescence. Nature. 1995 Feb 23;373(6516):663–664. doi: 10.1038/373663b0. [DOI] [PubMed] [Google Scholar]
  7. Hirokawa N. Kinesin and dynein superfamily proteins and the mechanism of organelle transport. Science. 1998 Jan 23;279(5350):519–526. doi: 10.1126/science.279.5350.519. [DOI] [PubMed] [Google Scholar]
  8. Hisatomi M., Hidaka H., Niki I. Ca2+/calmodulin and cyclic 3,5' adenosine monophosphate control movement of secretory granules through protein phosphorylation/dephosphorylation in the pancreatic beta-cell. Endocrinology. 1996 Nov;137(11):4644–4649. doi: 10.1210/endo.137.11.8895328. [DOI] [PubMed] [Google Scholar]
  9. Hurtley S. M. Recycling of a secretory granule membrane protein after stimulated secretion. J Cell Sci. 1993 Oct;106(Pt 2):649–655. doi: 10.1242/jcs.106.2.649. [DOI] [PubMed] [Google Scholar]
  10. Kreis T. E., Matteoni R., Hollinshead M., Tooze J. Secretory granules and endosomes show saltatory movement biased to the anterograde and retrograde directions, respectively, along microtubules in AtT20 cells. Eur J Cell Biol. 1989 Jun;49(1):128–139. [PubMed] [Google Scholar]
  11. Lacy P. E., Finke E. H., Codilla R. C. Cinemicrographic studies on beta granule movement in monolayer culture of islet cells. Lab Invest. 1975 Nov;33(5):570–576. [PubMed] [Google Scholar]
  12. Lang T., Wacker I., Steyer J., Kaether C., Wunderlich I., Soldati T., Gerdes H. H., Almers W. Ca2+-triggered peptide secretion in single cells imaged with green fluorescent protein and evanescent-wave microscopy. Neuron. 1997 Jun;18(6):857–863. doi: 10.1016/s0896-6273(00)80325-6. [DOI] [PubMed] [Google Scholar]
  13. Pouli A. E., Karagenc N., Wasmeier C., Hutton J. C., Bright N., Arden S., Schofield J. G., Rutter G. A. A phogrin-aequorin chimaera to image free Ca2+ in the vicinity of secretory granules. Biochem J. 1998 Mar 15;330(Pt 3):1399–1404. doi: 10.1042/bj3301399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Pouli A. E., Kennedy H. J., Schofield J. G., Rutter G. A. Insulin targeting to the regulated secretory pathway after fusion with green fluorescent protein and firefly luciferase. Biochem J. 1998 Apr 15;331(Pt 2):669–675. doi: 10.1042/bj3310669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Rorsman P. The pancreatic beta-cell as a fuel sensor: an electrophysiologist's viewpoint. Diabetologia. 1997 May;40(5):487–495. doi: 10.1007/s001250050706. [DOI] [PubMed] [Google Scholar]
  16. Rutter G. A., Burnett P., Rizzuto R., Brini M., Murgia M., Pozzan T., Tavaré J. M., Denton R. M. Subcellular imaging of intramitochondrial Ca2+ with recombinant targeted aequorin: significance for the regulation of pyruvate dehydrogenase activity. Proc Natl Acad Sci U S A. 1996 May 28;93(11):5489–5494. doi: 10.1073/pnas.93.11.5489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Rutter G. A., Theler J. M., Murgia M., Wollheim C. B., Pozzan T., Rizzuto R. Stimulated Ca2+ influx raises mitochondrial free Ca2+ to supramicromolar levels in a pancreatic beta-cell line. Possible role in glucose and agonist-induced insulin secretion. J Biol Chem. 1993 Oct 25;268(30):22385–22390. [PubMed] [Google Scholar]
  18. Sekine N., Cirulli V., Regazzi R., Brown L. J., Gine E., Tamarit-Rodriguez J., Girotti M., Marie S., MacDonald M. J., Wollheim C. B. Low lactate dehydrogenase and high mitochondrial glycerol phosphate dehydrogenase in pancreatic beta-cells. Potential role in nutrient sensing. J Biol Chem. 1994 Feb 18;269(7):4895–4902. [PubMed] [Google Scholar]
  19. Smith C. B., Betz W. J. Simultaneous independent measurement of endocytosis and exocytosis. Nature. 1996 Apr 11;380(6574):531–534. doi: 10.1038/380531a0. [DOI] [PubMed] [Google Scholar]
  20. Somers G., Blondel B., Orci L., Malaisse W. J. Motile events in pancreatic endocrine cells. Endocrinology. 1979 Jan;104(1):255–264. doi: 10.1210/endo-104-1-255. [DOI] [PubMed] [Google Scholar]
  21. Wacker I., Kaether C., Krömer A., Migala A., Almers W., Gerdes H. H. Microtubule-dependent transport of secretory vesicles visualized in real time with a GFP-tagged secretory protein. J Cell Sci. 1997 Jul;110(Pt 13):1453–1463. doi: 10.1242/jcs.110.13.1453. [DOI] [PubMed] [Google Scholar]
  22. Wasmeier C., Hutton J. C. Molecular cloning of phogrin, a protein-tyrosine phosphatase homologue localized to insulin secretory granule membranes. J Biol Chem. 1996 Jul 26;271(30):18161–18170. doi: 10.1074/jbc.271.30.18161. [DOI] [PubMed] [Google Scholar]

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Supplementary Materials

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