Secretory granules are recaptured largely intact after stimulated exocytosis in cultured endocrine cells

Justin W. Taraska, David Perrais, Mica Ohara-Imaizumi, Shinya Nagamatsu, Wolfhard Almers

Research output: Contribution to journalArticle

277 Scopus citations

Abstract

Classical cell biology teaches that exocytosis causes the membrane of exocytic vesicles to disperse into the cell surface and that a cell must later retrieve by molecular sorting whatever membrane components it wishes to keep inside. We have tested whether this view applies to secretory granules in intact PC-12 cells. Three granule proteins were labeled with fluorescent proteins in different colors, and two-color evanescent-field microscopy was used to view single granules during and after exocytosis. Whereas neuropeptide Y was lost from granules in seconds, tissue plasminogen activator (tPA) and the membrane protein phogrin remained at the granule site for over 1 min, thus providing markers for postexocytic granules. When tPA was imaged simultaneously with cyan fluorescent protein (CFP) as a cytosolic marker, the volume occupied by the granule appeared as a dark spot where it excluded CFP. The spot remained even after tPA reported exocytosis, indicating that granules failed to flatten into the cell surface. Phogrin was labeled with GFP at its luminal end and used to sense the pH in granules. When exocytosis caused the acidic granule interior to neutralize, GFP-phogrin at first brightened and later dimmed again as the interior separated from the extracellular space and reacidified. Reacidification and dimming could be reversed by application of NH4Cl. We conclude that most granules reseal in < 10 s after releasing cargo, and that these empty or partially empty granules are recaptured otherwise intact.

Original languageEnglish (US)
Pages (from-to)2070-2075
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume100
Issue number4
DOIs
StatePublished - Feb 18 2003

Keywords

  • Endocytosis
  • Evanescent-field microscopy
  • Kiss and run
  • PC-12 cells

ASJC Scopus subject areas

  • General

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