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

TLDR

Classical cell biology posits that exocytosis disperses vesicle membranes into the plasma membrane, requiring subsequent retrieval of desired components. This study tests whether secretory granules in intact PC‑12 cells follow that paradigm. Fluorescently labeled granule proteins (tPA, phogrin, neuro‑peptide Y) were imaged with two‑color evanescent‑field microscopy to track granule membrane and pH dynamics during and after exocytosis. tPA and phogrin remained at the granule site for over a minute, the granule did not flatten into the membrane, and reacidification of the granule interior (monitored by GFP‑phogrin) could be reversed by NH₄Cl, indicating that most granules reseal within <10 s and are recaptured largely intact.

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 neuro-peptide 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 NH 4 Cl. We conclude that most granules reseal in &lt;10 s after releasing cargo, and that these empty or partially empty granules are recaptured otherwise intact.