Enlargeosome Traffic: Exocytosis Triggered by Various Signals Is Followed by Endocytosis, Membrane Shedding or Both

Authors

  • Emanuele Cocucci,

    1. Center of Excellence in Cell Development, Vita-Salute San Raffaele University, DIBIT, via Olgettina 58, 20132 Milan, Italy
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  • Gabriella Racchetti,

    1. Scientific Institute San Raffaele, via Olgettina 58, 20132 Milan, Italy
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  • Paola Podini,

    1. Scientific Institute San Raffaele, via Olgettina 58, 20132 Milan, Italy
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  • Jacopo Meldolesi

    Corresponding author
    1. Center of Excellence in Cell Development, Vita-Salute San Raffaele University, DIBIT, via Olgettina 58, 20132 Milan, Italy
    2. Scientific Institute San Raffaele, via Olgettina 58, 20132 Milan, Italy
    3. Istituto Nazionale di Neuroscienze, via Olgettina 58, 20132 Milan, Italy
    4. IIT Network, Research Unit of Molecular Neuroscience, via Olgettina 58, 20132 Milan, Italy
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Jacopo Meldolesi, meldolesi.jacopo@hsr.it

Abstract

Enlargeosomes are cytoplasmic organelles discharged by regulated exocytosis, identified by immunofluorescence of their membrane marker, desmoyokin/Ahnak, but never revealed at the ultrastructural level. Among the numerous enlargeosome-positive cells, the richest and most extensively characterized are those of a PC12 clone, PC12-27, defective of classical neurosecretion. By using ultrastructural immunoperoxidase labeling of formaldehyde-fixed, Triton-X-100-permeabilized PC12-27 cells, we have now identified the enlargeosomes as small vesicles scattered in the proximity of, but never docked to, the plasma membrane. Upon stimulation, these vesicles undergo exocytosis [rapid after the Ca2+ ionophore, ionomycin, much slower after either the phorbol ester, phorbol myristate acetate (PMA), or ATP, working through a P2Y receptor], with appearance in the plasma membrane of typical desmoyokin/Ahnak (d/A)-positive, Ω-shaped and open profiles evolving into flat patches. Postexocytic removal of the exocytized d/A-positive membrane occurs by two processes: generation of endocytic vesicles, predominant after ionomycin and ATP 100–500 μM; and shedding of membrane-bound cytoplasmic bodies, predominant after PMA and 1 mM ATP, containing little or no trace of endoplasmic reticulum, Golgi, endo/lysosomes and also of a plasma membrane marker. Depending on the stimulation, therefore, the cell-surface expansion by enlargeosome exocytosis is not always recycled but can induce release of specific membranes, possibly important in the pericellular environment.

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