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Vol. 15, Issue 12, 5356-5368, December 2004

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Enlargeosome, an Exocytic Vesicle Resistant to Nonionic Detergents, Undergoes Endocytosis via a Nonacidic Route

Emanuele Cocucci ^*  , Gabriella Racchetti ^*, Paola Podini ^*, Marjan Rupnik , and Jacopo Meldolesi ^* 

^* Vita-Salute University, and San Raffaele Scientific Institute, Excellence Center in Cell Differentiation Pathophysiology, 20132 Milan, Italy; European Neuroscience Institute Goettingen, 37073 Goettingen, Germany; and Department of Pharmacology, University of Milan, 20133 Milan, Italy

Submitted July 9, 2004; Revised September 24, 2004; Accepted September 27, 2004
Monitoring Editor: Randy Schekman

Enlargeosomes, a new type of widely expressed cytoplasmic vesicles, undergo tetanus toxin-insensitive exocytosis in response to cytosolic Ca²⁺ concentration ([Ca²⁺]_i) rises. Cell biology of enlargeosomes is still largely unknown. By combining immunocytochemistry (marker desmoyokin-Ahnak, d/A) to capacitance electrophysiology in the enlargeosome-rich, neurosecretion-defective clone PC12-27, we show that 1) the two responses, cell surface enlargement and d/A surface appearance, occur with similar kinetics and in the same low micromolar [Ca²⁺]_i range, no matter whether induced by photolysis of the caged Ca²⁺ compound o-nitrophenyl EGTA or by the Ca²⁺ ionophore ionomycin. Thus, enlargeosomes seem to account, at least in large part, for the exocytic processes triggered by the two stimulations. 2. The enlargeosome membranes are resistant to nonionic detergents but distinct from other resistant membranes, rich in caveolin, Thy1, and/or flotillin1. 3. Cell cholesterol depletion, which affects many membrane fusions, neither disrupts enlargeosomes nor affects their regulated exocytosis. 4. The postexocytic cell surface decline is [Ca²⁺]_i dependent. 5. Exocytized d/A-rich membranes are endocytized and trafficked along an intracellular pathway by nonacidic organelles, distinct from classical endosomes and lysosomes. Our data define specific aspects of enlargeosomes and suggest their participation, in addition to cell differentiation and repair, for which evidence already exists, to other physiological and pathological processes.

Abbreviations used: antibody, mouse monoclonal antibody raised against d/A; [Ca²⁺]_i, cytosolic concentration of free calcium; cdx, methyl--cyclodextrin; d/A, desmoyokin/Ahnak; DG, dense granule; DM-nitrophen, dimethoxynitrophenamine tetrasodium salt; EEA1, early endosomal antigen 1; fF, femtoFarad, unit of capacitance; FITC, fluorescein isothiocyanate; TRITC, tetramethylrhodamine isothiocyanate; Lamp1, lysosomal membrane glycoprotein 1; NP-EGTA, o-nitrophenyl EGTA; PC12, rat pheochromocytoma cell line; SLMV, synaptic-like microvesicle; TeTx, tetanus toxin; TGN, trans-Golgi network; TX-100, Triton X-100; wt, wild-type.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Corresponding author. E-mail address: meldolesi.jacopo{at}hsr.it.

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