Annexin2 coating the surface of enlargeosomes is needed for their regulated exocytosis.
Enlargeosomes are small cytoplasmic vesicles that undergo rapid, Ca2+-dependent exo/endocytosis. The role of the cytoskeleton in these processes was unknown. In PC12-27 cells, microtubule disassembly had little effect on enlargeosomes, whereas microfilament disassembly increased markedly both their resting and stimulated exocytosis, and inhibited their endocytosis. Even at rest enlargeosomes are coated at their cytosolic surface by an actin-associated protein, annexin2, bound by a dual, Ca2+-dependent and Ca2+-independent mechanism. In contrast, the other enlargeosome marker, desmoyokin/Ahnak, is transported across the organelle membrane, apparently by an ABC transporter, and binds to its lumenal face. Annexin2-GFP expression revealed that, upon stimulation, the slow and random enlargeosome movement increases markedly and becomes oriented toward the plasma membrane. After annexin2 downregulation enlargeosome exocytosis induced by both [Ca2+]i rise and cytoskeleton disruption is inhibited, and the NGF-induced differentiation is blocked. Binding of annexin2 to the enlargeosome membrane, the most extensive ever reported (>50% annexin2 bound to approximately 3% of total membrane area), seems therefore to participate in the regulation of their exocytosis.
Lorusso
Anna
A
Scientific Institute San Raffaele, ALEMBIC, Advanced Light and Electron Microscopy BioImaging Center, Milan, Italy.
Covino
Cesare
C
Priori
Giuseppina
G
Bachi
Angela
A
Meldolesi
Jacopo
J
Chieregatti
Evelina
E
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D008841:Actin Cytoskeleton / Q000378:metabolism*; D000818:Animals; D017306:Annexin A2 / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D022162:Cytoplasmic Vesicles / Q000737:chemistry / Q000187:drug effects / Q000502:physiology*; D005089:Exocytosis / Q000187:drug effects / Q000235:genetics; D006801:Humans; D007425:Intracellular Membranes / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D009154:Mutation; D009363:Neoplasm Proteins / Q000378:metabolism*; D016716:PC12 Cells; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats
D018528:ATP-Binding Cassette Transporters; C489573:Ahnak protein, rat; D017306:Annexin A2; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D034741:RNA, Small Interfering