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 ∼3% of total membrane area), seems therefore to participate in the regulation of their exocytosis.
SummaryNeurite outgrowth is known as a slow (days) process occurring in nerve cells and neurons during neurotrophin treatment and upon transfer to culture, respectively. Using Y27632, a drug that induces activation of Rac1, a downstream step of the neurotrophin signaling cascade, we have identified a new form of outgrowth, which is rapid (<1 hour) and extensive (>500 mm 2 surface enlargement/single cell/first hour). However, this outgrowth takes place only in cells (PC12-27 and SH-SY5Y cells, and embryonic and neonatal neurons) rich in an exocytic organelle, the enlargeosome. Golgi vesicles, TGN vesicles and endosomes are not involved. The need for enlargeosomes for plasma-membrane expansion was confirmed by the appearance of their marker, Ahnak, at the cell surface and by the dependence of neurite outgrowth on VAMP4, the vSNARE of enlargeosome exocytosis. In enlargeosome-rich cells, VAMP4 downregulation also attenuated the slow outgrowth induced by nerve growth factor (NGF). Similar to NGF-induced neurite outgrowth in enlargeosomelacking cells, the new, rapid, Y27632-induced process required microtubules. Other properties of neurite outgrowth in cells lacking enlargeosomes -such as dependence on VAMP7, on microfilaments, on gene transcription and on protein synthesis, and blockade of mitoses and accumulation of neuronal markers -were not evident. The enlargeosome-sustained process might be useful for the rapid neurite outgrowth at peculiar stages and/or conditions of nerve and neuronal cells. However, its properties and its physiological and pathological role remain to be investigated.
Our data suggest that NK-1R antagonists, such as Fosaprepitant, could be a new, promising therapeutic tool to inhibit CNV after this has been established.
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