The regulated exocytosis of enlargeosomes is mediated by a SNARE machinery that includes VAMP4

Abstract

The mechanisms governing the fast, regulated exocytosis of enlargeosomes have been unknown, except for the participation of annexin-2 in a pre-fusion step. We investigated whether any SNAREs are involved. In PC12-27 cells, which are enlargeosome-rich, the expressed SNAREs exhibited various distributions (trans-Golgi network, scattered puncta, plasma membrane); however, only VAMP4 was colocalized in discrete puncta with the enlargeosome marker desmoyokin. The exocytosis of the organelle, revealed by capacitance increases and by surface appearance of desmoyokin, was largely inhibited by microinjection of anti-VAMP4, anti-syntaxin-6 and anti-SNAP23 antibodies, by incubation with botulinum toxin E, and by transfection of VAMP4 and syntaxin-6 siRNAs. Microinjection of the antibodies anti-VAMP7, anti-VAMP8 and anti-syntaxin-4, and transfection with the VAMP8 siRNA were ineffective. Inhibition of enlargeosome exocytosis by VAMP4 siRNA also occurred in a cell type that was competent for neurosecretion, SH-SY5Y. Moreover, in cells expressing a VAMP4-GFP construct, enlargeosome exocytosis and surface appearance of fluorescence occurred concomitantly, and many ensuing surface patches were co-labelled by GFP and desmoyokin. VAMP4, an R-SNARE that has never been shown to participate in regulated exocytoses, therefore appears to be harboured in the membrane of enlargeosomes and to be a member of the machinery mediating their regulated exocytosis. Syntaxin-6 and SNAP23 appear also to be needed for the process to occur; however, the mechanism of their participation, whether direct or indirect, remains undefined.

Role of VAMP8/endobrevin in Constitutive Exocytotic Pathway in HeLa Cells

“…The role of VAMP3 in TNFα trafficking from recycling endosomes to the plasma membrane was also strongly supported in macrophages (Murray et al, 2005). Furthermore VAMP4 has recently been demonstrated on enlargeosomes and was shown to be involved in Ca 2+ -dependent fusion of enlargeosomes to the plasma membrane (Cocucci et al, 2008). Thus the non-polarized cells, including HeLa cells, can have diverse options of v-SNARE for constitutive exocytosis, and VAMP8 may play a role as one of v-SNAREs in the constitutive pathways.…”

Myosin Vb controls biogenesis of post-Golgi Rab10 carriers during axon development

“…In line with this notion, the Golgi apparatus is associated with the sites of axon specification 55,56 . It seems that several types of vesicles provide the source of the membrane precursors 5,41,[57][58][59] . The possible interaction between these vesicular systems makes the dissection for the contribution of each system very difficult.…”

Rapid neurite outgrowth in neurosecretory cells and neurons is sustained by the exocytosis of a cytoplasmic organelle, the enlargeosome

“…Whether additional organelles, not yet identified, are also (or alternatively) involved (Tang, 2008;Bonanomi et al, 2008) remained to be established. Here we show that the enlargeosomes, exocytic organelles (Borgonovo et al, 2002;Cocucci et al, 2004;Cocucci et al, 2008) expressed by some nerve cells and embryonic and neonatal brain cortex neurons, sustain a new, very rapid (tens of minutes) form of neurite outgrowth induced by the activation of the small GTPase Rac1 (Nusser et al, 2002;Aoki et al, 2005;Takefuji et al, 2007). In nerve cells lacking enlargeosomes, a Rac1-induced response occurs but resembles that induced by nerve growth factor (NGF) inasmuch as it is less extensive and delayed.…”