Abstract
Multivesicular bodies (MVBs) are endocytic structures that contain small vesicles formed by the budding of an endosomal membrane into the lumen of the compartment. Fusion of MVBs with the plasma membrane results in secretion of the small internal vesicles termed exosomes. K562 cells are a hematopoietic cell line that releases exosomes. The application of monensin (MON) generated large MVBs that were labeled with a fluorescent lipid. Exosome release was markedly enhanced by MON treatment, a Na+/H+ exchanger that induces changes in intracellular calcium (Ca2+). To explore the possibility that the effect of MON on exosome release was caused via an increase in Ca2+, we have used a calcium ionophore and a chelator of intracellular Ca2+. Our results indicate that increasing intracellular Ca2+ stimulates exosome secretion. Furthermore, MON-stimulated exosome release was completely eliminated by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM), implying a requirement for Ca2+ in this process. We have observed that the large MVBs generated in the presence of MON accumulated Ca2+ as determined by labeling with Fluo3-AM, suggesting that intralumenal Ca2+ might play a critical role in the secretory process. Interestingly, our results indicate that transferrin (Tf) stimulated exosome release in a Ca2+-dependent manner, suggesting that Tf might be a physiological stimulus for exosome release in K562 cells.
Highlights
¶ Recipient of Programa de Cooperacion Argentino-Francesa de Formacion para la Investigacion Cientıfica y Tecnologica (SECYT-ECOS)Sud Joint Grant A98B04
Monensin Induces the Formation of Large Multivesicular bodies (MVBs) and Stimulates Exosome Secretion—K562 cells are human erythroleukemic cells that secrete exosomes (24), the small internal vesicles released into the extracellular media by fusion of MVBs with the plasma membrane (PM)
It has been shown by electron microscopy that treatment of K562 cells with the ionophore MON causes the formation of dilated MVBs (25, 12)
Summary
¶ Recipient of Programa de Cooperacion Argentino-Francesa de Formacion para la Investigacion Cientıfica y Tecnologica (SECYT-ECOS)Sud Joint Grant A98B04. In antigen-presenting cells, the fusion of these MVBs with the plasma membrane leads to the release of internal vesicles into the extracellular space (1). 17 and 18), is necessary to induce regulated secretion in most cell types (reviewed in Refs. and 20). Because MON generates large MVBs in K562 cells, the aim of the present study was to determine whether MON affects exosome release and establish whether Ca2ϩ is involved in this process. Our results indicate that both MON treatment and a rise in intracellular Ca2ϩ markedly stimulate exosome secretion. We have observed that MON induced the accumulation of Ca2ϩ in the enlarged MVBs, suggesting that intravesicular Ca2ϩ might be involved in the secretory step. Our results indicate that Tf stimulates exosome release in a Ca2ϩ-dependent manner
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