RESUMO
Morphological and biochemical studies have shown that autophagosomes fuse with endosomes forming the so-called amphisomes, a prelysosomal hybrid organelle. In the present report, we have analyzed this process in K562 cells, an erythroleukemic cell line that generates multivesicular bodies (MVBs) and releases the internal vesicles known as exosomes into the extracellular medium. We have previously shown that in K562 cells, Rab11 decorates MVBs. Therefore, to study at the molecular level the interaction of MVBs with the autophagic pathway, we have examined by confocal microscopy the fate of MVBs in cells overexpressing green fluorescent protein (GFP)-Rab11 and the autophagosomal protein red fluorescent protein-light chain 3 (LC3). Autophagy inducers such as starvation or rapamycin caused an enlargement of the vacuoles decorated with GFP-Rab11 and a remarkable colocalization with LC3. This convergence was abrogated by a Rab11 dominant negative mutant, indicating that a functional Rab11 is involved in the interaction between MVBs and the autophagic pathway. Interestingly, we presented evidence that autophagy induction caused calcium accumulation in autophagic compartments. Furthermore, the convergence between the endosomal and the autophagic pathways was attenuated by the Ca2+ chelator acetoxymethyl ester (AM) of the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), indicating that fusion of MVBs with the autophagosome compartment is a calcium-dependent event. In addition, autophagy induction or overexpression of LC3 inhibited exosome release, suggesting that under conditions that stimulates autophagy, MVBs are directed to the autophagic pathway with consequent inhibition in exosome release.
Assuntos
Autofagia/fisiologia , Vesículas Citoplasmáticas/fisiologia , Fusão de Membrana/fisiologia , Aminoácidos/deficiência , Autofagia/efeitos dos fármacos , Proteína 12 Relacionada à Autofagia , Cadaverina/análogos & derivados , Cadaverina/metabolismo , Cálcio/metabolismo , Quelantes/farmacologia , Meios de Cultura Livres de Soro/farmacologia , Vesículas Citoplasmáticas/efeitos dos fármacos , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Exocitose/efeitos dos fármacos , Exocitose/fisiologia , Proteínas de Choque Térmico HSC70/genética , Proteínas de Choque Térmico HSC70/metabolismo , Humanos , Células K562 , Fusão de Membrana/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Biológicos , Monensin/farmacologia , Nocodazol/farmacologia , Proteínas/genética , Proteínas/metabolismo , RNA Interferente Pequeno/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sirolimo/farmacologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina , Transfecção , Vimblastina/farmacologia , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
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.