INDUCTION OF AUTOPHAGY PROMOTES FUSION OF MULTIVESICULAR BODIES WITH AUTOPHAGIC VACUOLES IN K562 CELL

FADER, C. M.; SÁNCHEZ, D. AND COLOMBO M.I

Villa Carlos Paz. Córdoba. Argentina.

Workshop; Internetional Workshop on Membrane Trafficking; 2005

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 GFP-Rab11 and the autophagosomal protein RFP-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 autophagic pathways was attenuated by the Ca2+ chelator BAPTA-AM 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.