CALCIUM, A KEY COMPONENT IN THE REGULATION OF THE RAB11-DEPENDENT EXOSOME PATHWAY

CLAUDIO M. FADER, A. SAVINA AND M.I. COLOMBO

Pucon, Chile

Workshop; Protein trafficking: molecular mechanisms and disease; 2003

FONDAP-MIFAB

Multivesicular bodies (MVBs) are membranous structures that accumulate 60-100 nm vesicles internally. MVBs are generated after invagination and pinching off of the endosomal membrane in the lumen of the vacuole. We have previously demonstrated that Rabll is involved in exosorne secretion, regulating the interconnection between endocytic, recycling and secretory pathways. In the present report, we have examined how an increase in cytosolic calcium affects the development of MVBs and exosome release in K562 cells overexpressing wt GFP-Rabll or its mutants. We show that treatment of transfected cells with monensin, an agent that increases cytosolic calcium concentration, caused a marked enlargement of the MVBs, especially in mutant Rabl lQ70L and wt Rabl1 overexpressing cells. This effect was abrogated by the membrane permeant calcium chelator BAPTA-AM. We also examined the behavior of MVBs in living cells by time lapse confocal microscopy. Interestingly, we observed that many MVBs decorated by wt or Q70L mutant GFP-Rabl 1 are docked and ready to fuse in the presence of a calcium chelator. This observation suggests that Rabll is acting in tetherin-docking of MVBs to promote homotypic fusion, but that the final fusion reaction required the presence of calcium. Additionally, a rise in the intracellular calcium concentration enhanced exosome secretion in Rab11wt oaverexpressing cells and reverted the inhibition shown by the mutants. Taken together our results suggest that Rabl l is involved in MVB biogenesis and that calcium is required for the homotypic fusion of MVBs.