RAB11 PROMOTES DOCKING AND FUSION OF MULTIVESICULAR BODIES IN A CALCIUM-DEPENDENT MANNER

ARIEL SAVINA, CLAUDIO M. FADER, MARÍA T. DAMIANI AND MARÍA ISABEL COLOMBO

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Blackwell Publishing

Lugar: La Jolla, California. USA; Año: 2005 vol. 6 p. 131 - 143

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. In certain cell types fusion of MVBs with the plasma membrane results in the release of the internal vesicles called exosomes. In the present report, we have examined how an increase in cytosolic calcium affects the development of MVBs and exosome release in K562 cells overexpresing wt GFP-Rab11 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 Rab11Q70L and wt Rab11 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-Rab11 are docked and ready to fuse in the presence of a calcium chelator. This observation suggests that Rab11 is acting in tethering/docking of MVBs to promote homotypic fusion, but that the final fusion reaction requires the presence of calcium. Additionally, a rise in the intracellular calcium concentration enhanced exosome secretion in Rab11 wt overexpressing cells and reverted the inhibition shown by the mutants. Taken together our results suggest that Rab11 is involved in MVB biogenesis and that calcium is required for the homotypic fusion of MVBs.