Rab11 promotes docking and fusion of multivesicular bodies in a calcium-dependent manner.
Multivesicular bodies (MVBs) are membranous structures within 60-100 nm diameter vesicles accumulate. 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 this report we have examined how an increase in cytosolic calcium affects the development of MVBs and exosome release in K562 cells overexpressing GFP-Rab11 wt or its mutants. In cells overexpressing the Rab11Q70 L mutant or Rab11 wt, an increase in the cytosolic calcium concentration induced by monensin caused a marked enlargement of the MVBs. 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. Many MVBs, decorated by wt or Q70L mutant GFP-Rab11, were docked and ready to fuse in the presence of a calcium chelator. This observation suggests that Rab11 is acting in the tethering/docking of MVBs to promote homotypic fusion, but that the final fusion reaction requires the presence of calcium. Additionally, a rise in intracellular calcium concentration enhanced exosome secretion in Rab11 wt overexpressing cells and reversed the inhibition of the mutants. The results suggest that both Rab11 and calcium are involved in the homotypic fusion of MVBs.
Savina
Ariel
A
Laboratorio de Biología Celular y Molecular-Instituto de Histología y Embriología, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo--CONICET, Mendoza, 5500, Argentina.
Fader
Claudio M
CM
Damiani
María T
MT
Colombo
María Isabel
MI
D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D003600:Cytosol / Q000737:chemistry / Q000187:drug effects; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D011992:Endosomes / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D020014:K562 Cells; D007700:Kinetics; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D008561:Membrane Fusion; D018613:Microscopy, Confocal; D018715:Microscopy, Video; D008985:Monensin / Q000494:pharmacology; D009154:Mutation; D022161:Transport Vesicles / Q000378:metabolism*; D014966:Xanthenes; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000099193:rab11 GTP-Binding Proteins
D002118:Calcium; D002614:Chelating Agents; D004533:Egtazic Acid; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D007476:Ionophores; D008985:Monensin; D014966:Xanthenes; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; C034657:Texas red