Rab11 promotes docking and fusion of multivesicular bodies in a calcium-dependent manner
Rab11 promotes docking and fusion of multivesicular bodies in a calcium-dependent manner
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DOI：
10.1111/j.1600-0854.2004.00257.x
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发表时间：
2005-02-01
期刊：
影响因子：
4.5
通讯作者：
Colombo, MI
中科院分区：
文献类型：
--
作者：
Savina, A;Fader, CM;Colombo, MI
多泡体（Multivesicular bodies，MVB）是聚集在直径为60-100 nm的囊泡内的膜结构。MVB是在液泡腔中的内体膜内陷和夹断后产生的。在某些细胞类型中，MVB与质膜的融合导致称为外泌体的内部囊泡的释放。在这份报告中，我们已经研究了如何增加胞浆钙影响的发展MVB和外泌体释放K562细胞过表达GFP-Rab 11野生型或其突变体。在过表达Rab 11 Q70 L突变体或Rab 11 wt的细胞中，莫能菌素诱导的胞浆钙浓度增加导致MVB显著增大。这种作用被膜渗透钙螯合剂BAPTA-AM所消除。我们还通过延时共聚焦显微镜检查了活细胞中MVB的行为。许多由wt或Q70 L突变体GFP-Rab 11修饰的MVB被对接并准备在钙螯合剂的存在下融合。这一观察结果表明Rab 11在MVB的拴系/对接中起作用以促进同型融合，但最终的融合反应需要钙的存在。此外，细胞内钙离子浓度的升高增强了Rab 11 wt过表达细胞的外泌体分泌，并逆转了突变体的抑制作用。结果表明，Rab 11和钙离子都参与了MVB的同型融合。
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.