Induction of autophagy promotes fusion of multivesicular bodies with autophagic vacuoles in K562 cells
Induction of autophagy promotes fusion of multivesicular bodies with autophagic vacuoles in K562 cells
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DOI：
10.1111/j.1600-0854.2007.00677.x
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发表时间：
2008-02-01
期刊：
影响因子：
4.5
通讯作者：
Colombo, Maria I.
中科院分区：
文献类型：
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作者：
Fader, Claudio M.;Sanchez, Diego;Colombo, Maria I.
形态学和生化研究表明，自噬体与内体融合，形成所谓的安菲体，一种前溶酶体混合细胞器。在本报告中，我们分析了 K562 细胞中的这一过程，K562 细胞是一种红白血病细胞系，可产生多囊泡体 (MVB) 并将称为外泌体的内部囊泡释放到细胞外介质中。我们之前已经证明，在 K562 细胞中，Rab11 修饰 MVB。因此，为了在分子水平上研究 MVB 与自噬途径的相互作用，我们通过共聚焦显微镜检查了 MVB 在过表达绿色荧光蛋白（GFP）-Rab11 和自噬体蛋白红色荧光蛋白-轻链 3（LC3）的细胞中的命运。自噬诱导剂（例如饥饿或雷帕霉素）导致 GFP-Rab11 装饰的液泡增大，并与 LC3 显着共定位。这种收敛被 Rab11 显性失活突变体消除，表明功能性 Rab11 参与了 MVB 和自噬途径之间的相互作用。有趣的是，我们提供的证据表明自噬诱导导致自噬室中钙积累。此外，内体和自噬途径之间的融合被钙螯合剂 1,2-双（邻氨基苯氧基）乙烷-N,N,N',N'-四乙酸（BAPTA）的 Ca2+ 螯合剂乙酰氧基甲酯（AM）减弱，表明 MVB 与自噬体区室的融合是钙依赖性事件。此外，自噬诱导或 LC3 过度表达会抑制外泌体释放，这表明在刺激自噬的条件下，MVB 会定向至自噬途径，从而抑制外泌体释放。
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 green fluorescent protein (GFP)-Rab11 and the autophagosomal protein red fluorescent protein-light chain 3 (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 the autophagic pathways was attenuated by the Ca2+ chelator acetoxymethyl ester (AM) of the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), 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.