Modulation of intracellular calcium homeostasis blocks autophagosome formation
Modulation of intracellular calcium homeostasis blocks autophagosome formation
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DOI：
10.4161/auto.25900
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发表时间：
2013-10-01
期刊：
影响因子：
13.3
通讯作者：
Mills, Ian G.
中科院分区：
文献类型：
--
作者：
Engedal, Nikolai;Torgersen, Maria L.;Mills, Ian G.
细胞应激反应通常涉及胞质钙水平的升高，并且这已经被认为刺激自噬。然而，在这里，我们证明了改变细胞内钙离子稳态和诱导ER应激的药物--钙离子载体A23187和肌质/内质网Ca 2 +-ATP酶抑制剂thapsigargin（TG）--有效地抑制自噬。这种抗自噬作用发生在营养丰富和氨基酸饥饿的条件下，并反映了长寿命的蛋白质的自噬降解的强烈减少。此外，我们发现钙调节剂在获得WIPI 1后但在自噬体关闭之前抑制自噬体生物合成。后者是明显的，从几乎完全不能A23187或TG处理的细胞，以螯合胞质乳酸脱氢酶。此外，我们观察到饥饿诱导的EGFP-LC 3斑点的数量和大小减少，以及串联荧光mRFP-EGFP-LC 3细胞系中mRFP-LC 3斑点的数量减少。A23187和TG的抗自噬作用不依赖于ER应激，因为化学或siRNA介导的未折叠蛋白反应的抑制并不改变钙调节剂阻断自噬的能力。最后，值得注意的是，我们发现钙调节剂的抗自噬活性不需要细胞溶质钙水平的持续或大量变化。总之，我们认为细胞内钙离子水平的局部扰动可以在自噬体扩张和关闭阶段对自噬产生抑制作用。
Cellular stress responses often involve elevation of cytosolic calcium levels, and this has been suggested to stimulate autophagy. Here, however, we demonstrated that agents that alter intracellular calcium ion homeostasis and induce ER stressthe calcium ionophore A23187 and the sarco/endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin (TG)potently inhibit autophagy. This anti-autophagic effect occurred under both nutrient-rich and amino acid starvation conditions, and was reflected by a strong reduction in autophagic degradation of long-lived proteins. Furthermore, we found that the calcium-modulating agents inhibited autophagosome biogenesis at a step after the acquisition of WIPI1, but prior to the closure of the autophagosome. The latter was evident from the virtually complete inability of A23187- or TG-treated cells to sequester cytosolic lactate dehydrogenase. Moreover, we observed a decrease in both the number and size of starvation-induced EGFP-LC3 puncta as well as reduced numbers of mRFP-LC3 puncta in a tandem fluorescent mRFP-EGFP-LC3 cell line. The anti-autophagic effect of A23187 and TG was independent of ER stress, as chemical or siRNA-mediated inhibition of the unfolded protein response did not alter the ability of the calcium modulators to block autophagy. Finally, and remarkably, we found that the anti-autophagic activity of the calcium modulators did not require sustained or bulk changes in cytosolic calcium levels. In conclusion, we propose that local perturbations in intracellular calcium levels can exert inhibitory effects on autophagy at the stage of autophagosome expansion and closure.