Microtubules facilitate autophagosome formation and fusion of autophagosomes with endosomes
Microtubules facilitate autophagosome formation and fusion of autophagosomes with endosomes
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DOI：
10.1111/j.1600-0854.2005.00368.x
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发表时间：
2006-02-01
期刊：
影响因子：
4.5
通讯作者：
Tooze, SA
中科院分区：
文献类型：
--
作者：
Köchl, R;Hu, XW;Tooze, SA
真核细胞的营养剥夺引起各种应激反应，包括自噬。自噬是由自噬体进行的，自噬体在与含有蛋白酶的内体融合后，将细胞质成分和细胞器隔离以进行降解。为了确定微管在自噬中的作用，我们使用诺可唑和长春碱破坏微管，并独立测量了原代大鼠肝细胞自噬体的形成和融合。通过测量自噬体标志物GFP-LC3在自噬体上的易位和GFP-LC3的脂化，我们量化了自噬体形成的速度和幅度。饥饿增加了基础水平上的自噬体形成速率和每个细胞的自噬体总数。最大的自噬体形成需要一个完整的微管网络。自噬体与核内体的融合，通过获得蛋白酶抑制剂的敏感性以及与LysoTracker red阳性核内体的重叠，需要完整的微管。活细胞成像显示自噬体是可运动的结构，其运动也需要微管。有趣的是，在观察到微管网络的任何可识别的变化之前，长春碱刺激自噬体形成超过两倍。vinblastine对自噬体形成的刺激不依赖于营养物质和mTOR活性，但被自噬蛋白Atg5和Atg6的消耗所抑制，Atg5和Atg6是自噬所必需的。
Nutrient deprivation of eukaryotic cells provokes a variety of stress responses, including autophagy. Autophagy is carried out by autophagosomes which sequester cytosolic components and organelles for degradation after fusion with protease-containing endosomes. To determine the role of microtubules in autophagy, we used nocodazole and vinblastine to disrupt microtubules and independently measured formation and fusion of autophagsosomes in primary rat hepatocytes. By measuring the translocation of GFP-LC3, an autophagosomal marker, to autophagosomes and the lipidation of GFP-LC3, we quantified the rate and magnitude of autophagosome formation. Starvation increased both the rate of autophagosome formation over the basal level and the total number of autophagosomes per cell. Maximal autophagosome formation required an intact microtubule network. Fusion of autophagosomes with endosomes, assayed by acquisition of protease-inhibitor sensitivity as well as overlap with LysoTracker Red-positive endosomes, required intact microtubules. Live-cell imaging demonstrated that autophagosomes were motile structures, and their movement also required microtubules. Interestingly, vinblastine stimulated autophagosome formation more than twofold before any discernable change in the microtubule network was observed. Stimulation of autophagosome formation by vinblastine was independent of nutrients and mTOR activity but was inhibited by depletion of the Autophagy proteins Atg5 and Atg6, known to be required for autophagy.