Köchl Robert, Hu Xiao Wen, Chan Edmond Y W, Tooze Sharon A
Cancer Research UK, London Research Institute, Secretory Pathways Laboratory, 44 Lincoln's Inn Fields, London, WC2A 3PX, UK.
Traffic. 2006 Feb;7(2):129-45. doi: 10.1111/j.1600-0854.2005.00368.x.
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.
真核细胞的营养剥夺会引发多种应激反应，包括自噬。自噬由自噬体执行，自噬体隔离胞质成分和细胞器，在与含蛋白酶的内体融合后进行降解。为了确定微管在自噬中的作用，我们使用诺考达唑和长春花碱破坏微管，并独立测量原代大鼠肝细胞中自噬体的形成和融合。通过测量自噬体标记物GFP-LC3向自噬体的转位以及GFP-LC3的脂化，我们量化了自噬体形成的速率和程度。饥饿增加了自噬体形成速率超过基础水平以及每个细胞中自噬体的总数。最大自噬体形成需要完整的微管网络。通过获得蛋白酶抑制剂敏感性以及与溶酶体追踪红阳性内体的重叠来检测自噬体与内体的融合，这需要完整的微管。活细胞成像表明自噬体是动态结构，它们的移动也需要微管。有趣的是，在观察到微管网络有任何可辨别的变化之前，长春花碱刺激自噬体形成超过两倍。长春花碱对自噬体形成的刺激与营养和mTOR活性无关，但被自噬蛋白Atg5和Atg6的缺失所抑制，已知这些蛋白是自噬所必需的。
