微丝在植物细胞自噬中的功能研究

Autophagy is an evolutionarily conserved process for bulk degradation and recycling of cytoplasmic components, such as protein aggregates and organelles, in the vacuole or lysosome. The cytoskeleton, as a highly dynamic cellular scaffold, has been proven to play roles in the multiple processes of autophagy in yeast and mammalian cells, such as autophagosome formation, trafficking, fusion process of autophagosome with lysosome and cargo selection during selective autophagy. Recently, we reported that microtubules play a facilitative role in autophagosome formation in plants. However, little is known about the involvement of actin filament, another major cytoskeleton component, in plants. In this project, we propose to disturb actin filaments and study the autophagic response to actin disorganization in the model plant, Nicotiana benthamiana and evaluate its contribution to plant autophagy, which will greatly advance our knowledge of crosstalk between autophagy, actin filaments and even the cytoskeleton. This will be accomplished by using a combination of divergent approaches, including pharmacological, biochemical, genetic and molecular biological methods. So far, some preliminary data have been obtained. First, we disrupted actin filaments successfully in N. benthamiana by either shor- or long-term treatment with actin polymerization inhibitors, cytochalasin D and latrunculin B or silencing of Actin genes. Second, we observed a larger number of autophagosomes in Actin-silenced plants than the control, which were further increased by treatment with E64d, a protease inhibitor widely used to block cargo degradation in vacuole, suggesting autophagy activation in Actin-silenced plants. Further experiments are needed to confirm the effect of actin disorganization on various types of autophagy, investigate the molecular links between actin cytoskeleton and autophagy and uncover the role of actin filaments in plant autophagy and its underlying mechanism.

细胞自噬是真核生物中进化保守的经液泡或溶酶体介导的物质降解过程。作为细胞内具有高度动态活性的支架结构，细胞骨架被证明在酵母和哺乳动物细胞中的细胞自噬的多个生物步骤中起作用。我们最近的研究首次揭示了微管在植物自噬小泡形成中的促进作用。然而，关于微丝在植物细胞自噬中的作用鲜有报道。本项目中，我们将在本生烟中干扰微丝网络后研究其对细胞自噬的影响，进而对其在植物细胞自噬中的作用进行评估。目前为止，我们已经取得了一些初步的结果：通过在叶片中短时段、长时段地施加微丝解聚剂或在烟草中Actin基因的方法成功实现了对微丝网络的干扰；我们在沉默Actin的烟草叶片中观察到了自噬流的激活。未来，我们需要在多种细胞自噬诱导系统中去检测微丝网络受损对细胞自噬的影响，并且对微丝与细胞自噬的分子联系进行研究。该项目的立项和顺利开展将大大有助于增进我们对细胞自噬与微丝甚至整个细胞骨架系统之间联系的了解。

微丝是植物骨架的重要组成部分，在多种生命过程中均有重要的作用。此前的研究表明，微丝对于酵母选择性自噬的发生及哺乳动物细胞中自噬体的形成都非常重要。这让许多人相信，微丝骨架在植物细胞自噬的发生中也起着非常重要的作用。然而令人意外的是，我们的研究发现微丝对于植物的非选择性自噬不是必需的。使用微丝聚合抑制剂细胞松弛素D、拉春库林B 或过表达Profilin3在烟草中解聚微丝，均不影响本生烟中基础自噬、夜间发生的自噬上调以及盐胁迫诱导的自噬。使用微丝聚合抑制剂处理拟南芥，也不影响基础自噬和盐胁迫诱导的自噬发生。另外，沉默Actin7或微丝聚合抑制剂处理24h长期损伤丝骨架，会导致内质网异常，激活细胞自噬并介导内质网降解，这一过程依赖于自噬关键蛋白ATG2、ATG3、ATG5、ATG6 和ATG7。以上结果表明，和动物中不同，植物微丝并不参与非选择性细胞自噬的发生和调控。