Examining Autophagy in Plant by Transmission Electron Microscopy (TEM)
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Author:
Date:
2018-10-20
[Abstract] In plants, macroautophagy, here referred as autophagy, is a degradation pathway during which the double-membrane structure named autophagosome engulfs the cargo and then fuses with vacuole for material recycling.
To investigate the process of autophagy, transmission electron microscopy (TEM) was used to monitor the ultrastructure of autophagic structures and identify the cargo during this process due to its high resolution. Compared to other autophagy examination methods including biochemical assays and confocal microscopy, TEM is the only method that indicates the morphology of autophagic structures in nanoscale, which is considered to be one of the best ways to illustrate the morphology of autophagic intermediates and the substrate of autophagy. Here, we describe the ...
[摘要] 在植物中,巨自噬,这里称为自噬,是一种降解途径,在此期间,称为自噬体的双膜结构吞噬货物,然后与液泡融合以进行材料回收。
为了研究自噬过程,透射电子显微镜(TEM)用于监测自噬结构的超微结构,并由于其高分辨率在此过程中识别货物。 与其他自噬检测方法(包括生化检测和共聚焦显微镜)相比,TEM是唯一能够显示纳米级自噬结构形态的方法,被认为是阐明自噬中间体形态和自噬基质的最佳方法之一。。 在这里,我们描述了使用TEM在 Nicotiana benthamiana >叶细胞中的自噬检测分析。
【背景】自噬是真核生物中高度保守的大分子降解途径(Dikic,2017)。在植物中,自噬是由几种胁迫条件诱导的,包括饥饿,氧化应激,盐胁迫和衰老(Doelling et al。>,2002; Hanaoka et al。>,2002; Liu et al。>,2005; Bassham,2007; Liu and Bassham,2009; Luo et al。>,2017)。在自噬期间,称为自噬体的双膜囊泡在细胞质中形成并转运到中央液泡中,其中自噬体的外膜与液泡膜融合。然后被称为自噬体的单膜结构进入液泡腔并最终降解(Ohsumi,2001; Liu和Bassham,2012)。
到目前为止,已经建立了许多检测植物自噬的方法。常用的检测方法是共聚焦显微镜,电子显微镜和生化方法。至于共聚焦显微镜检测,自噬标记包括与荧光蛋白融合的ATG8,ATG5和SH3P2用于标记自噬相关结构(Zhuang ...
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Autophagy Assays (LC3B immunofluorescence, LC3B western blot, acridine orange assay)
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Author:
Date:
2013-12-20
[Abstract] Autophagy is a dynamic cellular event that is involved in the degradation of long lived proteins and organelles in cells. Biochemical methods such as western blot to measure autophagic proteins have been increasingly used in autophagy studies because it is convenient and objective. Among them, the total amount of Microtubule-associated protein light chain 3 (LC3), the mammalian homologue of the autophagy-related Atg8 in yeast, is a very useful and most commonly used tool in autophagy studies. Other methods such as electron microscopy and immunofluorescence are also available to measure autophagy. The following protocol describes three simple and commonly used protocols for measuring autophagy in cells: LC3B immunofluorescence, western blot and acridine orange assay. Although these three ...
[摘要] 自噬是参与细胞中长寿蛋白和细胞器的降解的动态细胞事件。生物化学方法如蛋白质印迹测量自噬蛋白已被越来越多地用于自噬研究,因为它是方便和客观的。其中,微管相关蛋白轻链3(LC3),酵母中自噬相关Atg8的哺乳动物同源物的总量是自噬研究中非常有用和最常用的工具。其他方法如电子显微镜和免疫荧光也可用于测量自噬。以下方案描述了用于测量细胞中自噬的三种简单且常用的方案:LC3B免疫荧光,western印迹和吖啶橙测定。虽然这三种方法经常用于提供关于自噬的基本信息,但是人们应该记住,由于这种动态过程的复杂性,它们不足以给出关于自噬通量的确切细节。此外,吖啶橙测定法仅是检测自噬的补充方法,因为其也对其他细胞器如溶酶体具有高亲和力。对于化合物对自噬通量的影响的进一步研究,推荐另外的和更复杂的测定。这里的协议提供了一个起点,人们得到一个化合物是否可以影响组织培养细胞自噬的快照。
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