Systematic Transmission Electron Microscopy-Based Identification and 3D Reconstruction of Cellular Degradation Machinery-猫眼课题宝

DOI：

10.1101/2021.09.26.461841

发表时间：

2021-09

期刊：

bioRxiv

影响因子：

通讯作者：

中科院分区：

其他

文献类型：

作者：

Kit Neikirk;Z. Vue;Prasanna Katti;Benjamin Rodriguez;Salem A. Omer;J. Shao;T. Christensen;E. Garza Lopez;A. Marshall;Caroline B. Palavicino-Maggio;Jessica M. Ponce;Ahmad F Alghanem;Lar L. Vang;Taylor Barongan;H. Beasley;Taylor A Rodman;Dominique C. Stephens;M. Mungai;M. Correia;V. Exil;S. Damo;Sandra A. Murray;Amber Crabtree;B. Glancy;R. Pereira;E. Abel;Antentor O. Hinton

许多相互关联的降解机制，包括自噬、溶酶体和内吞体，协同工作来进行自噬，这是一种对细胞内平衡至关重要的细胞内降解系统。改变的自噬有助于各种疾病的病理生理学，包括癌症和代谢性疾病。虽然许多研究已经对自噬进行了研究，以阐明疾病的发病机制，但识别自噬机制的特定组件一直是具有挑战性的。这篇论文的目的是描述一种方法，以重复性地识别和区分与宏观自噬有关的亚细胞结构。我们提供了帮助避免常见陷阱的方法，包括详细解释区分溶酶体和脂滴，并讨论自噬体和包涵体之间的差异。这些方法是基于使用透射电子显微镜(TEM)，能够在固定样本中生成细胞降解成分的纳米级显微图像。我们还利用连续块面扫描电子显微镜(SBF-SEM)提供了一种可视化降解机械的3D形态的方案。除了透射电子显微镜和3D重建，我们还讨论了其他成像技术，如免疫荧光和免疫金标记法，这些技术可以用于可靠和准确地对细胞器进行分类。我们的结果表明，这些方法可以用来准确地量化不同条件下的细胞降解机制，例如内质网应激源thapsigargin的治疗或动力蛋白相关蛋白1的消融。

Many interconnected degradation machineries including autophagosomes, lysosomes, and endosomes work in tandem to conduct autophagy, an intracellular degradation system that is crucial for cellular homeostasis. Altered autophagy contributes to the pathophysiology of various diseases, including cancers and metabolic diseases. Although many studies have investigated autophagy to elucidate disease pathogenesis, identification of specific components of the autophagy machinery has been challenging. The goal of this paper is to describe an approach to reproducibly identify and distinguish subcellular structures involved in macro autophagy. We provide methods that help avoid common pitfalls, including a detailed explanation for distinguishing lysosomes and lipid droplets and discuss differences between autophagosomes and inclusion bodies. These methods are based on using transmission electron microscopy (TEM), capable of generating nanometer-scale micrographs of cellular degradation components in a fixed sample. We also utilize serial block face-scanning electron microscopy (SBF-SEM) to offer a protocol for visualizing 3D morphology of degradation machinery. In addition to TEM and 3D reconstruction, we discuss other imaging techniques, such as immunofluorescence and immunogold labeling that can be utilized to reliably and accurately classify cellular organelles. Our results show how these methods may be used to accurately quantify the cellular degradation machinery under various conditions, such as treatment with the endoplasmic reticulum stressor thapsigargin or ablation of the dynamin-related protein 1.

Systematic Transmission Electron Microscopy-Based Identification and 3D Reconstruction of Cellular Degradation Machinery

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