The autophagy research in electron microscopy
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Autophagy, a highly conserved process of eukaryotic cellular recycling, plays an important role in cell survival and maintenance. Dysfunctional autophagy contributes to the pathologies of many human diseases. Many studies have attempted to clarify the process of autophagy. Here, we review morphological studies of autophagy involving electron microscopy.
KeywordsElectron microscopy Autophagic flux CLEM Immuno-gold Cryo-EM
In the late 1950s, electron microscopy (EM) studies identified the autophagosome as a mitochondria surrounded by a larger vesicular structure (Rhodin 1954). Since this discovery, EM has been the main tool used to study autophagy (Rhyu 2017). Autophagy is a well-known lysosomal degradation pathway and a major factor in cellular clearance mechanisms. The ubiquitin–proteasome system degrades short-lived or abnormally folded proteins, while the lysosome-autophagy process targets long-lived macromolecular complexes and organelles. Defects in autophagy decrease the removal of potential sources of genotoxic stress, such as reactive oxygen species (ROS), from damaged and leaky mitochondria or other organelles (White 2012). Accumulated ROS may be an important cause of DNA damage and genetic instability. Neurodegenerative disease is a main consequence of autophagic failure. The definition of autophagy covers three general types of mechanisms, depending on the pathway used to deliver cargo: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). Macroautophagy and microautophagy are conserved in all eukaryotes, whereas CMA seems to be specific to higher eukaryotes. In macroautophagy, a double- or multi-membraned autophagosome fuses with a lysosome to enable nonspecific degradation. In contrast, microautophagy involves direct engulfment by the lysosome. Although these two systems were visualized in an EM image of the rat liver in 1966 (De Duve and Wattiaux 1966), microautophagy was not as well clarified. In this review, we describe several techniques used to understand various autophagic processes.
Electron microscopy with conventional fixation and plastic sectioning
Correlative light and electron microscopy
Immuno-gold labeling of proteins in autophagy flux
Negative staining and 2D averaging of protein complexes in autophagy
Negative staining is the most common EM technique used to determine protein structures (Scarff et al. 2018). This method enables the relatively simple and rapid observation of macromolecules and macromolecular complexes using a contrast-enhanced staining solution, such as uranyl acetate or uranyl formate. However, this technique has a limited resolution and is mainly used to assess samples before cryo-TEM. However, negative staining is a powerful EM technique for the structural studies of protein and protein complexes when combined with 2D class averaging. Negative stained EM was also used to visualize the core mechanistic factors of autophagy, such as the Atg17–Atg31–Atg29 complex. The S-shape of this complex suggested that it may contribute to high vesicular curvature. This S-shaped structure could be visualized using 2D classification (Chew et al. 2013; Mao et al. 2013), which is used to increase the signal-to-noise ratio and enhance finer details via image averaging (Ohi et al. 2004). The S-shaped Atg17–Atg31–Atg29 complex suggested PAS organization and autophagy induction. Chew and colleagues also used negative staining EM and 2D averaging to demonstrate that Atg17 mediates dimerization and conformational flexibility (Chew et al. 2013). Later, they reported that Atg13 serves as a bridge to the catalytic Atg1 subunit in the Atg17–Atg31–Atg29 complex (Chew et al. 2015). Negative stained EM, 2D averaging and 3D reconstruction were also used to reveal the V-shaped structure of the phosphatidylinositol 3-kinase complex 1 (PI3KC3–C1 complex), which is involved in autophagy initiation (Baskaran et al. 2014). This complex comprises the lipid kinase VPS34, scaffolding protein VPS15, tumor suppressor BECN1 and autophagy-specific subunit ATG14. The two arms of the V shape consist of the largest protein, VPS15, which acts as a bridge between BECN1 and VPS34. This connection forms a flexible structure with one inflexible arm, suggesting that this structural characteristic may be useful for drug design. A detailed higher-resolution structure was also studied using cryo-EM.
Cryo-EM of protein related to autophagy
Cryo-EM provides higher-resolution images because proteins are not covered in a contrast-enhanced staining solution (Cressey and Callaway 2017). The Nobel prize winner Dubochet and colleagues developed a liquid ethane-based technique for the rapid freezing of proteins that prevents the dehydration of water-soluble biomolecules in the vacuum of an electron microscope. Subsequently, Henderson and Frank, also Nobel prize winners, developed software to reconstruct the Cryo-EM images into 3D structures. This software allowed researchers to use EM to determine the structures of proteins at much higher resolutions than were previously available, and it is considered as a resolution revolution (Kuhlbrandt 2014). The PI3KC3–C1 complex was studied using cryo-EM and a single particle analysis (Ma et al. 2017). Specifically, Ma et al. reported the cryo-EM structures of human PI3KC3–C1 and PI3KC3–C2 at a subnanometer resolution. These authors also visualized the orientations of the complexes on membranes by deleting ATG14L or the C terminus of VPS34. The study results demonstrated that the C terminus of ATG14L is responsible for anchoring C1 on membranes, while the C-terminal VPS34 deletion mutant determined the orientation of the complex. Autophagosomes are thought to emerge from omegasomes in the ER. One report suggested that omegasome formation may be related to the phosphorylation of ER via PI3KC3 kinases (Nascimbeni et al. 2017). Structural data based on cryo-TEM suggested the process by which the kinases were recruited to the ER. This process required ATG14L-BATs and mediated the binding of PI3KC3 to the membranes, suggesting that the complex interacts directly with the ER membrane. Purified proteins are useful for cryo-EM-based structural studies. However, more advanced EM techniques, such as cryo-electron tomography (cryo-ET) using cryo-sectioning (CEMOVIS), and new approaches involving combinations with the cryo-Focused Ion Beam (cryo-FIB) are available for studies of unknown autophagic processes within the cell.
Autophagy was originally described using electron microscopy approximately 50 years ago, when electron microscopy and sample preparation methods for biological materials had just emerged. The field has expanded exponentially since the discovery of autophagy genes, and this biological process has increasingly gained attention. Despite major developments in various methods used to monitor autophagy in cells and organisms, EM provides necessary qualitative and quantitative information that cannot be obtained using other methods. In the field of EM, cryofixation and tomography are likely to provide high-resolution 3D images of autophagic compartments that are free of artifacts caused by chemical fixation. These characteristics are very likely to elucidate unanswered questions in the field of autophagy field. Higher-resolution imaging and specific labeling techniques based on advanced EM, including CLEM and CEMOVIS, are needed to investigate various unanswered questions regarding the mechanisms of autophagy regulation.
TEM data were acquired at Brain Research Core Facilities in KBRI.
MKJ and HC contributed to data acquisition and preparation of figures. JYM was a major contributor in writing the manuscript. All authors read and approved the final manuscript.
This research was supported by KBRI Basic Research Program through Korea Brain Research Institute Funded by Ministry of Science and ICT, Grant Number: 19-BR-01-08, and National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2019R1A2C1010634).
The authors declare that they have no competing interests.
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