Correlative light-electron microscopy of autophagosome biogenesis

Session

LSA.8 - Imaging self eating - autophagy under the microscope

Authors

Sigurdur Runar Gudmundsson (3), Helena Vihinen (3), Eija Jokitalo (3), Shun Kageyama (2), Nicholas Ktistakis (1), Masaaki Komatsu (2), Eeva-Liisa Eskelinen (4, 3)

Affiliations

1. Babraham Institute
2. Juntendo University
3. University of Helsinki
4. University of Turku

Keywords

Autophagy, Correlative Light - Electron Microscopy

Abstract text

Macroautophagy is a crucial pathway of transporting unwanted or harmful cellular material to the lysosome. The material is isolated by a double membraned structure named the phagophore. When the phagophore closes it forms an autophagosome. The autophagosomes then fuse with endosomes and finally with lysosomes where the isolated material is degraded and recycled into the cytoplasm. The origin of the phagophore membrane and the biogenesis of autophagosomes are long-lasting questions that are still not fully understood. 

Our goal is to image forming phagophores of nonselective and selective autophagy at different time points to establish the 3D structure of the phagophore assembly site and identify potential membrane donors that would supply lipids to the phagophore. To be able to locate early phagophores we established a Correlative Light and Electron Microscopy (CLEM) protocol that enables us to track the phagophore formation over time by live cell imaging of early autophagy proteins such as ATG13 and DFCP1. We then fix the cells and prepare them for electron microscopy in order to capture 3D electron micrographs.

Currently we have collected data on starvation induced autophagy, and on selective autophagy of mitochondria (mitophagy) and SQSTM1/p62 aggregates (aggrephagy). From these datasets we have modelled phagophores at different stages of formation, visualizing the phagophore and surrounding organelles in 3D.

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Autophagy is a catabolic pathway that delivers cytoplasmic material to the lysosome and is activated by cellular stress. This pathway protects the cell by degrading and recycling non-essential cytoplasmic material and damaged organelles, aggregated proteins and intracellular pathogens. If autophagy is not functional, these substrates accumulate in cells, which leads to disturbed cellular functions and cell death. Post-mitotic cells like neurons are particularly dependent on the autophagic clearance. In autophagy, the cellular material being degraded is first isolated by a membrane cistern known as the phagophore. When the phagophore is fully closed it forms an autophagosome that will fuse with endosomes and later with lysosomes where the cargo is degraded and recycled back into the cytoplasm. Autophagy can either be selective or nonselective as is the case during amino-acid starvation.  

During starvation the mechanistic target of rapamycin complex 1 (mTORC1) inactivates, causing the translocation of the ULK1 protein kinase complex to the endoplasmic reticulum (ER), which leads to the sequential recruitment of the autophagic machinery to the ER. These events initiate phagophore membrane formation. During selective autophagy the harmful substrate is identified and labeled by ubiquitin that is recognized by autophagy receptors, which initiates phagophore formation. 

Currently it is unclear how the phagophore forms and where the membranes forming the phagophore originate from. There is however a consensus that the ER and different types of vesicles deliver lipids to the growing phagophore membrane. It has also been shown that the phagophore can form connections or membrane contact sites with mitochondria.

The phagophore is a very short-lived, small and complex structure that rapidly changes, making it technically challenging to locate and image, especially in electron microscopy. To be able to find and image the phagophores at different time points during their formation, I established a live-cell imaging CLEM protocol (Gudmundsson et al. Methods in Molecular Biology 2018). This method allows me to image nascent phagophores labelled with fluorescent proteins in living cells and then fix the cells and continue visualizing the same structures with three-dimensional electron microscopy (3D-EM). Using transmission electron microscopy (TEM) to image the membrane structure in cells embedded in epoxy resin, I can achieve high resolution in either 2D by standard thin sections, or 3D using serial sections or electron tomography.

Currently we have obtained CLEM datasets of phagophores in both selective and nonselective autophagy. During nonselective autophagy the phagophore forms from the ER as a small round structure in close proximity of a multivesicular body – type endosome. At a later stage the phagophore forms multiple contact sites with the ER, both ER inside the phagophore and ER outside the phagophore. In mitophagy the phagophore forms, in association with the ER, as a sheet and wraps around the mitochondrion. Additionally, multiple phagophores can simultaneously form around a single mitochondrion, which then fuse together to form an autophagosome. 

 

During selective autophagy of p62 aggregates the phagophore forms as sheets similar to those observed in mitophagy, in association with the ER. The p62 aggregate is typically first split into smaller pieces which are subsequently isolated by multiple phagophores. Additionally, there are phagophores that form next to the aggregates in certain orientation but do not contain p62, suggesting that the aggregates also induce formation of nonselective autophagosomes. 

 

It is also of note that while phagophores in starvation induced autophagy or surrounding p62 aggregates form membrane contact sites with mitochondria on the outside of the phagophore, the only mitochondrial membrane contact that we identified in mitophagy is with the cargo mitochondrion.

 

In conclusion, we have visualized the forming phagophore at a higher resolution than has been done before and shown that the main membrane donor is the ER in both selective and starvation induced autophagy. While the membrane donor for both selective and starvation induced autophagy is the same, the shape of the phagophore vastly differs depending on the situation. Under starvation the early phagophore is a small round structure, while in selective autophagy its shape is determined by the cargo. A selective phagophore forms as one or several sheets that wrap around and enclose the cargo.