Three-dimensional snapshots of phagophores in near-native state
Joanna Biazik1, Helena Vihinen2, Eija Jokitalo2 and Eeva-Liisa Eskelinen1
University of Helsinki; 1Department of Biosciences; Division of Biochemistry and Biotechnology, and 2Institute of Biotechnology; Electron Microscopy Unit
Autophagy is a fundamental housekeeping and survival pathway that recycles organelles and aggregate-prone proteins in eukaryotic cells. Nascent autophagosomes, also called phagophores, nucleate from a subdomain of the endoplasmic reticulum (ER). Several other organelles such as mitochondria, Golgi complex, plasma membrane and recycling endosomes have also been linked with phagophore biogenesis.
We have shown using electron tomography (ET) of aldehyde-fixed cells, that phagophores form membrane contacts with several other organelles, including ER, ER exit sites, Golgi cisternae, endosomes/lysosomes, and mitochondria (Biazik et al. 2015). We currently focus on improving the ultrastructural preservation of our electron microscopy samples. To achieve this, we are using electron tomography of high-pressure frozen, freeze substituted cells to visualize phagophores and their surrounding organelles in near-native state. The preliminary results have revealed that high-pressure freezing and freeze substitution preserve small vesicles better than conventional aldehyde fixation.
Further, better preservation of filamentous projections such as microtubules is achieved. This will allow us to analyze the relationship of the phagophores and small vesicles and cytoskeletal filaments at high resolution.
Biazik J, Ylä-Anttila P, Vihinen H, Jokitalo E, Eskelinen
EL: Ultrastructural relationship of the phagophore with surrounding organelles. Autophagy 2015, DOI 10.1080/15548627.2015.1017178