3D tomography reveals connections between the phagophore and endoplasmic reticulum

TLDR

Autophagosomes are reported to form near the endoplasmic reticulum, but it is unclear whether the phagophore membrane is directly connected to adjacent ER cisternae. The study aimed to investigate the relationship between the phagophore membrane and the ER. Electron microscopic tomography of serum‑ and amino‑acid‑starved normal rat kidney cells was performed, with cells fixed, embedded, and dual‑axis tilt series acquired to model 3D phagophore and ER morphology. Tomographic reconstructions revealed narrow extensions connecting the phagophore/autophagosome membrane to closely apposed ER cisternae, especially those inside the autophagosome, and also showed lipid droplets in close contact, suggesting implications for autophagosome membrane origin and phagophore extension.

Abstract

Autophagosomes have been reported to form in the vicinity of the endoplasmic reticulum (ER). In many cases, the phagophore membrane is observed between two cisternae of rough ER, but it is not known whether these two membranes are directly connected. To investigate the relationship of the phagophore membrane and the ER, we used electron microscopic tomography of serum and amino acid starved normal rat kidney cells. The cells were fixed in glutaraldehyde and reduced osmium tetroxide and embedded in Epon. Dual axis tilt image series were acquired from two successive 250-nm sections. To analyze the three-dimensional (3D) morphology of phagophores and the associated rough ER, 3D tomograms were used to model the ER and phagophore membranes. The tomographic reconstructions revealed connections between the phagophore/autophagosome membrane and the closely located ER cisternae, especially with the ER located inside the autophagosome. The connections were typically formed by narrow extensions from the phagophore/autophagosome to the ER. This finding has potential implications on the origin of autophagosome membranes, and on the mechanism of phagophore membrane extension. In addition, we observed lipid droplets in very close contact with the phagophores/autophagosomes.