In situ visualization of autophagy reveals the presence of vesicles at the expanding phagophore rim.

The autophagy core machinery mediates the enclosure of cytosolic cargo destined for degradation in the lysosome. The Atg9-Atg2-Atg18 complex coordinates phagophore expansion via directed lipid transfer until closure of the phagophore rim. Using an Atg2 variant (Atg2-PM4) as a model of decelerated autophagosome biogenesis, we visualized the morphological states prior to autophagosome closure by cryogenic correlative light and electron microscopy in S. cerevisiae. Using in situ cryo-electron tomography, we find an enlarged rim morphology of an expanding phagophore in Atg2-PM4 cells in comparison with Atg2 wildtype condition. Analysis of segmented rim membrane features reveal surrounding and membrane-attached vesicles. The dimensions of the enlarged rims are consistent with fusion of cytosolic vesicles with the growing phagophore. High-resolution imaging in this study suggests that, apart from the initial nucleation phase, vesicle fusion can also contribute to phagophore expansion during later stages of autophagosome biogenesis.