Fig. 6.
Autophagic structures at ERESs are engulfed by lysosomal membranes. (A and B) COPII-positive procollagen autophagic structures marked with GFP-proα2G610C(I), TagBFP2-LC3, and Apple-Sec23 were imaged by Airyscan microscopy in MC3T3 cells without (A) and with (B) 6 h 100 µM leupeptin treatment. The cells were also transfected with a lysosomal membrane marker, LAMP1-Halo. In zoomed panels, yellow outlines of LAMP1-positive puncta are projected in white onto other channels. (A, Bottom) Outlines show proα2G610C(I), LC3, and Sec23 surrounded by LAMP1-positive membranes; the large structure appears to be only partially surrounded. (B, Bottom) Outlines show multiple proα2/LC3/LAMP1 puncta that also contain Sec23. All images are single Airyscan slices; blue channels are displayed in cyan. [Scale bars: 10 µm (whole cell) and 2 µm (zoom).] (C–E) Effect of 6-h leupeptin treatment on procollagen autophagic structures. Bar charts display mean values ± SEM; ***P < 0.001. (C and D) Fraction of proα2/LC3 (C) and proα2/LC3/Sec23 (D) puncta that are also positive for LAMP1. (E) Fraction of proα2/LC3/LAMP1 puncta that are also positive for Sec23.