Figure 6. Relationship of PI(3)P-containing omegasomes with the ER and autophagosomes by live imaging and in fixed cells. (A) Clone 201 cells were transfected with mRFP-MAP-LC3 (red) and CFP-ER (blue) and starved for 60 min. Imaging was performed at 1 frame per 10 s, and a selected interval within this sequence is shown, starting at 33 min after starvation. Arrows indicate the first discernible omegasome (green) and autophagosome (red) occurrences. The enlarged panels are from the two boxed areas and represent views of (1) ER, (2) DFCP1, (3) MAP-LC3, (4) ER-MAPLC3, (5) DFCP1-MAPLC3, and (6) MAPLC3-ER, in that order. Also see Video 3 (available). (B) Dynamic relation of omegasomes and the ER using TIRFM imaging. Note that omegasomes form in regions containing ER and collapse there. Also see Video 4. (C) Thawed cryosection of clone 201 cells 45 min after starvation and double-labeled for anti–GFP-DFCPI (arrowheads, 10 nm gold) and anti-PDI (arrows, 5 nm gold). Note the putative autophagic-like vacuoles (asterisks) labeled for DFCPI-GFP adjacent to PDI-labeled ER membranes. This is one panel of a multipanel supplemental figure (Fig. S4). (D) Selected frames from live imaging of GFP-DFCP1 and mRFP-MAP-LC3 during starvation, whereby an intermediate is formed in which MAP-LC3 appears to bud from the omegasome while still being outlined with a DFCP1-staining membrane. At the bottom of each panel are line drawings of these structures using magnified photographs of the relevant frames. Also see Video 6. (E) Colocalization of omegasomes with a PI(3)P-binding protein applied exogenously. Clone 201 cells were starved for 60 min, perforated using nitrocellulose, and stained with purified GST-PX domain from p40phox. The majority of GSP-PX domain stains early endosomes (not depicted) but a substantial amount of the protein also binds to DFCP1 omegasomes (F and G). (F and G) Relationship of omegasomes to autophagy proteins and the ER. Clone 201 cells counterstained with antibodies to endogenous ER and endogenous MAP-LC3 or Atg5 as indicated. Selected examples from such cells (in addition to the one shown) where the ER was in a single layer and well resolved from cytosol, to allow evaluation of colocalizations, are shown in magnified panels 1–5. Bars: (A, B, and D) 1 μm; (E–G) 20 μm.
Figure 6.

Relationship of PI(3)P-containing omegasomes with the ER and autophagosomes by live imaging and in fixed cells. (A) Clone 201 cells were transfected with mRFP-MAP-LC3 (red) and CFP-ER (blue) and starved for 60 min. Imaging was performed at 1 frame per 10 s, and a selected interval within this sequence is shown, starting at 33 min after starvation. Arrows indicate the first discernible omegasome (green) and autophagosome (red) occurrences. The enlarged panels are from the two boxed areas and represent views of (1) ER, (2) DFCP1, (3) MAP-LC3, (4) ER-MAPLC3, (5) DFCP1-MAPLC3, and (6) MAPLC3-ER, in that order. Also see Video 3 . (B) Dynamic relation of omegasomes and the ER using TIRFM imaging. Note that omegasomes form in regions containing ER and collapse there. Also see Video 4. (C) Thawed cryosection of clone 201 cells 45 min after starvation and double-labeled for anti–GFP-DFCPI (arrowheads, 10 nm gold) and anti-PDI (arrows, 5 nm gold). Note the putative autophagic-like vacuoles (asterisks) labeled for DFCPI-GFP adjacent to PDI-labeled ER membranes. This is one panel of a multipanel supplemental figure (Fig. S4). (D) Selected frames from live imaging of GFP-DFCP1 and mRFP-MAP-LC3 during starvation, whereby an intermediate is formed in which MAP-LC3 appears to bud from the omegasome while still being outlined with a DFCP1-staining membrane. At the bottom of each panel are line drawings of these structures using magnified photographs of the relevant frames. Also see Video 6. (E) Colocalization of omegasomes with a PI(3)P-binding protein applied exogenously. Clone 201 cells were starved for 60 min, perforated using nitrocellulose, and stained with purified GST-PX domain from p40phox. The majority of GSP-PX domain stains early endosomes (not depicted) but a substantial amount of the protein also binds to DFCP1 omegasomes (F and G). (F and G) Relationship of omegasomes to autophagy proteins and the ER. Clone 201 cells counterstained with antibodies to endogenous ER and endogenous MAP-LC3 or Atg5 as indicated. Selected examples from such cells (in addition to the one shown) where the ER was in a single layer and well resolved from cytosol, to allow evaluation of colocalizations, are shown in magnified panels 1–5. Bars: (A, B, and D) 1 μm; (E–G) 20 μm.

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