Supplemental Materials
This article contains the following supporting material:
- Supplemental Materials
- Movie01 - Video S1: 3D model of Huh-7 ER. The video shows the SB-EM dataset and the modeled ER (yellow) and NE (blue) of a control Huh-7 cell and a representative sheet at the end of the video (see Figure 2A). The cell was co-transfected with ssHRP-KDEL and CMVTag1 and cytochemically stained (dark precipitate). Images were acquired using FEG-SEM Quanta 250 (FEI) equipped with 3View system (Gatan Inc.). Voxel size is 14.3 × 14.3 × 30 nm and the dimensions of the bounding box are 14.9 × 14.8 × 2.5 μm. Bar 5μm.
- Movie02 - Video S2: 3D model of ER in actin-depolymerized Huh-7 cell reveal the uneven ER network distribution and sheet remnants. The video shows the SB-EM dataset and the modeled ER (yellow) and NE (blue) of a Huh-7 cell treated with latrunculin A and a representative sheet remnant at the end of the video (see Figure 2B). Huh-7 cell expressing ssHRP-KDEL was cytochemically stained (dark precipitate). Images were acquired using FEG-SEM Quanta 250 (FEI) equipped with 3View system (Gatan Inc.). Voxel size is 17.3 × 17.3 × 40 nm and the dimensions of the bounding box are 24.3 × 23.1 × 2.0 μm. Bar 10 μm.
- Movie03 - Video S3: Actin filament arrays localize to polygons defined by the surrounding ER sheets and tubules. Confocal frames of live Huh-7 cell expressing Hsp47-GFP (green) and mCherry-Actin (magenta) (see Figure 3A) showing short actin filament arrays and foci localizing to ER network polygons. While dynamic actin arrays localize in close proximity with ER, the longer actin filaments i.e. cortical actin and stress fibers do not have a clear interaction with ER. Images were acquired using an inverted TCS SP5II HCS A laserscanning confocal microscope (Leica). The imaging frame rate was 1 frame/0.54 s and the playback frame rate is 10 frames/ s. The images were filtered with rotationally symmetric Gaussian lowpass filter, hsize is 3; 3, sigma is 0.6. Bar 5 μm.
- Movie04 - Video S4: Relocation or disappearance of actin arrays from ER network polygons precedes ER transformations. A cropped area of confocal acquisition of live Huh-7 cell expressing Hsp47-GFP (green) and mCherry-Actin (magenta). Images were acquired using an inverted TCS SP5II HCS A laser-scanning confocal microscope (Leica). The 00:00 time in Figure 3B corresponds to the first frame in the video. The imaging frame rate was 1 frame/0.50 s and the playback frame rate is 10 frames/s. The images were filtered with rotationally symmetric Gaussian lowpass filter, hsize is 3; 3, sigma is 0.6. Bar 5 μm. See also Video S5.
- Movie05 - Video S5: Formation of short actin filament arrays leads to the subsequent opening of a polygon in ER sheets. A cropped area of confocal acquisition of live Huh-7 cell expressing Hsp47-GFP (green) and mCherry-Actin (magenta). Images were acquired using an inverted TCS SP5II HCS A laser-scanning confocal microscope (Leica). The 00:00 time in Figure 3C corresponds to the first frame in the video. The imaging frame rate was 1 frame/0.50 s and the playback frame rate is 10 frames/s. The images were filtered with rotationally symmetric Gaussian lowpass filter, hsize is 3; 3, sigma is 0.6. Bar 5 μm. See also Video S4.
- Movie06 - Video S6: Actin filament arrays present a pool of transforming dynamic structures positive for myo1c. Confocal frames of live Huh-7 cell expressing myo1c-EGFP (green) and mCherry-Actin (magenta) are shown in combination of merged channels. Images were acquired using an inverted TCS SP5II HCS A laser-scanning confocal microscope (Leica). Static (arrow) and dynamic (arrowhead) actin filaments are indicated. An actin filament array transforming into foci (circle) and vice versa (two dashed circles) are shown. The images were filtered with rotationally symmetric Gaussian lowpass filter, hsize is 3; 3, sigma is 0.6. The imaging frame rate was 1 frames/0.50 s and the playback frame rate is 10 frames/s. Bar 10 μm. See Figure 4B.
- Movie07 - Video S7: 3D model of ER in myo1c-depleted Huh-7 cell reveal ER network distribution defect and sheet remnants. The video shows the SB-EM dataset and the modeled ER (yellow) and NE (blue) of myo1c depleted Huh-7 cell and a representative sheet remnant at the end of the video (Video S1 for comparison). Huh-7 cell expressing ssHRP-KDEL was cytochemically stained (dark precipitate). The model consists of ER membranes (yellow) and NE (blue). See Figure 6D. Images were acquired using FEG-SEM Quanta 250 (FEI) equipped with 3View system (Gatan Inc.). Voxel size is 14.6 × 14.6 × 30 nm and the dimensions of the bounding box are 20.14 × 19.03 × 1.71 μm. Bar 5 μm.