Category: Endoplasmic Reticulum

Endoplasmic reticulum & nuclear envelope via Sec61β/Lamin B1

2/20/2019

Movie. High magnification Z-stack of a live hiPS cell colony expressing mEGFP-tagged Sec61 beta and mTagRFP-T-tagged lamin B1. Panels show individual channels for Sec61 beta (left) and lamin B1 (middle) and the overlay of the two (right). Cells were imaged in 3D on a spinning-disk confocal microscope. Movie starts at the bottom of the cells and ends at the top. Scale bar, 5µm.

Movie. Time-lapse in high magnification movie of a live hiPS cell colony expressing mEGFP-tagged Sec61 beta and mTagRFP-T-tagged lamin B1. Panels show individual channels for Sec61 beta (left) and lamin B1 (middle) and the overlay of the two (right). A single, mid-level plane was imaged on a spinning-disk confocal microscope every 5 min. Movie plays at 3000x real time. Scale bar, 5 µm.

Movie. Time-lapse in low magnification movie of a live hiPS cell colony expressing mEGFP-tagged Sec61 beta and mTagRFP-T-tagged lamin B1. Panels show individual channels for Sec61 beta (left) and lamin B1 (middle) and the overlay of the two (right). Cells were imaged in 3D on a spinning-disk confocal microscope every 5 min. A single mid-level z-section is shown. Movie plays at 1200x real time. Scale bar, 20 µm.

Observations

We previously tagged Sec61 beta and Lamin B1 with mEGFP in two separate cell lines (AICS-0010 and AICS-0013, respectively). The localization of mEGFP-tagged Sec61 beta and mTagRFP-T-tagged lamin B1 in our dual-edited line does not differ from the localization of mEGFP-tagged Sec61 beta and mEGFP-tagged lamin B1 in our previously validated lines.
In interphase, the two tagged proteins co-localize to the nuclear envelope, and only Sec61 beta is found in the ER sheets and tubules throughout the cytoplasm. During cell division, both proteins co-localize to a dim extended wavy morphology. At the end of division, Sec61 beta and lamin B1 localize around the re-forming nucleus. Lamin B1 appears as bright segments along the ER near the re-forming nucleus; the segments disappear by early G1. Just after division, lamin B1 and Sec61 beta co-localize within many invaginations within the nuclear envelope, which decrease in number with time.

Endoplasmic Reticulum (ER) via Sec61-ß

3/15/2017

Z-stack

High magnification timelapse (cell division)

Figure 1. Movies of Sec61-ß in ER. Top left: Z-stack of live hiPS cells expressing mEGFP tagged Sec61-ß imaged on a spinning-disk confocal microscope. Images start from the bottom of the cells and end at the top. Top right: Timelapse movie of live hiPS cells expressing mEGFP tagged Sec61-ß. Images were collected in 3D every 2 minutes for 3 hours on a spinning-disk confocal microscope. Image is a single slice through the center of the cells. Playback speed is 600x real time. Bottom image panel: live hiPSC cells expressing mEGFP tagged Sec61-ß imaged on a Zeiss LSM 880 AiryScan FAST in super-resolution mode.

Figure 2. Images of Sec61-ß in ER. Left, middle, and right images represent a single slice at the bottom, center, and top of cells with AiryScanFast SuperRes

Observations

Sec61-ß is a member of the Sec61 complex, which is involved in protein translocation and insertion into the ER membrane.
In hiPS cells, the ER is localized to the nuclear periphery and in tubules and sheet-like structures throughout the cytoplasm.
ER morphology differs at the top vs. the bottom of cells. The ER is more densely packed near the top of cells such that the tubules have a highly branched appearance. The tubules appear longer and less branched at the bottom of cells. This pattern of increased organelle density near the top and decreased near the bottom of cells is similar to that of mitochondria and may also be due to apical-basal variation in microtubule positioning within cells.
During cell division, the ER stays mostly intact but the peripheral ER takes on a wavy morphology very similar to that of the nuclear envelope. After division, as the peripheral ER reforms, similar membrane invaginations are seen as in LaminB1 tagged cells. This suggests these invaginations might be composed of both nuclear envelope and ER. These invaginations disappear with time during interphase.

Cell Structure Observations

Endoplasmic reticulum & nuclear envelope via Sec61β/Lamin B1

Endoplasmic Reticulum (ER) via Sec61-ß

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