Endoplasmic reticulum & nuclear envelope via Sec61β/Lamin B1

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.

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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.

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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.

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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.