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Chromatin visualized via SMC protein 1A

2/20/2019

Movie. High magnification z-stack of a live hiPS cell colony expressing mEGFP-tagged SMC protein 1A. 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 SMC protein 1A. Cells were imaged in 3D on a spinning-disk confocal microscope every 3 min. A single mid-level plane is shown. Frames were histogram matched to adjust for photobleaching. Movie plays at 900x real time. Scale bar, 5 µm.

Movie. Time-lapse in low magnification movie of a live hiPS cell colony expressing mEGFP-tagged SMC protein 1A. Cells were imaged in 3D on a spinning-disk confocal microscope every 5 min. A single mid-level plane is shown. Movie plays at 3000x real time. Scale bar, 20 µm.

Observations

SMC protein 1A is encoded by the X-linked gene SMC1A and escapes X-chromosome inactivation.
SMC protein 1A is part of the cohesin complex. Cohesin is important for controlling chromosomal shape and organization in interphase and mitosis. Cohesin is best known for its role in joining sister chromatids during part of the cell cycle between DNA replication and anaphase so that that chromatids are properly distributed between daughter cells.
In hiPS cells, SMC protein 1A has a granular appearance throughout the nucleus and is largely excluded from the nucleolus. SMC protein 1A reorganizes during cell division, forming puncta that align at the center of the spindle (consistent with localization to centromeres), localizing in a diffuse haze at anaphase, and reappearing in a granular distribution in the nucleus as the nucleus is reassembled.

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.

Sarcomeric thick filaments via MLC-2v

2/20/2019

Movie. High magnification Z-stack of live hiPSC-derived cardiomyocytes expressing mEGFP-tagged MLC-2v. Twelve days after the onset of differentiation, cells were plated on PEI- and laminin-coated glass and imaged in 3D on a spinning-disk confocal microscope 28 days later (40 days total after the onset of differentiation). Cells were treated with 20 mM of the myosin inhibitor 2,3-butanedione monoxime (BDM) to prevent beating during image acquisition. Inset is a 3x enlargement of the boxed region to show detail of MLC-2v in myofibrils. Movie starts at the bottom of the cells and ends at the top. Scale bar, 20 µm.

Movie. Time-lapse in high magnification movie of live hiPSC-derived cardiomyocytes expressing mEGFP-tagged MLC-2v protein. Twelve days after the onset of differentiation, cells were plated on PEI and laminin coated glass and imaged on a spinning-disk confocal microscope 28 days later (40 days total after the onset of differentiation). A single plane of cells was imaged continuously with a 100 ms exposure time. Inset is a 3x enlargement of the boxed region to show detail of MLC-2v in myofibrils. Movie plays in real time.Scale bar, 20 µm.

Observations

MLC-2v is the cardiac ventricular isoform of the Myosin Light Chain 2 (MLC-2) protein. It localizes to the thick filament of the sarcomere where it binds to the myosin heavy chain and functions to regulate myosin-based contractility. The expression of MLC-2v is developmentally regulated; it is frequently used as a marker of cardiac development due to its up-regulation with ventricular development.
In hiPSC-derived cardiomyocytes, we observed mEGFP-tagged MLC-2v in a striated appearance along myofilaments, reflecting its localization to the thick filaments of sarcomeres, and absence from the Z-disk and I-band. During cardiomyocyte beating, the contraction of sarcomeres can be seen in the changes in spacing between striations, and some myofibrils buckle.

Paraspeckles and stress granules via RNA-binding protein FUS

2/20/2019

Movie. High magnification Z-stack of a live hiPS cell colony expressing mEGFP-tagged RNA-binding protein FUS in control cells (left panel) and in the presence of 500 µM sodium arsenite for 15 min (right panel). 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. High magnification Time-lapse movie of a live hiPS cell colony expressing mEGFP-tagged RNA-binding protein FUS. Six minutes after the introduction of 500 µM sodium arsenite, cells were imaged every 5 sec in 3D on a spinning-disk confocal microscope. A single mid-level plane is shown. The inset is a 2.5x enlargement of the boxed region to show detail of aggregate formation. Frames were histogram matched to adjust for photobleaching. Movie plays at 25x real time. Scale bar, 5 µm.

Observations

RNA-binding protein FUS (Fused In Sarcoma) is a DNA/RNA binding protein involved in transcription, mRNA splicing and transport, and DNA repair.
FUS forms various condensed-phase compartments in cells. In the absence of a stressor, FUS compartments form in the nucleus, including at sites of active genes, DNA damage, and paraspeckles (RNA-protein bodies in the interchromatin space). Stressful conditions (e.g. generation of reactive oxygen species (ROS)) lead to a redistribution of FUS from the nucleus to the cytoplasm, where it localizes to stress granules.
In unstressed hiPS cells imaged with spinning-disk light microscopy, mEGFP-tagged FUS has a granular appearance within the nucleoplasm including some relatively bright spots which may be paraspeckles. In the absence of a stressor, there is no mEGFP-tagged FUS in the cytoplasm.

After the application of the stressor sodium arsenite to hIPS cells, mEGFP-tagged FUS appears as puncta in the cytoplasm that join together, reflecting stress granule nucleation and coalescence. Concurrently, the intensity of FUS decreases in the nucleoplasm.

Cell structure observations

Chromatin visualized via SMC protein 1A

Endoplasmic reticulum & nuclear envelope via Sec61β/Lamin B1

Sarcomeric thick filaments via MLC-2v

Paraspeckles and stress granules via RNA-binding protein FUS

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