Cell structure observations

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.

Histones visualized via Histone H2B type 1-J

8/1/2018

Movie. Z-stack of live hiPS cell colony expressing mEGFP-tagged Histone H2B type 1-J protein. 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 movie of live hiPS cell colony expressing mEGFP-tagged Histone H2B type 1-J protein. Cells were imaged in 3D on a spinning-disk confocal microscope every 3 min. A single mid-level z-section is shown. Movie plays at 1260x real time. Scale bar, 5 µm.

Movie. Z-stack of live hiPS cell colony expressing mEGFP-tagged Histone H2B type 1-J protein. Cell Mask Deep Red Plasma Membrane dye was applied to mark cell boundaries (magenta). NucBlue Live Ready Probe Reagent was used to compare mEGFP-tagged H2B localization (left panel) to a NucBlue Live (Hoechst) DNA stain (left 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.

Observations

Histone H2B type 1-J is a protein that participates in organizing DNA. It and other histone proteins form a core around which DNA wraps. These structures, called nucleosomes, are the fundamental repeating units within chromatin (DNA and histones). The packing of these structures impacts DNA accessibility, affecting functions like transcription, replication, and repair of DNA.
In hiPS cells imaged with spinning-disk light microscopy, mEGFP-tagged Histone H2B type 1-J appears within the nucleus as puncta of varying size and intensity, likely reflecting more densely packed regions of nucleosomes, and in a “haze”, where nucleosomes cannot be resolved.
The localization of mEGFP-tagged Histone H2B type 1-J is similar to the pattern of the DNA-binding dye Hoechst. Hoechst binds the minor grove of DNA (with preference for A-T rich regions), while mEGFP-tagged Histone H2B type 1-J localizes to the nucleosomal cores around which the DNA wraps.
Histone H2B type 1-J pattern of localization and intensity changes through the cell cycle. During interphase, Histone H2B type 1-J localizes throughout the nucleus, with some regions of greater intensity (reflecting a greater density of protein) near the nuclear periphery and the nucleolus. During cell division, Histone H2B type 1-J localizes to chromosomes.

Sarcoplasmic reticulum visualized via SERCA2

8/1/2018

Movie. Z-stack of live hiPS cell colony expressing mEGFP-tagged SERCA2 protein. 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.

Movies. Z-stack of live hiPSC-derived cardiomyocytes expressing mEGFP-tagged SERCA2 protein. 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 29 days later (41 days total after the onset of differentiation). Cells were treated with 15 mM of the myosin inhibitor 2,3-Butanedione monoxime (BDM) to prevent beating during image acquisition. Movie starts at the bottom of the cells and ends at the top. Inset shows detail of SERCA2 in longitudinal sarcoplasmic reticulum (SR). Scale bars, 10 µm.

Movie. Time-lapse movie of live beating hiPSC-derived cardiomyocytes expressing mEGFP-tagged SERCA2 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 8 days later (20 days total after the onset of differentiation). A single plane of cells was imaged continuously with a 100 ms exposure time. Inset shows detail of SERCA2 in longitudinal sarcoplasmic reticulum (SR). Scale bars, 10 µm. Movie plays in real time.

Observations

SERCA2 (sarco/endoplasmic reticulum calcium ATPase) proteins form pumps that use the energy from ATP hydrolysis to import calcium from the cytosol into the endoplasmic and/or sarcoplasmic reticula (ER and/or SR). This regulates cytosolic calcium concentration, important for many functions, including contractility in cardiomyocytes.
In hiPSC, mEGFP-taged SERCA2 localizes to the nuclear periphery and to ER tubules. In contrast, while both mEGFP-tagged SERCA2 and Sec61-beta proteins localize to the ER membrane, Sec61 beta localizes to the nuclear periphery as well as both ER tubules and sheets. This difference in localization may reflect a preference for SERCA2 to localize to smooth ER over rough ER.
In hiPSC-derived cardiomyocytes, mEGFP-tagged SERCA2 localizes to the nuclear periphery and to a tubular network throughout the rest of the cytoplasm. The linear alignment of SERCA2 labeling along presumable myofilaments suggests localization to longitudinal SR.
mEGFP-tagged SERCA2 moves when cardiomyocytes beat, presumably moving along with the contraction and buckling of myofilaments.

Nuclear pores visualized via Nucleoporin Nup153

8/1/2018

Movie. Z-stack of live hiPS cell colony expressing mEGFP-tagged Nucleoporin Nup153 protein. 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 movie of live hiPS cell colony expressing mEGFP-tagged Nucleoporin Nup153 protein. Cells were imaged in a 3D volume spanning ~1.5 µm through the middle of the cells on a spinning-disk confocal microscope every 3 min. A single mid-level z-section is shown. Movie plays at 1800x real time. Scale bar, 5 µm.

Observations

Nucleoporin Nup153 is a protein within the nuclear pore complex (NPC). The NPC bridges the double membrane of the nuclear envelope to regulate the exchange of molecules between the nucleus and cytoplasm. Nucleoporin Nup153 is located on the nuclear side of the NPC. It is critically involved in the ability of the NPC to transport molecules between the nucleus and cytoplasm; but it also plays other roles in nuclear properties and functions, including the shape of the NPC and the nucleus, and regulation of transcription.
In hiPS cells, Nucleoporin Nup153 appears as numerous dense puncta along the nuclear envelope, including within invaginations.
During cell division, Nucleoporin Nup153 localization changes with the reorganization of the nuclear envelope. During mitosis, Nuceloporin Nup153 localizes in a dim wavy structure, likely the ER. At the end of mitosis, Nucleoporin Nup153 appears on chromosomes before finally reappearing at the nuclear envelope.

Sarcomere M-line visualized via Titin

8/1/2018

Movie. Z-stack of live hiPSC-derived cardiomyocytes expressing mEGFP-tagged titin protein. 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 23 days later (35 days total after the onset of differentiation). Cells were treated with 15 mM of the myosin inhibitor 2,3-Butanedione monoxime (BDM) to prevent beating during image acquisition. Movie starts at the bottom of the cells and ends at the top. Inset shows detail of titan in myofibrils. Scale bars, 10 µm.

Movie. Time-lapse movie of live hiPSC-derived cardiomyocytes expressing mEGFP-tagged titin 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 23 days later (35 days total after the onset of differentiation). A single plane of cells was imaged continuously with a 100 ms exposure time. Inset shows detail of titan in myofibrils. Scale bars, 10 µm. Movie plays in real time.

Observations

Titin is the largest known protein. It stretches across the sarcomere from the Z-disk (at its N-terminus) to the M-line (at its C-terminus). Titin functions like a spring when under tension with the contraction of sarcomeres. Titin also plays a structural role in regulating the length of the thick filament within sarcomeres.
In hiPSC-derived cardiomyocytes, titin tagged at its C-terminus with mEGFP localizes to thin lines that striate myofilaments, consistent with localization to the M-line of sarcomeres.
In hiPSC-derived cardiomyocytes from the mEGFP tagged Titin line, the fluorescent signal localizes to thin lines that striate myofilaments. While Titin spans the entire length from the Z-disk to the M-line, the much smaller mEGFP only highlights the location of the tagged Titin C-terminus. During cardiomyocyte beating, the contraction of sarcomeres can be seen in the changes in spacing between striations, and some myofibrils buckle.

Sarcomere thick filaments visualized via MLC-2a

8/1/2018

Movie. Z-stack of live hiPSC-derived cardiomyocytes expressing mEGFP-tagged MLC-2a. 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 20 days later (32 days total after the onset of differentiation). Cells were treated with 15 mM of the myosin inhibitor 2,3-Butanedione monoxime (BDM) to prevent beating during image acquisition. Movie starts at the bottom of the cells and ends at the top. Inset shows detail of MLC-2a in myofibrils. Scale bars, 10 µm.

Movie. Time-lapse movie of live hiPSC-derived cardiomyocytes expressing mEGFP-tagged MLC-2a 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 19 days later (31 days total after the onset of differentiation). A single plane of cells was imaged continuously with a 100 ms exposure time. Inset shows detail of MLC-2a in myofibrils. Scale bars, 10 µm. Movie plays in real time.

Observations

MLC-2a is the cardiac atrial isoform of the Myosin Light Chain 2 (MLC-2) protein. It localizes to the thick filament of the sarcomere where it functions to regulate myosin-based contractility. The expression of MLC-2a is developmentally regulated; it is frequently used as a marker of cardiac development due to its down-regulation with ventricular development.
In hiPSC-derived cardiomyocytes, we observed mEGFP-tagged MLC-2a 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.

Sarcomere visualized via Troponin I, slow skeletal muscle

4/24/2018

Movie. Timelapse movie of sarcomeres in beating cells. Time-lapse movie of live hiPSC-derived cardiomyocytes expressing mEGFP-tagged troponin I. Cells were continuously imaged on a spinning-disk confocal microscope with a 100ms exposure time. Inset shows detail of sarcomeres in myofibrils. Scale bars, 5 µm and 10 µm for inset and larger field respectively. Movie plays in real time.

Observations

Troponin I, slow skeletal muscle labels the thin filament of sarcomeres (actin-based), the contractile apparatus in muscle cells, and regulates myosin-based contraction. Sarcomeres align end-to-end to form myofibrils, striated filaments that generate force when sarcomeres contract.
Troponin I is absent from Z-disks, which results in a striated appearance along a myofibril. The spacing of this striation shortens as the cardiomyocytes beat. Sometimes buckling of the myofibril can be seen during beating.

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