Category: Time Lapse

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.

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.

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.

Centrioles via Centrin

10/3/2017

Figure. Centrin and DNA through cell cycle. Single plane images of hiPS cells expressing mTagRFP-T–tagged centrin (green) and labeled with Hoechst dye (DNA; blue) imaged on a spinning-disk confocal microscope. Cells labeled A-F represent different stages of the cell cycle (see diagram). A) G1-phase, B) early S-phase, C) later S-phase, D) G2/M-phase and E–F) M-phase contain centrioles at distinguishable stages of duplication (see zoomed in images and cell cycle diagram).

Observations

Centrin is a key protein in centrioles, which are small cylindrical structures made primarily of tubulin. Centrioles are important components of centrosomes, the structures that form microtubule organizing centers. Centrioles are also found at the base of cilia and flagella.
Centrioles localize to the very top of the hiPS cells in interphase, consistent with the presence of primary cilia, which emanate from the centrioles. Centriole duplication and separation is observed throughout the cell cycle consistent with models of cell cycle regulation of centriole behavior. Small daughter centrioles are seen to appear next to their mother centrioles and grow in size.
See FAQs for reasoning behind on our choice of red-fluorescent protein tagging.

Cell–cell contacts: Tight junctions visualized via ZO-1

4/1/2017

High magnification timelapse

Low magnification timelapse

Figure. Timelapse movies of ZO-1 in tight junctions. Timelapse movies of live hiPS cells expressing mEGFP-tagged tight junction protein ZO-1 imaged on a spinning-disk confocal microscope. Images were collected in 3D every 3 min for 1.5 hrs (left) or for 15 hrs (right). Images are maximum intensity projections; playback speed is 910x (left) and 1800x (right) real time.

Observations

ZO-1 is a tight junction-associated protein that connects adjacent epithelial cells near the apical surface. The tight junctions form a continuous ring around the cell, limiting passage of molecules between the top and bottom of the cells or the tissues they comprise.
In hiPS cells, ZO-1 forms a ring around each cell near the apical surface. Due to this localization, it is a good marker for the apical cell periphery as the cells grow, divide and move around within the colony.
The ZO-1 band becomes less coherent and widens during cell division, perhaps due to a release of tension between cells.
During division, ZO-1 forms rosette-like structures as neighboring cells maintain tight contact with each other when another cell is expelled from the epithelial ‘sheet’. This rosette is resolved as cells move within the colony, grow and divide.

Cell–cell contacts: desmosomes visualized via desmoplakin

3/21/2017

Z-stack with overlay

Low magnification timelapse

Figure. Movies of desmoplakin in desmosomes. Top: Z-stack of live hiPS cells expressing mEGFP-tagged desmoplakin imaged on a spinning-disk confocal microscope. Images start from the bottom of the cells and end at the top. The right panel shows the left panel overlaid onto the equivalent transmitted light image. Bottom: timelapse movie of a hiPS cell colony expressing mEGFP-tagged desmoplakin. Images were collected in 3D every 4 minutes for 8 hours on a spinning-disk confocal microscope. Images are maximum intensity projections; playback speed is 2400x real time.

Observations

Desmoplakin is involved in the linkage of intermediate filaments to cell-cell adhesion sites (desmosomes) in epithelial cells. These desmosomes are seen as small puncta at apical cell-cell boundaries.
In hiPS cells, desmoplakin puncta are not visible in all cells. However, when present there are between 1 and ~20 puncta present per cell.
There may be position-dependent differences in number of desmosomes depending on the spatial location of a cell within a colony. For example, we observe differences between the number of desmosomes in the tightly-packed centers of colonies vs. the flatter, less epithelial-like cells at the edges of colonies; however, this is a casual rather than a rigorous observation.
Desmosomes stay intact during cell division

Nucleolus via Fibrillarin

3/16/2017

Z-stack

High magnification timelapse (cell division)

Figure 1. Movies of fibrillarin in Nucleoli. Left: Z-stack of live hiPS cells expressing mEGFP tagged fibrillarin imaged on a spinning-disk confocal microscope. Images start from the bottom of the cells and end at the top. Right: Timelapse movie of live hiPS cells expressing mEGFP tagged fibrillarin. Images were collected in 3D every 3 minutes for 1.5 hours on a spinning-disk confocal microscope. Image is a maximum intensity projection. Playback speed is 900x real time.

Figure 2. Time series of cell division. A single cell going through cell division taken from the movie on the right.

Observations

Fibrillarin marks the dense fibrillar component (DFC) of the nucleolus, the nuclear subcompartment where ribosome biogenesis occurs.
During much of interphase the nucleolus exists in 1-2 large, textured clusters within the nucleus of hiPS cells. During cell division, the nucleolus appears to ‘melt’ and then dissociate. After cell division, the nucleolus reassembles, first into small particles and progressing into the larger textured clusters observed during interphase. Low levels of fibrillarin are visible on chromosomes during chromosome segregation in mitosis.

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.

Mitochondria visualized via Tom20

3/14/2017

Z-stack

High magnification timelapse (cell division)

Figure. Live cell movies of Tom20 in Mitochondria. Left: Z-stack of live hiPS cells expressing mEGFP-tagged Tom20 imaged on a spinning-disk confocal microscope. Images start from the bottom of the cells and end at the top. Right: Timelapse movie of hiPS cells expressing mEGFP-tagged Tom20 imaged on a Zeiss LSM880 Airyscan FAST in super-resolution mode. Images were collected in 3D every 30 seconds for 30 minutes. Images show a single slice near the bottom of the cell; playback speed is 150x real time.

Observations

Tom20 is a member of the TOM (translocase of the outer membrane) complex, which permits movement of proteins through the outer mitochondrial membrane and into the intermembrane space of mitochondria.
In hiPS cells, mitochondria are localized primarily to the top of cells in the nucleus-free ‘cytoplasmic pocket.’ In the center of cells, they localize perpendicular to the substrate and appear like hollow tubes in cross section, as expected for an outer mitochondrial membrane protein. There are fewer mitochondria at the bottom of cells. This observation is consistent with mitochondrial positioning in the cell being primarily dependent on microtubule positioning during interphase.
Mitochondria form long, interconnected tubules as well as smaller separated structures. The longest tubules are most visible at the bottom of cells where mitochondria are less crowded. Mitochondria are dynamic, jiggling due to Brownian motion, moving (presumably along microtubules) and exhibiting fission and fusion dynamics.
In mitotic cells mitochondria are more evenly distributed throughout the cell and tend to cluster towards the cell periphery, outside of the mitotic spindle.

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Cell Structure Observations

Chromatin visualized via SMC protein 1A

Sarcomeric thick filaments via MLC-2v

Paraspeckles and stress granules via RNA-binding protein FUS

Sarcomere thick filaments visualized via MLC-2a

Centrioles via Centrin

Cell–cell contacts: Tight junctions visualized via ZO-1

Cell–cell contacts: desmosomes visualized via desmoplakin

Nucleolus via Fibrillarin

Endoplasmic Reticulum (ER) via Sec61-ß

Mitochondria visualized via Tom20

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