Category: Mitosis

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.

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: 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.

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.

Microtubules visualized via α-tubulin in both green (GFP) & red (mTagRFP-T)

3/13/2017

Z-stack

High magification (mitosis)

Low magnification (mitosis)

3D rotation

Figure. Movies of α-tubulin in microtubules. Top left: Z-stack of live hiPS cells expressing mEGFP-tagged α-tubulin imaged on a spinning-disk confocal microscope. Images start from the bottom of the cells and end at the top. Top center and right: Timelapse movies of a hiPSC colony expressing mEGFP-tagged α-tubulin imaged on a spinning disk confocal microscope. Center: images were collected in 3D every 4 minutes for 400 minutes. Images are maximum intensity projections; playback speed is 1200x real time. Top right: images were collected as a single slice near the top of the cell every 1 minute for 65 minutes; playback speed is 900x real time. Bottom row: 3D reconstructions of hiPS cells expressing mEGFP-tagged α-tubulin to visualize both the general organization of microtubules within the cell and the primary cilia at the top of cells.

Observations:

α-tubulin polymerizes with ß-tubulin into microtubules, which are a component of the cell’s cytoskeleton. They are important in a number of cellular processes including intracellular transport of organelles and chromosome separation during mitosis.

Most of the structures we observe are likely bundles of microtubules instead of individual microtubules. In dividing cells we can observe weak astral microtubules (originating from the spindle poles but not connected to chromosome kinetochores), which could include individual microtubules. Therefore, all brighter tubulin structures are likely bundles of microtubules.
In hiPS cells, microtubules localize throughout the cytoplasm. More microtubules are seen near the top of cells with fewer near the bottom; in general microtubules seem to be oriented along the apical-basal axis throughout the center planes of the cell. This suggests microtubule nucleation occurs near the top of cells; however, a clear microtubule organizing center is not consistently seen. In some cells microtubules do seem to radiate from a more central location, which may be cell cycle related.
During cell division, cells form bipolar spindles that are most often oriented in the same plane as the cells. However, we do frequently see spindles rotating in all 3 directions during division.
After division, sister cells remain connected by their cytoplasmic bridges for 1-2 hours. These bridges often localize to the tops of colonies where they span across multiple cells due the sister cells intercalating to non-adjacent positions within the colony. Tubulin-rich midbodies are present in these cytoplasmic bridges.
Bright spots near the top of cells seen in the z-stack represent primary cilia, which are seen in most cells; their absence may be cell cycle related.
See FAQs for reasoning behind on our choice of red-fluorescent protein tagging.

Cell Structure Observations

Histones visualized via Histone H2B type 1-J

Centrioles via Centrin

Cell–cell contacts: desmosomes visualized via desmoplakin

Nucleolus via Fibrillarin

Mitochondria visualized via Tom20

Microtubules visualized via α-tubulin in both green (GFP) & red (mTagRFP-T)

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Cell Structure Observations

Histones visualized via Histone H2B type 1-J

Centrioles via Centrin

​Cell–cell contacts: desmosomes visualized via desmoplakin

Nucleolus via Fibrillarin

​Mitochondria visualized via Tom20

Microtubules visualized via α-tubulin in both green (GFP) & red (mTagRFP-T)

About

Archives

Categories

The Institute

Legal

Help & contact

Cell–cell contacts: desmosomes visualized via desmoplakin

Mitochondria visualized via Tom20