Category: Colony

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

Cell-substrate adhesions via Paxillin

3/17/2017

High magnification edge of colony (with overlay)

Low magnification of moving colony

Figure. Movies of paxillin in cell-substrate adhesions. Timelapse movies of hiPS cells expressing EGFP-tagged paxillin imaged on a spinning-disk confocal microscope. Left: images were collected as a partial z-stack near the bottom of the cell every 5 minutes for 160 minutes. Image is a maximum intensity projection and movie is sped up 1500x over real time. Right: single slice images near the bottom of the cell taken every 5 minutes for 400 minutes; playback speed is 3000x real time.

Observations

Paxillin is a signaling adaptor that is present in most integrin-mediated sites of adhesions between the cell and the extracellular matrix.
In hiPS cell colonies, paxillin primarily localizes to small adhesions which form puncta at the bottom of cells that are less than 0.5 microns in diameter. These adhesions are dynamic, forming and turning over as the edge of the cell protrudes and retracts. Larger, elongated adhesions are also present in these cells particularly along the more protrusive edges of colonies.

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.

Nuclear envelope visualized via lamin B1

3/10/2017

Low magnification timelapse

High magnification timelapse

Figure. Timelapse movies of hiPSC cells expressing mEGFP tagged Lamin B1. Images were collected in 3D every 3 minutes for 12 hours (left) or every 35 seconds for 23 minutes (right) on a spinning-disk confocal microscope. Images are maximum intensity projection (left) or single slices from the middle of the z-stack (right). Playback speed is 1800x (left) and 350x (right) real time.

Observations

Lamin B1 is a member of the lamin family of proteins that make up the nuclear lamina, located just inside the inner nuclear envelope. - In hiPS cells, nuclei in these cells occupy 30-50% of the cell volume making them very prominent. In the center of cells the nuclei occupy almost the entire cytoplasm such that a ‘bird’s eye view’ of the cell monolayer shows nuclei that appear tightly packed together.
As the cells enter mitosis, the lamin B1-containing nuclear envelope is seen to ruffle and take on a wavy morphology as it begins to breakdown. However the nuclear envelope does not completely breakdown during mitosis. As the nuclear envelope reforms, invaginations are seen that can look like spots within the nucleus. These spots decrease and disappear over time suggesting that they may be cell cycle related. These invaginations are also seen with Sec61B, which labels the ER including the peripheral ER surrounding the nucleus (See ER section).

Cell Structure Observations

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

Cell–cell contacts: desmosomes visualized via desmoplakin

Cell-substrate adhesions via Paxillin

Nucleolus via Fibrillarin

Mitochondria visualized via Tom20

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

Nuclear envelope visualized via lamin B1

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