Category: Cell Contacts

Cell–cell contacts: Gap junctions visualized via connexin-43

4/24/2018

A. Z-stack

B. Timelapse

Figures. Z-stack and timelapse movies of gap junctions. A) Z-stack of a live hiPS cell colony expressing mEGFP-tagged connexin-43. 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 = 5 µm. B) Timelapse movie of a live hiPS cell colony expressing mEGFP-tagged connexin-43. Cells were imaged on a spinning-disk confocal microscope every 3.25 min. Image is a maximum intensity projection of 20 z-slices acquired at the top of the cells. Contrast is enhanced to show localization of connexin-43 to internal structures. Frames were histogram matched to adjust for photobleaching. Movie plays at 975x real time. Scale bar = 5 µm.

Observations

Connexin-43 is a key component of gap junctions, which are channels between cells that allow for small molecules and ions to pass directly between the cytoplasm of adjacent cells. Gap junctions often cluster in larger gap junction plaques.
In hiPS cells, connexin-43 localizes most prominently to many puncta (presumably clusters of individual channels) at apical cell-cell junctions near the very top of the cells. Some puncta are also seen near the bottom and middle of the cell although in smaller numbers.
Connexin-43 is also localized to the plasma membrane and in internal structures that resemble the Golgi. This localization likely reflects the assembly and trafficking of connexin into gap junctions.
As hiPS cells progress though mitosis, the apical puncta at cell-cell junctions remain intact, but the connexin-43 associated with internal organelles, e.g. the Golgi, becomes diffuse throughout the cell.

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 Structure Observations

Cell–cell contacts: Gap junctions visualized via connexin-43

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

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Cell Structure Observations

Cell–cell contacts: Gap junctions visualized via connexin-43​

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

About

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Categories

The Institute

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Cell–cell contacts: Gap junctions visualized via connexin-43

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