Plasma Membrane visualized via CAAX domain of K-Ras

Figure 1. Timelapse movie of plasma membrane. Timelapse movie of a live hiPS cell colony expressing the CAAX domain of K-Ras tagged with mTagRFP-T. Cells were imaged in 3D on a spinning-disk confocal microscope every 5 min. Panels show the same colony at different z-positions. Left) The bottom z-section of the cells. Middle) A maximum intensity projection through the middle 5 z-sections of the cells. Right) A maximum intensity projection through the top 12 z-sections of the cells. Frames in each panel were histogram matched to adjust for photobleaching. Movie plays at 1500x real time. Scale bar = 5 µm.

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Figure 2. Timelapse movie of plasma membrane. Timelapse movie of a live hiPS cells in an epithelial sheet containing both unedited cells (which remain unseen in black), and cells expressing the CAAX domain of K-Ras tagged with mTagRFP-T. Cells were imaged in 3D on a spinning-disk confocal microscope every 3 min; the top and bottom z-sections are shown pseudocolored in green and magenta, respectively. Movie plays at 1800x real time. Scale bar = 10 µm.
 
Observations

• The plasma membrane of the cell forms a barrier between the cytoplasm and the extracellular space and neighboring cells. We labeled CAAX, the membrane-targeting domain of K-Ras with mTagRFP-T to label the plasma membrane.
• In hiPS cells the plasma membrane exhibits differing behavior depending on the location in the cell. At the bottom of the cell, the membrane surrounds dynamic lamellar protrusions, which overlap with neighboring cells. At the middle region of cells, the plasma membrane clearly outlines the boundaries between cells. At the very top of cells, the membrane surrounds dynamic microvilli, seen as small textured puncta.
• In addition, bright puncta are present throughout the cell, with a greater number near the top of the cell. These are likely endosomes created by plasma membrane taken into the cell.
• By mixing the cells expressing this FP-tagged CAAX domain with unedited cells, the behavior of individual cells within a colony can be observed more clearly. Pseudocoloring a frame from the bottom of a z-stack in a different color from a frame from the top of the same stack permits visualization of the different behavior of the cell at each location. The bottom of the cell changes shape and explores more space than the top of the cell. The tops of cells appear anchored in space, consistent with the localization of cell junctional proteins to apical cell-cell boundaries. ​