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.

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

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.

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

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.