Category: Time Lapse

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

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

Nuclear envelope visualized via lamin B1

About

Archives

Categories

Home

Terms of Use

Citation Policy

Privacy Policy

FAQs

Help

Send us a message