Behavior of hiPS Cells During Mitosis
hiPS cells were manually categorized into 7 stages of mitosis based on the appearance (shape and texture) of the fluorescence signal of the DNA labeled with NucBlue Live (Figure 1 below).
Figure 1. Representative examples of hiPS cells in seven stages of mitosis and one category representing interphase categorized by shape and texture patterns of the DNA. Live hiPS cells were labeled with NucBlue Live (DNA; cyan; maximum intensity projection) and CellMask Deep Red (cell membrane; magenta; single slice near middle of the cell) and imaged on a spinning-disk confocal microscope. Top row shows DNA and bottom row shows DNA and membrane.
These stages were not selected based on prior biological knowledge of standard mitotic stages. However, examination of hiPS cells expressing mEGFP-tagged alpha-tubulin (Figure 2 below) showed good correspondence between these stages and some of the classic features of canonical mitotic stages including prophase, prometaphase, metaphase, anaphase, telophase and cytokinesis. In addition, non-mitotic cells were considered as one ensemble interphase stage.
Figure 2. Representative examples of hiPS cells expressing mEGFP-tagged alpha-tubulin in seven stages of mitosis and one stage of interphase categorized by shape and texture patterns of the DNA. Live hiPS cells were labeled with NucBlue Live (DNA; cyan;) and CellMask Deep Red (cell membrane; magenta) and imaged on a spinning-disk confocal microscope. Images are a single slice near middle of the cell. Bottom row shows an illustration of mitosis stages as depicted in the listed reference.
These 8 categories were used for manual annotation of ~700 mitotic cells from the Allen Cell Collection. These cells represent data from 14 gene-edited clonal hiPSC cell lines with FP-tagged structures. The data used for this analysis are available in the 3D Cell Viewer or in bulk on the data downloads page.
An integrated view of the cell at each stage of mitosis
For each cell line and mitotic stage category, all assigned cells were examined for the location and morphology of the intracellular structure. One representative example of each is shown in Figure 3 below, as well as in the 3D Visual Summary tool. To simplify the presentation in the 3D Visual Summary tool, several of these mitotic categories were removed and the remaining categories were named after canonical mitotic stages.
Figure 3. Representative examples of hiPS cells expressing 14 different FP-tagged intracellular structures (listed on left, more information in the Cell Catalog) in seven stages of mitosis and one stage of interphase categorized by shape and texture patterns of the DNA. Live hiPS cells were labeled with NucBlue Live (DNA; cyan;) and CellMask Deep Red (cell membrane; magenta) and imaged on a spinning-disk confocal microscope. Cells were rotated to align the axis of cell division.
For any given structure, general changes in localization of the protein and the intracellular structure it represents can be followed through mitosis (Figure 3 left to right columns within one row). In addition, for any given mitotic stage, the combined set of structure localizations can be examined (Figure 3 all rows within one column), thereby creating an integrated view of the cell at each stage of mitosis.
Some interesting observations include:
- reorganization of microtubules to form the mitotic spindle and segregate the chromosomes, validating the DNA appearance-based classification.
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Z-stack of microtubules visualized via α-tubulin during M5
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Z-stack of microtubules visualized via α-tubulin during M7
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- reorganization of the nuclear envelope: lamin B1 associates with the peripheral ER during interphase and with the ER more generally during mitosis, including nuclear invaginations after cell division. These observations are based on close examination of patterns of both lamin B1 and Sec61B during mitosis.
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Z-stack of nuclear envelope visualized via lamin B1 during M0
Z-stack of endoplasmic reticulum visualized via Sec61ß during M0
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Z-stack of nuclear envelope visualized via lamin B1 during M5
Z-stack of endoplasmic reticulum visualized via Sec61ß during M5
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Z-stack of nuclear envelope visualized via lamin B1 during M7
Z-stack of endoplasmic reticulum visualized via Sec61ß during M7
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- fragmentation and disassembly of Golgi and redistribution of sialyltransferase 1 before cell division followed its reassembly to form the Golgi after cell division.
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Z-stack of Golgi visualized via sialyltransferase 1 during M5
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Z-stack of Golgi visualized via sialyltransferase 1 during M7
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- disassembly of the nucleolus and redistribution of fibrillarin. Disassembly includes a stage where the nucleolus seems to ‘melt’. Reassembly occurs around the chromosomes after cell division.
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Z-stack of nucleolus visualized via fibrillarin during M5
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Z-stack of nucleolus visualized via fibrillarin during M7
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- redistribution of mitochondria to surround the mitotic spindle during cell division.
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Z-stack of mitochondria visualized via TOM20 during M5
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Z-stack of mitochondria visualized via TOM20 during M7
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- structures that do not change their localization during most of mitosis include many of the structures localized to and representing the cell-cell contacts near the apical side of the hiPS cells, permitting cells to maintain their position within the colony during cell division.
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Z-stack of mitochondria visualized via ZO1 during M5
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Z-stack of mitochondria visualized via ZO1 during M5
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