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Support for the immortal strand hypothesis: neural stem cells partition DNA asymmetrically in vitro.

Karpowicz P, Morshead C, Kam A, Jervis E, Ramunas J, Ramuns J, Cheng V, van der Kooy D - J. Cell Biol. (2005)

Bottom Line: The immortal strand hypothesis proposes that asymmetrically dividing stem cells (SCs) selectively segregate chromosomes that bear the oldest DNA templates.We investigated cosegregation in neural stem cells (NSCs).It was confirmed that some BrdU-retaining cells divided actively, and that these cells exhibited some characteristics of SCs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Science, University of Toronto, Toronto, M5R 1A8, Canada. phillip.karpowicz@utoronto.ca

ABSTRACT
The immortal strand hypothesis proposes that asymmetrically dividing stem cells (SCs) selectively segregate chromosomes that bear the oldest DNA templates. We investigated cosegregation in neural stem cells (NSCs). After exposure to the thymidine analogue 5-bromo-2-deoxyuridine (BrdU), which labels newly synthesized DNA, a subset of neural precursor cells were shown to retain BrdU signal. It was confirmed that some BrdU-retaining cells divided actively, and that these cells exhibited some characteristics of SCs. This asymmetric partitioning of DNA then was demonstrated during mitosis, and these results were further supported by real time imaging of SC clones, in which older and newly synthesized DNA templates were distributed asymmetrically after DNA synthesis. We demonstrate that NSCs are unique among precursor cells in the uneven partitioning of genetic material during cell divisions.

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Cell division inhibition of BrdU-retaining cells suggests asymmetric DNA partitioning. (A) BrdU distribution in a cell arrested during cytokinesis, 10 d after BrdU. Arrows indicate symmetric BrdU(+) nuclei in same cell. (i) Bright field shows binucleate cell; (ii) histone-labeled nuclei are red; (iii) BrdU is green; (iv) merge of histone and BrdU. (B) BrdU distribution in a cell arrested during cytokinesis, 10 d after BrdU. Arrows indicate BrdU(+) nucleus, adjacent to BrdU(−) nucleus, in same cell. (i) Bright field shows binucleate cell; (ii) histone-labeled nuclei are red; (iii) BrdU is green; (iv) shows merge of histone and BrdU. (C) BrdU distribution in binucleate cell population treated with cytochalasin D. Uneven segregation of labeled DNA to daughter nuclei occurs in 10% of the binucleate cell population. (D) Confocal microscopy of BrdU-exposed cells arrested during karyokinesis, 10 d after BrdU exposure. Upon removal of inhibitor, cells were timed for fixation at late anaphase or telophase. Mitotic cells were observed segregating labeled DNA nonrandomly to one daughter in top row (arrows), as opposed to the even segregation of BrdU in bottom examples (arrowheads). BrdU labeling was confirmed at all focal planes. (i) Bright field shows mitotic cells; (ii) histone-labeled nuclei are red; (iii) BrdU is green; (iv) merge of histone and BrdU. (E) Cell doublets arising from 10 d after BrdU cells inhibited during karyokinesis. Cells released from inhibition were allowed to complete mitosis. Uneven labeling of BrdU(+/−) daughter nuclei was again apparent (arrows). (i) Bright field shows two cells; (ii) histone-labeled nuclei are red; (iii) BrdU is indicated by green; (iv) merge of histone and BrdU. (F) Cell doublets arising from 10 d after BrdU cells inhibited during karyokinesis. Cells released from inhibition were allowed to complete mitosis. Some doublets displayed evenly labeled BrdU(+) daughters (arrows) or unlabeled, BrdU(−) doublets (arrowheads). (i) Bright field shows binucleate cells; (ii) merge of histone-labeled nuclei (red) and BrdU (green).
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fig6: Cell division inhibition of BrdU-retaining cells suggests asymmetric DNA partitioning. (A) BrdU distribution in a cell arrested during cytokinesis, 10 d after BrdU. Arrows indicate symmetric BrdU(+) nuclei in same cell. (i) Bright field shows binucleate cell; (ii) histone-labeled nuclei are red; (iii) BrdU is green; (iv) merge of histone and BrdU. (B) BrdU distribution in a cell arrested during cytokinesis, 10 d after BrdU. Arrows indicate BrdU(+) nucleus, adjacent to BrdU(−) nucleus, in same cell. (i) Bright field shows binucleate cell; (ii) histone-labeled nuclei are red; (iii) BrdU is green; (iv) shows merge of histone and BrdU. (C) BrdU distribution in binucleate cell population treated with cytochalasin D. Uneven segregation of labeled DNA to daughter nuclei occurs in 10% of the binucleate cell population. (D) Confocal microscopy of BrdU-exposed cells arrested during karyokinesis, 10 d after BrdU exposure. Upon removal of inhibitor, cells were timed for fixation at late anaphase or telophase. Mitotic cells were observed segregating labeled DNA nonrandomly to one daughter in top row (arrows), as opposed to the even segregation of BrdU in bottom examples (arrowheads). BrdU labeling was confirmed at all focal planes. (i) Bright field shows mitotic cells; (ii) histone-labeled nuclei are red; (iii) BrdU is green; (iv) merge of histone and BrdU. (E) Cell doublets arising from 10 d after BrdU cells inhibited during karyokinesis. Cells released from inhibition were allowed to complete mitosis. Uneven labeling of BrdU(+/−) daughter nuclei was again apparent (arrows). (i) Bright field shows two cells; (ii) histone-labeled nuclei are red; (iii) BrdU is indicated by green; (iv) merge of histone and BrdU. (F) Cell doublets arising from 10 d after BrdU cells inhibited during karyokinesis. Cells released from inhibition were allowed to complete mitosis. Some doublets displayed evenly labeled BrdU(+) daughters (arrows) or unlabeled, BrdU(−) doublets (arrowheads). (i) Bright field shows binucleate cells; (ii) merge of histone-labeled nuclei (red) and BrdU (green).

Mentions: We asked whether in vitro neural precursors could distribute DNA asymmetrically using cytokinetic and karyokinetic inhibitors and immunofluorescence. 10 d after BrdU cells were exposed to an actin binding protein, cytochalasin D, to arrest them during cytokinesis, although karyokinesis had already occurred. Such treatment resulted in the recovery of many binucleate cells, composing approximately half of the total cell population. The complete dissociation of cells, including binucleate cells, into a single cell suspension was verified on a hemocytometer. Though most BrdU(+) binucleate cells displayed equivalent BrdU signal in Fig. 6 A we found instances of cells that had one labeled nucleus and one unlabeled nucleus (Fig. 6 B). Such cells had been arrested by mitotic inhibition over a period of 24 h, meaning that it is likely that many of these cells had a cell cycle of <24 h. Thus, it is likely that at least 10 divisions occurred in these cells over 10 d in neurosphere culture. Notwithstanding 10 consecutive divisions, a subset of cells cosegregated BrdU-labeled chromosomes into one nucleus and remained positively labeled in contrast to the majority of cells examined at this time point. Quantification revealed that 78.3 ± 4.5 binucleate cells had two unlabeled nuclei, 11.4 ± 2.7% had equally labeled nuclei, and 10.3 ± 1.9% exhibited BrdU signal in only one of the daughter nuclei (Fig. 6 C). No evidence of uneven BrdU(+) signal in fibroblast cells treated with cytochalasin D was found (unpublished data), in contrast to neural cells.


Support for the immortal strand hypothesis: neural stem cells partition DNA asymmetrically in vitro.

Karpowicz P, Morshead C, Kam A, Jervis E, Ramunas J, Ramuns J, Cheng V, van der Kooy D - J. Cell Biol. (2005)

Cell division inhibition of BrdU-retaining cells suggests asymmetric DNA partitioning. (A) BrdU distribution in a cell arrested during cytokinesis, 10 d after BrdU. Arrows indicate symmetric BrdU(+) nuclei in same cell. (i) Bright field shows binucleate cell; (ii) histone-labeled nuclei are red; (iii) BrdU is green; (iv) merge of histone and BrdU. (B) BrdU distribution in a cell arrested during cytokinesis, 10 d after BrdU. Arrows indicate BrdU(+) nucleus, adjacent to BrdU(−) nucleus, in same cell. (i) Bright field shows binucleate cell; (ii) histone-labeled nuclei are red; (iii) BrdU is green; (iv) shows merge of histone and BrdU. (C) BrdU distribution in binucleate cell population treated with cytochalasin D. Uneven segregation of labeled DNA to daughter nuclei occurs in 10% of the binucleate cell population. (D) Confocal microscopy of BrdU-exposed cells arrested during karyokinesis, 10 d after BrdU exposure. Upon removal of inhibitor, cells were timed for fixation at late anaphase or telophase. Mitotic cells were observed segregating labeled DNA nonrandomly to one daughter in top row (arrows), as opposed to the even segregation of BrdU in bottom examples (arrowheads). BrdU labeling was confirmed at all focal planes. (i) Bright field shows mitotic cells; (ii) histone-labeled nuclei are red; (iii) BrdU is green; (iv) merge of histone and BrdU. (E) Cell doublets arising from 10 d after BrdU cells inhibited during karyokinesis. Cells released from inhibition were allowed to complete mitosis. Uneven labeling of BrdU(+/−) daughter nuclei was again apparent (arrows). (i) Bright field shows two cells; (ii) histone-labeled nuclei are red; (iii) BrdU is indicated by green; (iv) merge of histone and BrdU. (F) Cell doublets arising from 10 d after BrdU cells inhibited during karyokinesis. Cells released from inhibition were allowed to complete mitosis. Some doublets displayed evenly labeled BrdU(+) daughters (arrows) or unlabeled, BrdU(−) doublets (arrowheads). (i) Bright field shows binucleate cells; (ii) merge of histone-labeled nuclei (red) and BrdU (green).
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fig6: Cell division inhibition of BrdU-retaining cells suggests asymmetric DNA partitioning. (A) BrdU distribution in a cell arrested during cytokinesis, 10 d after BrdU. Arrows indicate symmetric BrdU(+) nuclei in same cell. (i) Bright field shows binucleate cell; (ii) histone-labeled nuclei are red; (iii) BrdU is green; (iv) merge of histone and BrdU. (B) BrdU distribution in a cell arrested during cytokinesis, 10 d after BrdU. Arrows indicate BrdU(+) nucleus, adjacent to BrdU(−) nucleus, in same cell. (i) Bright field shows binucleate cell; (ii) histone-labeled nuclei are red; (iii) BrdU is green; (iv) shows merge of histone and BrdU. (C) BrdU distribution in binucleate cell population treated with cytochalasin D. Uneven segregation of labeled DNA to daughter nuclei occurs in 10% of the binucleate cell population. (D) Confocal microscopy of BrdU-exposed cells arrested during karyokinesis, 10 d after BrdU exposure. Upon removal of inhibitor, cells were timed for fixation at late anaphase or telophase. Mitotic cells were observed segregating labeled DNA nonrandomly to one daughter in top row (arrows), as opposed to the even segregation of BrdU in bottom examples (arrowheads). BrdU labeling was confirmed at all focal planes. (i) Bright field shows mitotic cells; (ii) histone-labeled nuclei are red; (iii) BrdU is green; (iv) merge of histone and BrdU. (E) Cell doublets arising from 10 d after BrdU cells inhibited during karyokinesis. Cells released from inhibition were allowed to complete mitosis. Uneven labeling of BrdU(+/−) daughter nuclei was again apparent (arrows). (i) Bright field shows two cells; (ii) histone-labeled nuclei are red; (iii) BrdU is indicated by green; (iv) merge of histone and BrdU. (F) Cell doublets arising from 10 d after BrdU cells inhibited during karyokinesis. Cells released from inhibition were allowed to complete mitosis. Some doublets displayed evenly labeled BrdU(+) daughters (arrows) or unlabeled, BrdU(−) doublets (arrowheads). (i) Bright field shows binucleate cells; (ii) merge of histone-labeled nuclei (red) and BrdU (green).
Mentions: We asked whether in vitro neural precursors could distribute DNA asymmetrically using cytokinetic and karyokinetic inhibitors and immunofluorescence. 10 d after BrdU cells were exposed to an actin binding protein, cytochalasin D, to arrest them during cytokinesis, although karyokinesis had already occurred. Such treatment resulted in the recovery of many binucleate cells, composing approximately half of the total cell population. The complete dissociation of cells, including binucleate cells, into a single cell suspension was verified on a hemocytometer. Though most BrdU(+) binucleate cells displayed equivalent BrdU signal in Fig. 6 A we found instances of cells that had one labeled nucleus and one unlabeled nucleus (Fig. 6 B). Such cells had been arrested by mitotic inhibition over a period of 24 h, meaning that it is likely that many of these cells had a cell cycle of <24 h. Thus, it is likely that at least 10 divisions occurred in these cells over 10 d in neurosphere culture. Notwithstanding 10 consecutive divisions, a subset of cells cosegregated BrdU-labeled chromosomes into one nucleus and remained positively labeled in contrast to the majority of cells examined at this time point. Quantification revealed that 78.3 ± 4.5 binucleate cells had two unlabeled nuclei, 11.4 ± 2.7% had equally labeled nuclei, and 10.3 ± 1.9% exhibited BrdU signal in only one of the daughter nuclei (Fig. 6 C). No evidence of uneven BrdU(+) signal in fibroblast cells treated with cytochalasin D was found (unpublished data), in contrast to neural cells.

Bottom Line: The immortal strand hypothesis proposes that asymmetrically dividing stem cells (SCs) selectively segregate chromosomes that bear the oldest DNA templates.We investigated cosegregation in neural stem cells (NSCs).It was confirmed that some BrdU-retaining cells divided actively, and that these cells exhibited some characteristics of SCs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Science, University of Toronto, Toronto, M5R 1A8, Canada. phillip.karpowicz@utoronto.ca

ABSTRACT
The immortal strand hypothesis proposes that asymmetrically dividing stem cells (SCs) selectively segregate chromosomes that bear the oldest DNA templates. We investigated cosegregation in neural stem cells (NSCs). After exposure to the thymidine analogue 5-bromo-2-deoxyuridine (BrdU), which labels newly synthesized DNA, a subset of neural precursor cells were shown to retain BrdU signal. It was confirmed that some BrdU-retaining cells divided actively, and that these cells exhibited some characteristics of SCs. This asymmetric partitioning of DNA then was demonstrated during mitosis, and these results were further supported by real time imaging of SC clones, in which older and newly synthesized DNA templates were distributed asymmetrically after DNA synthesis. We demonstrate that NSCs are unique among precursor cells in the uneven partitioning of genetic material during cell divisions.

Show MeSH
Related in: MedlinePlus