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Thrombopoietin-induced polyploidization of bone marrow megakaryocytes is due to a unique regulatory mechanism in late mitosis.

Nagata Y, Muro Y, Todokoro K - J. Cell Biol. (1997)

Bottom Line: It has been postulated that polyploidization is due to a skipping of mitosis after each round of DNA replication.We further noted that the pair of spindle poles in anaphase were located in close proximity to each other, probably because of the lack of outward movement of spindle poles during anaphase B.Thus, the reassembling nuclear envelope may enclose all the sister chromatids in a single nucleus at anaphase and then skip telophase and cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), Tsukuba, Ibaraki 305, Japan.

ABSTRACT
Megakaryocytes undergo a unique differentiation program, becoming polyploid through repeated cycles of DNA synthesis without concomitant cell division. However, the mechanism underlying this polyploidization remains totally unknown. It has been postulated that polyploidization is due to a skipping of mitosis after each round of DNA replication. We carried out immunohistochemical studies on mouse bone marrow megakaryocytes during thrombopoietin- induced polyploidization and found that during this process megakaryocytes indeed enter mitosis and progress through normal prophase, prometaphase, metaphase, and up to anaphase A, but not to anaphase B, telophase, or cytokinesis. It was clearly observed that multiple spindle poles were formed as the polyploid megakaryocytes entered mitosis; the nuclear membrane broke down during prophase; the sister chromatids were aligned on a multifaced plate, and the centrosomes were symmetrically located on either side of each face of the plate at metaphase; and a set of sister chromatids moved into the multiple centrosomes during anaphase A. We further noted that the pair of spindle poles in anaphase were located in close proximity to each other, probably because of the lack of outward movement of spindle poles during anaphase B. Thus, the reassembling nuclear envelope may enclose all the sister chromatids in a single nucleus at anaphase and then skip telophase and cytokinesis. These observations clearly indicate that polyploidization of megakaryocytes is not simply due to a skipping of mitosis, and that the megakaryocytes must have a unique regulatory mechanism in anaphase, e.g., factors regulating anaphase such as microtubule motor proteins might be involved in this polyploidization process.

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Centromere movement during polyploidizing megakaryocytes. TPO-treated primary megakaryocytes were stained with anticentromere antibody (red, first column), anti–α-tubulin antibody (green, second column), DAPI (blue, third column), and triple staining  (fourth column) during mitosis. (A) Megakaryocyte in interphase. (B) Megakaryocytes in prometaphase. (C) Megakaryocyte with  ploidy 8N at the stage just before metaphase. (D) Megakaryocyte with ploidy 8N in metaphase. (E) Megakaryocyte with ploidy 8N in  anaphase A. (F) Megakaryocyte with ploidy 16N in anaphase A.
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Figure 4: Centromere movement during polyploidizing megakaryocytes. TPO-treated primary megakaryocytes were stained with anticentromere antibody (red, first column), anti–α-tubulin antibody (green, second column), DAPI (blue, third column), and triple staining (fourth column) during mitosis. (A) Megakaryocyte in interphase. (B) Megakaryocytes in prometaphase. (C) Megakaryocyte with ploidy 8N at the stage just before metaphase. (D) Megakaryocyte with ploidy 8N in metaphase. (E) Megakaryocyte with ploidy 8N in anaphase A. (F) Megakaryocyte with ploidy 16N in anaphase A.

Mentions: To see the chromosome movement in more detail, we looked further at the polyploidizing megakaryocytes stained with anticentromere antibody, anti–α-tubulin antibody, and DAPI at all stages of mitosis (Fig. 4). In interphase, the centromeres were dispersed in the entire area of the nucleus (Fig. 4 A). In prophase and prometaphase, the chromatin condensed into well-defined chromosomes, and staining of the centromeres (Fig. 4 B) showed that the chromosomes began to align for metaphase. The multiple mitotic spindle poles were formed at this stage. Fig. 4, C–E, shows a megakaryocyte polyploidizing from ploidy 4N to 8N at the stage just before metaphase, metaphase, and anaphase A, respectively. At the stage just before metaphase, two planes of the aligned chromosomes crossed at right angles, and two pairs of spindle poles were symmetrically located in close proximity to this crossing on either side of each face of the plate (Fig. 4 C). The centromeres were located around the crossing and moving to points equidistant from the poles. In metaphase, two pairs of spindle poles moved outward and stretched the sister chromatids tightly toward the poles; thus, the centromeres were located just in the middle of the crossed chromosome planes between the two poles (Fig. 4 D). In anaphase A, the sister chromatids were pulled toward the spindle poles so that four sets of centromeres were moving toward each pole (Fig. 4 E). No set, however, could be separated far enough to be enclosed by individual nuclear envelopes. Fig. 4 F shows a megakaryocyte polyploidizing from ploidy 8N to 16N in anaphase A. The sets of centromeres were located close to each centrosome, and none of the sets of chromosomes was separated completed. We found no megakaryocytes in telophase or cytokinesis.


Thrombopoietin-induced polyploidization of bone marrow megakaryocytes is due to a unique regulatory mechanism in late mitosis.

Nagata Y, Muro Y, Todokoro K - J. Cell Biol. (1997)

Centromere movement during polyploidizing megakaryocytes. TPO-treated primary megakaryocytes were stained with anticentromere antibody (red, first column), anti–α-tubulin antibody (green, second column), DAPI (blue, third column), and triple staining  (fourth column) during mitosis. (A) Megakaryocyte in interphase. (B) Megakaryocytes in prometaphase. (C) Megakaryocyte with  ploidy 8N at the stage just before metaphase. (D) Megakaryocyte with ploidy 8N in metaphase. (E) Megakaryocyte with ploidy 8N in  anaphase A. (F) Megakaryocyte with ploidy 16N in anaphase A.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139799&req=5

Figure 4: Centromere movement during polyploidizing megakaryocytes. TPO-treated primary megakaryocytes were stained with anticentromere antibody (red, first column), anti–α-tubulin antibody (green, second column), DAPI (blue, third column), and triple staining (fourth column) during mitosis. (A) Megakaryocyte in interphase. (B) Megakaryocytes in prometaphase. (C) Megakaryocyte with ploidy 8N at the stage just before metaphase. (D) Megakaryocyte with ploidy 8N in metaphase. (E) Megakaryocyte with ploidy 8N in anaphase A. (F) Megakaryocyte with ploidy 16N in anaphase A.
Mentions: To see the chromosome movement in more detail, we looked further at the polyploidizing megakaryocytes stained with anticentromere antibody, anti–α-tubulin antibody, and DAPI at all stages of mitosis (Fig. 4). In interphase, the centromeres were dispersed in the entire area of the nucleus (Fig. 4 A). In prophase and prometaphase, the chromatin condensed into well-defined chromosomes, and staining of the centromeres (Fig. 4 B) showed that the chromosomes began to align for metaphase. The multiple mitotic spindle poles were formed at this stage. Fig. 4, C–E, shows a megakaryocyte polyploidizing from ploidy 4N to 8N at the stage just before metaphase, metaphase, and anaphase A, respectively. At the stage just before metaphase, two planes of the aligned chromosomes crossed at right angles, and two pairs of spindle poles were symmetrically located in close proximity to this crossing on either side of each face of the plate (Fig. 4 C). The centromeres were located around the crossing and moving to points equidistant from the poles. In metaphase, two pairs of spindle poles moved outward and stretched the sister chromatids tightly toward the poles; thus, the centromeres were located just in the middle of the crossed chromosome planes between the two poles (Fig. 4 D). In anaphase A, the sister chromatids were pulled toward the spindle poles so that four sets of centromeres were moving toward each pole (Fig. 4 E). No set, however, could be separated far enough to be enclosed by individual nuclear envelopes. Fig. 4 F shows a megakaryocyte polyploidizing from ploidy 8N to 16N in anaphase A. The sets of centromeres were located close to each centrosome, and none of the sets of chromosomes was separated completed. We found no megakaryocytes in telophase or cytokinesis.

Bottom Line: It has been postulated that polyploidization is due to a skipping of mitosis after each round of DNA replication.We further noted that the pair of spindle poles in anaphase were located in close proximity to each other, probably because of the lack of outward movement of spindle poles during anaphase B.Thus, the reassembling nuclear envelope may enclose all the sister chromatids in a single nucleus at anaphase and then skip telophase and cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), Tsukuba, Ibaraki 305, Japan.

ABSTRACT
Megakaryocytes undergo a unique differentiation program, becoming polyploid through repeated cycles of DNA synthesis without concomitant cell division. However, the mechanism underlying this polyploidization remains totally unknown. It has been postulated that polyploidization is due to a skipping of mitosis after each round of DNA replication. We carried out immunohistochemical studies on mouse bone marrow megakaryocytes during thrombopoietin- induced polyploidization and found that during this process megakaryocytes indeed enter mitosis and progress through normal prophase, prometaphase, metaphase, and up to anaphase A, but not to anaphase B, telophase, or cytokinesis. It was clearly observed that multiple spindle poles were formed as the polyploid megakaryocytes entered mitosis; the nuclear membrane broke down during prophase; the sister chromatids were aligned on a multifaced plate, and the centrosomes were symmetrically located on either side of each face of the plate at metaphase; and a set of sister chromatids moved into the multiple centrosomes during anaphase A. We further noted that the pair of spindle poles in anaphase were located in close proximity to each other, probably because of the lack of outward movement of spindle poles during anaphase B. Thus, the reassembling nuclear envelope may enclose all the sister chromatids in a single nucleus at anaphase and then skip telophase and cytokinesis. These observations clearly indicate that polyploidization of megakaryocytes is not simply due to a skipping of mitosis, and that the megakaryocytes must have a unique regulatory mechanism in anaphase, e.g., factors regulating anaphase such as microtubule motor proteins might be involved in this polyploidization process.

Show MeSH