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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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Multiple mitotic  spindle poles formation during  TPO-induced polyploidization  of primary megakaryocytes.  Mitotic spindle poles were  detected by immunofluorescent light microscopy in TPO-induced primary mouse megakaryocytes. Megakaryocytes  cultured with TPO were fixed  in methanol for probing with  anti–α-tubulin antibody (A),  anti–γ-tubulin antibody (B),  and anticentriole antibody  (C), followed by incubation  with an FITC-labeled F(ab′)2  fragment (A) or a Cy3-conjugated F(ab′)2 fragment (B  and C).
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Figure 1: Multiple mitotic spindle poles formation during TPO-induced polyploidization of primary megakaryocytes. Mitotic spindle poles were detected by immunofluorescent light microscopy in TPO-induced primary mouse megakaryocytes. Megakaryocytes cultured with TPO were fixed in methanol for probing with anti–α-tubulin antibody (A), anti–γ-tubulin antibody (B), and anticentriole antibody (C), followed by incubation with an FITC-labeled F(ab′)2 fragment (A) or a Cy3-conjugated F(ab′)2 fragment (B and C).

Mentions: To study the TPO-induced polyploidization process of primary megakaryocytes, we immunostained a number of bone marrow megakaryocytes, which were cultured in the presence of TPO for 2 wk, with antibodies against various kinds of intracellular proteins regulating mitosis. A normal cell division requires a functional bipolar spindle, but how mitotic spindle poles are organized during polyploidization of megakaryocytes has not been characterized. Therefore, first of all, we stained the megakaryocytes with anti– α-tubulin antibody to learn the organization of mitotic spindles. Surprisingly, we found that multiple mitotic spindle poles were formed in all megakaryocytes in mitosis. The photograph in Fig. 1 A shows a megakaryocyte forming 32 spindle poles. The number of spindle poles in a megakaryocyte varies from 4 to 64, or even much more, but we were unable to count the exact number formed when it exceeded 32 because of the abundance.


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)

Multiple mitotic  spindle poles formation during  TPO-induced polyploidization  of primary megakaryocytes.  Mitotic spindle poles were  detected by immunofluorescent light microscopy in TPO-induced primary mouse megakaryocytes. Megakaryocytes  cultured with TPO were fixed  in methanol for probing with  anti–α-tubulin antibody (A),  anti–γ-tubulin antibody (B),  and anticentriole antibody  (C), followed by incubation  with an FITC-labeled F(ab′)2  fragment (A) or a Cy3-conjugated F(ab′)2 fragment (B  and C).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Multiple mitotic spindle poles formation during TPO-induced polyploidization of primary megakaryocytes. Mitotic spindle poles were detected by immunofluorescent light microscopy in TPO-induced primary mouse megakaryocytes. Megakaryocytes cultured with TPO were fixed in methanol for probing with anti–α-tubulin antibody (A), anti–γ-tubulin antibody (B), and anticentriole antibody (C), followed by incubation with an FITC-labeled F(ab′)2 fragment (A) or a Cy3-conjugated F(ab′)2 fragment (B and C).
Mentions: To study the TPO-induced polyploidization process of primary megakaryocytes, we immunostained a number of bone marrow megakaryocytes, which were cultured in the presence of TPO for 2 wk, with antibodies against various kinds of intracellular proteins regulating mitosis. A normal cell division requires a functional bipolar spindle, but how mitotic spindle poles are organized during polyploidization of megakaryocytes has not been characterized. Therefore, first of all, we stained the megakaryocytes with anti– α-tubulin antibody to learn the organization of mitotic spindles. Surprisingly, we found that multiple mitotic spindle poles were formed in all megakaryocytes in mitosis. The photograph in Fig. 1 A shows a megakaryocyte forming 32 spindle poles. The number of spindle poles in a megakaryocyte varies from 4 to 64, or even much more, but we were unable to count the exact number formed when it exceeded 32 because of the abundance.

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
Related in: MedlinePlus