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Updated: Aug 17, 2026
In Situ Exploration of Murine Megakaryopoiesis using Transmission Electron Microscopy
Published on: September 8, 2021
Kinetics of endomitosis in primary murine megakaryocytes
C E Carow1, N E Fox, K Kaushansky
1Department of Medicine, University of Washington, Seattle 98195-7710, USA. cecarow@u.washington.edu
Abstract:
Megakaryocytes (MKs) develop from diploid progenitor cells via successive rounds of DNA synthesis in the absence of cell division, a process termed endomitosis (EnM). While the mechanism underlying EnM is not known, studies in yeast and leukemic cell lines have suggested that it may be due to reduced levels of cyclin B1 or cdc2, leading to a decrease in mitotic kinase activity. Using flow cytometry to study EnM highly purified marrow-derived MK precursors, we found that: (1) on average, 36% of 8N-32N MKs expressed abundant cyclin B during G2/M. The percentage of cells in G2/M decreased in >64N MKs, suggesting the limit of EnM, (2) the level of cyclin B per G2/M MK increased linearly with ploidy, (3) cyclin B expression oscillated normally in polyploid MKs, (4) MPM-2, a phosphoepitope created by the action of mitotic kinases and specific to M-phase cells, was expressed in a significant fraction of polyploid MKs, and (5) there was an apparent increase of cyclin B in G1-phase in polyploid MKs. This study provides the first qualitative kinetic data regarding the cell cycle status of MKs within individual ploidy classes. It also demonstrates the feasibility of using anti-cyclin B antibody and flow cytometry to resolve G1 from G2/M populations in polyploid MKs. Finally, these findings establish that neither a relative nor absolute deficiency of mitotic kinase components is responsible for EnM, suggesting that the departure from normal cell division kinetics seen in polyploid MKs is likely due to alterations in other cell cycle regulators.
Insights
Megakaryocyte endomitosis (EnM) involves DNA replication without cell division. This study shows that mitotic kinase components are not deficient in polyploid megakaryocytes, suggesting other cell cycle regulators are involved.
Area of Science:
- Cell Biology
- Hematopoiesis
- Molecular Biology
Background:
- Megakaryocytes (MKs) undergo endomitosis (EnM), a unique cell division process where DNA replicates without cell division.
- The molecular mechanisms driving EnM remain largely unknown.
- Previous hypotheses suggested reduced mitotic kinase activity, involving cyclin B1 and cdc2, might cause EnM.
Purpose of the Study:
- To investigate the cell cycle status and mitotic kinase activity in megakaryocyte precursors during endomitosis.
- To determine the role of cyclin B and mitotic kinases in the process of EnM.
- To provide kinetic data on cell cycle progression in polyploid megakaryocytes.
Main Methods:
- Utilized flow cytometry to analyze highly purified marrow-derived MK precursors.
- Employed anti-cyclin B antibody to assess cyclin B expression levels and cell cycle phase distribution (G1 vs. G2/M).
- Used MPM-2 antibody to detect mitotic kinase activity in polyploid MKs.
Main Results:
- Cyclin B expression was abundant in G2/M phase MKs up to 32N ploidy, with expression increasing linearly with ploidy.
- The percentage of cells in G2/M decreased in MKs >64N, indicating a potential limit to EnM.
- MPM-2 epitope expression confirmed mitotic kinase activity in a significant fraction of polyploid MKs.
- Observed an apparent increase of cyclin B in G1 phase of polyploid MKs, with normal oscillation of cyclin B expression.
Conclusions:
- Findings refute the hypothesis that EnM is caused by a deficiency in mitotic kinase components like cyclin B or cdc2.
- The study demonstrates the feasibility of using anti-cyclin B and flow cytometry to differentiate G1 and G2/M phases in polyploid MKs.
- Alterations in other cell cycle regulators, rather than mitotic kinase deficiency, likely underlie the unique cell division kinetics observed in polyploid megakaryocytes.
