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Updated: Jun 1, 2026
In Situ Exploration of Murine Megakaryopoiesis using Transmission Electron Microscopy
Published on: September 8, 2021
Distinct functional effects for dynamin 3 during megakaryocytopoiesis
Wenjing Wang1, Diana M Gilligan, Sijie Sun
1Puget Sound Blood Center, Seattle, Washington 98104, USA. wenjingw@psbcresearch.org
Abstract:
Dynamin 3 (DNM3) is a member of a family of motor proteins that participate in a number of membrane rearrangements such as cytokinesis, budding of transport vesicles, phagocytosis, and cell motility. Recently, DNM3 was implicated as having a role in megakaryocyte (MK) development. To further investigate the functional role of DNM3 during megakaryocytopoiesis, we introduced sequence-specific short hairpin RNAs (shRNAs) into developing MKs. The results showed that knockdown of DNM3 inhibited a stage of MK development that involved progenitor amplification. This was evident by significant decreases in the number of colony forming unit-megakaryocytes, the total number of nucleated cells, and the number of CD41(+) and CD61(+) MKs produced in culture. Using a styrl membrane dye to quantify the demarcation membrane system (DMS) of terminally differentiated MKs, we found that DNM3 co-localized with the DMS and that DNM3 lentiviral shRNAs precluded the formation of the DMS. Knockdown of dynamin 3 in murine MKs also caused a decrease in the number of morphologically large MKs and the overall size of large MKs was decreased relative to controls. MK protein lysates were used in overlay blots to show that both DNM3 and actin bind to nonmuscle myosin IIA (MYH9). Consistent with these observations, immunofluorescence studies of MKs and proplatelet processes showed co-localization of DNM3 with MYH9. Overall, these studies demonstrate that DNM3 not only participates in MK progenitor amplification, but is also involved in cytoplasmic enlargement and the formation of the DMS.
Insights
Dynamin 3 (DNM3) is crucial for megakaryocyte (MK) development, impacting progenitor amplification and cell size. DNM3 is essential for forming the demarcation membrane system (DMS) in mature MKs.
Area of Science:
- Cell Biology
- Hematopoiesis
- Protein Function
Background:
- Dynamin 3 (DNM3) is a motor protein involved in membrane dynamics.
- Emerging evidence suggests a role for DNM3 in megakaryocyte (MK) development.
Purpose of the Study:
- To investigate the functional role of DNM3 in megakaryocytopoiesis.
- To elucidate DNM3's contribution to MK progenitor amplification, cytoplasmic growth, and demarcation membrane system (DMS) formation.
Main Methods:
- Short hairpin RNAs (shRNAs) were used to knock down DNM3 expression in developing MKs.
- Quantification of progenitor cells (CFU-MKs), nucleated cells, and MK surface markers (CD41, CD61).
- Styryl membrane dye and immunofluorescence were used to assess DMS formation and protein co-localization (DNM3, nonmuscle myosin IIA/MYH9).
Main Results:
- DNM3 knockdown significantly inhibited MK progenitor amplification, reducing CFU-MKs, total nucleated cells, and CD41/CD61 positive MKs.
- DNM3 co-localized with the DMS, and its knockdown precluded DMS formation in terminally differentiated MKs.
- DNM3 depletion led to smaller MKs and impaired cytoplasmic enlargement, with DNM3 and actin shown to bind MYH9.
Conclusions:
- DNM3 plays a critical role in MK progenitor amplification.
- DNM3 is essential for cytoplasmic enlargement and the formation of the DMS during megakaryopoiesis.
- DNM3 interacts with MYH9, suggesting a mechanism for its role in MK development.
