Direct visualization of the endomitotic cell cycle in living megakaryocytes: Differential patterns in low and high ploidy cells

Papadantonakis, Nicholas; Makitalo, Maria; McCrann, Donald J.; Liu, Kenian; Nguyen, Hao G.; Martin, Greg V.; Patel–Hett, Sunita; Italiano, Joseph E.; Ravid, Katya

doi:10.4161/cc.6325

Cited by 34 publications

(28 citation statements)

References 19 publications

(22 reference statements)

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“…At the 2N to 4N transition, cleavage furrow ingression occurs, but subsequently regresses with a backward movement between the two daughter cells 5,6 . A similar phenomenon is observed at higher ploidy with a less profound furrow ingression 7,8 . The cleavage furrow is related to the development at the equator of a contractile ring consisting of myosin II and F-actin, an actomyosin complex generating the forces necessary for furrow ingression and cell separation 9 .…”

supporting

confidence: 69%

RUNX1-induced silencing of non-muscle myosin heavy chain IIB contributes to megakaryocyte polyploidization

et al. 2012

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megakaryocytes are unique mammalian cells that undergo polyploidization (endomitosis) during differentiation, leading to an increase in cell size and protein production that precedes platelet production. Recent evidence demonstrates that endomitosis is a consequence of a late failure in cytokinesis associated with a contractile ring defect. Here we show that the non-muscle myosin IIB heavy chain (mYH10) is expressed in immature megakaryocytes and specifically localizes in the contractile ring. mYH10 downmodulation by short hairpin RnA increases polyploidization by inhibiting the return of 4n cells to 2n, but other regulators, such as of the G1/s transition, might regulate further polyploidization of the 4n cells. Conversely, re-expression of mYH10 in the megakaryocytes prevents polyploidization and the transition of 2n to 4n cells. During polyploidization, mYH10 expression is repressed by the major megakaryocyte transcription factor RunX1. Thus, RunX1-mediated silencing of mYH10 is required for the switch from mitosis to endomitosis, linking polyploidization with megakaryocyte differentiation.

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confidence: 69%

RUNX1-induced silencing of non-muscle myosin heavy chain IIB contributes to megakaryocyte polyploidization

et al. 2012

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“…Their maturation involves unusual processes of polyploidisation1 and proplatelet formation2 regulated by dynamic changes in transcription factors such as GATA1, NF‐E2, FLI1, and RUNX1 3, 4. Thrombopoietin (TPO) is the main humoral driver of megakaryopoiesis 5, 6.…”

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confidence: 99%

N‐methyl‐d‐aspartate receptor mediated calcium influx supports in vitro differentiation of normal mouse megakaryocytes but proliferation of leukemic cell lines

Kamal

Green

Hearn

et al. 2018

Research and Practice in Thrombosis and Haemostasis

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Essentials Intracellular calcium pathways regulate megakaryopoiesis but details are unclear.We examined effects of NMDAR‐mediated calcium influx on normal and leukemic cells in culture.NMDARs facilitated differentiation of normal but proliferation of leukemic megakaryocytes.NMDAR inhibitors induced differentiation of leukemic Meg‐01 cells. Background N‐methyl‐d‐aspartate receptors (NMDARs) contribute calcium influx in megakaryocytic cells but their roles remain unclear; both pro‐ and anti‐differentiating effects have been shown in different contexts.ObjectivesThe aim of this study was to clarify NMDAR contribution to megakaryocytic differentiation in both normal and leukemic cells.MethodsMeg‐01, Set‐2, and K‐562 leukemic cell lines were differentiated using phorbol‐12‐myristate‐13‐acetate (PMA, 10 nmol L−1) or valproic acid (VPA, 500 μmol L−1). Normal megakaryocytes were grown from mouse marrow‐derived hematopoietic progenitors (lineage‐negative and CD41a‐enriched) in the presence of thrombopoietin (30‐40 nmol L−1). Marrow explants were used to monitor proplatelet formation in the native bone marrow milieu. In all culture systems, NMDARs were inhibited using memantine and MK‐801 (100 μmol L−1); their effects compared against appropriate controls.ResultsThe most striking observation from our studies was that NMDAR antagonists markedly inhibited proplatelet formation in all primary cultures employed. Proplatelets were either absent (in the presence of memantine) or short, broad and intertwined (with MK‐801). Earlier steps of megakaryocytic differentiation (acquisition of CD41a and nuclear ploidy) were maintained, albeit reduced. In contrast, in leukemic Meg‐01 cells, NMDAR antagonists inhibited differentiation in the presence of PMA and VPA but induced differentiation when applied by themselves.Conclusions NMDAR‐mediated calcium influx is required for normal megakaryocytic differentiation, in particular proplatelet formation. However, in leukemic cells, the main NMDAR role is to inhibit differentiation, suggesting diversion of NMDAR activity to support leukemia growth. Further elucidation of the NMDAR and calcium pathways in megakaryocytic cells may suggest novel ways to modulate abnormal megakaryopoiesis.

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“…Molecular candidates for the endomitotic switch include cyclin B1 and cyclin E (Carow et al, 2001;Bermejo et al, 2002;Eliades et al, 2010). In addition, live-imaging experiments were performed using the histone2B (H2B)-GFP or YFP-tubulin marker to monitor how mitosis aborted (Geddis et al, 2007;Papadantonakis et al, 2008).…”

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confidence: 99%

Visualizing developmentally programmed endoreplication in mammals using ubiquitin oscillators

Sakaue-Sawano

Hoshida

et al. 2013

Development

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The majority of mammalian somatic cells maintain a diploid genome. However, some mammalian cell types undergo multiple rounds of genome replication (endoreplication) as part of normal development and differentiation. For example, trophoblast giant cells (TGCs) in the placenta become polyploid through endoreduplication (bypassed mitosis), and megakaryocytes (MKCs) in the bone marrow become polyploid through endomitosis (abortive mitosis). During the normal mitotic cell cycle, geminin and Cdt1 are involved in ‘licensing’ of replication origins, which ensures that replication occurs only once in a cell cycle. Their protein accumulation is directly regulated by two E3 ubiquitin ligase activities, APCCdh1 and SCFSkp2, which oscillate reciprocally during the cell cycle. Although proteolysis-mediated, oscillatory accumulation of proteins has been documented in endoreplicating Drosophila cells, it is not known whether the ubiquitin oscillators that control normal cell cycle transitions also function during mammalian endoreplication. In this study, we used transgenic mice expressing Fucci fluorescent cell-cycle probes that report the activity of APCCdh1 and SCFSkp2. By performing long-term, high temporal-resolution Fucci imaging, we were able to visualize reciprocal activation of APCCdh1 and SCFSkp2 in differentiating TGCs and MKCs grown in our custom-designed culture wells. We found that TGCs and MKCs both skip cytokinesis, but in different ways, and that the reciprocal activation of the ubiquitin oscillators in MKCs varies with the polyploidy level. We also obtained three-dimensional reconstructions of highly polyploid TGCs in whole, fixed mouse placentas. Thus, the Fucci technique is able to reveal the spatiotemporal regulation of the endoreplicative cell cycle during differentiation.

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Direct visualization of the endomitotic cell cycle in living megakaryocytes: Differential patterns in low and high ploidy cells

Cited by 34 publications

References 19 publications

RUNX1-induced silencing of non-muscle myosin heavy chain IIB contributes to megakaryocyte polyploidization

RUNX1-induced silencing of non-muscle myosin heavy chain IIB contributes to megakaryocyte polyploidization

N‐methyl‐d‐aspartate receptor mediated calcium influx supports in vitro differentiation of normal mouse megakaryocytes but proliferation of leukemic cell lines

Visualizing developmentally programmed endoreplication in mammals using ubiquitin oscillators

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