RhoA/Cdc42 signaling drives cytoplasmic maturation but not endomitosis in megakaryocytes

Heib, Tobias; Hermanns, Heike M.; Manukjan, Georgi; Englert, Maximilian; Kusch, Charly; Becker, Isabelle C.; Gerber, Annika; Wackerbarth, Lou Martha; Burkard, Philipp; Dandekar, Thomas; Balkenhol, Johannes; Jahn, Daniel; Beck, Sarah; Meub, Mara; Dütting, Sebastian; Stigloher, Christian; Sauer, Markus; Cherpokova, Deya; Schulze, Harald; Brakebusch, Cord; Nieswandt, Bernhard; Nagy, Z.; Pleines, Irina

doi:10.1016/j.celrep.2021.109102

Cited by 17 publications

(11 citation statements)

References 95 publications

(120 reference statements)

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“…The reduced surface expression of GPIbα, α2 integrin, and GPVI agrees with data from Mpig6b −/− and Mpig6b fl/fl ;Pf4-Cre + mice 2,11 ; however, the reduction was attributed to shedding of these receptors. 2 Interestingly, our finding resembles other mouse lines, where reduced MK maturation is associated with normal ploidy, including Gfi1b −/− , 12 Nfe2 −/− , 13 or Rhoa fl/f l ; Cdc42 fl/fl ; Pf4-Cre + 14 mice. The accumulation of immature MKs can lead to myelofibrosis, osteosclerosis, or both, as reported for the Gata1 low or Nfe2 −/− mouse lines 15-17 and may explain the phenotypes in G6b-B–null mice.…”

Section: Resultssupporting

confidence: 83%

G6b-B regulates an essential step in megakaryocyte maturation

et al. 2022

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G6b-B is a megakaryocyte lineage-specific immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor, essential for platelet homeostasis. Mice with a genomic deletion of the entire Mpig6b locus develop severe macrothrombocytopenia and myelofibrosis, which is reflected in humans with null-mutations in MPIG6B. The current model proposes that megakaryocytes lacking G6b-B develop normally, while proplatelet release is hampered, but the underlying molecular mechanism remains unclear. Here, we report on a spontaneous recessive single nucleotide mutation in C57BL/6 mice, localized within the intronic region of the Mpig6b locus that abolishes G6b-B expression and reproduces macrothrombocytopenia, myelofibrosis and osteosclerosis. As the mutation is based on a single nucleotide exchange, Mpig6bmut mice represent an ideal model to study the role of G6b-B. Megakaryocytes from these mice were smaller in size, displayed a less developed demarcation membrane system and reduced expression of receptors. RNA sequencing revealed a striking global reduction in the level of megakaryocyte-specific transcripts, in conjunction with decreased protein levels of the transcription factor GATA-1, and impaired thrombopoietin signaling. The reduced number of mature MKs in the bone marrow was corroborated on a newly developed Mpig6b null mouse strain. Our findings highlight an unexpected essential role of G6b-B in the early differentiation within the megakaryocytic lineage.

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Section: Resultssupporting

confidence: 83%

G6b-B regulates an essential step in megakaryocyte maturation

et al. 2022

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“…In fact, conditional double knock-out mice for the two small GTPases, RhoA and Cdc42, have megakaryocytes with increased LIMK and cofilin expression levels. These megakaryocytes are still able to undergo endomitoses but the normal maturation process of their cytoplasm does not take place, which results in a macrothrombocytopenia phenotype in these mice [109]. This observation reinforces previous studies in which phosphorylated, inactive cofilin or cofilin deficiency correlates with altered proplatelet formation and macrothrombocytopenia [110,111].…”

Section: Blood Cellssupporting

confidence: 86%

The Role of LIM Kinases during Development: A Lens to Get a Glimpse of Their Implication in Pathologies

Ribba

Fraboulet

Sadoul

et al. 2022

Cells

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The organization of cell populations within animal tissues is essential for the morphogenesis of organs during development. Cells recognize three-dimensional positions with respect to the whole organism and regulate their cell shape, motility, migration, polarization, growth, differentiation, gene expression and cell death according to extracellular signals. Remodeling of the actin filaments is essential to achieve these cell morphological changes. Cofilin is an important binding protein for these filaments; it increases their elasticity in terms of flexion and torsion and also severs them. The activity of cofilin is spatiotemporally inhibited via phosphorylation by the LIM domain kinases 1 and 2 (LIMK1 and LIMK2). Phylogenetic analysis indicates that the phospho-regulation of cofilin has evolved as a mechanism controlling the reorganization of the actin cytoskeleton during complex multicellular processes, such as those that occur during embryogenesis. In this context, the main objective of this review is to provide an update of the respective role of each of the LIM kinases during embryonic development.

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“…Protein levels of MLC2, myosin IIA, and MYPT1, and phosphorylation levels of MLC2 were analyzed using an automated capillary-based immunoassay platform ( 37 ), Jess (ProteinSimple). A total of 5 × 10 8 platelets/ml were lysed by the addition of equal volume ice-cold 2× lysis buffer (300 mM NaCl, 20 mM tris, 2 mM EGTA, 2 mM EDTA, 10 mM NaF, 4 mM Na 3 VO 4 , and 1% IGEPAL CA-630).…”

Section: Methodsmentioning

confidence: 99%

Reduced platelet forces underlie impaired hemostasis in mouse models of MYH9 -related disease

et al. 2022

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MYH9 -related disease patients with mutations in the contractile protein nonmuscle myosin heavy chain IIA display, among others, macrothrombocytopenia and a mild-to-moderate bleeding tendency. In this study, we used three mouse lines, each with one point mutation in the Myh9 gene at positions 702, 1424, or 1841, to investigate mechanisms underlying the increased bleeding risk. Agonist-induced activation of Myh9 mutant platelets was comparable to controls. However, myosin light chain phosphorylation after activation was reduced in mutant platelets, which displayed altered biophysical characteristics and generated lower adhesion, interaction, and traction forces. Treatment with tranexamic acid restored clot retraction in the presence of tPA and reduced bleeding. We verified our findings from the mutant mice with platelets from patients with the respective mutation. These data suggest that reduced platelet forces lead to an increased bleeding tendency in patients with MYH9 -related disease, and treatment with tranexamic acid can improve the hemostatic function.

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RhoA/Cdc42 signaling drives cytoplasmic maturation but not endomitosis in megakaryocytes

Cited by 17 publications

References 95 publications

G6b-B regulates an essential step in megakaryocyte maturation

G6b-B regulates an essential step in megakaryocyte maturation

The Role of LIM Kinases during Development: A Lens to Get a Glimpse of Their Implication in Pathologies

Reduced platelet forces underlie impaired hemostasis in mouse models of MYH9 -related disease

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