Targeted disruption of mouse ortholog of the human MYH9 responsible for macrothrombocytopenia with different organ involvement: hematological, nephrological, and otological studies of heterozygous KO mice

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We have generated 3 mouse lines, each with a different mutation in the nonmuscle myosin II-A gene, Myh9 (R702C, D1424N, and E1841K). Each line develops MYH9-related disease similar to that found in human patients. R702C mutant human cDNA fused with green fluorescent protein was introduced into the first coding exon of Myh9, and D1424N and E1841K mutations were introduced directly into the corresponding exons. Homozygous R702C mice die at embryonic day 10.5-11.5, whereas homozygous D1424N and E1841K mice are viable. All heterozygous and homozygous mutant mice show macrothrombocytopenia with prolonged bleeding times, a defect in clot retraction, and increased extramedullary megakaryocytes. Studies of cultured megakaryocytes and live-cell imaging of megakaryocytes in the BM show that heterozygous R702C megakaryocytes form fewer and shorter proplatelets with less branching and larger buds. The results indicate that disrupted proplatelet formation contributes to the macrothrombocytopenia in mice and most probably in humans. We also observed premature cataract formation, kidney abnormalities, including albuminuria, focal segmental glomerulosclerosis and progressive kidney disease, and mild hearing loss. Our results show that heterozygous mice with mutations in the myosin motor or filament-forming domain manifest similar hematologic, eye, and kidney phenotypes to humans with MYH9-related disease. (Blood. 2012;119(1): 238-250) IntroductionPoint mutations in MYH9, the gene encoding nonmuscle myosin heavy chain II-A (NMHCII-A), underlie autosomal dominant syndromes in humans (incidence, ϳ 1 in 500 000). 1-3 The human abnormalities manifest as macrothrombocytopenia, granulocyte inclusions, progressive proteinuric renal disease, cataracts, and sensorineural deafness. Most patients have a mild bleeding tendency. Patients may develop early or late onset deafness, cataracts, and progressive glomerulosclerosis, leading to kidney failure. These syndromes, now referred to as MYH9-related diseases (MYH9-RDs), were formerly called May-Hegglin, Fechtner, Sebastian, and Epstein syndromes. 1,4 Each nonmuscle myosin II (NMII) molecule is composed of a pair of heavy chains (M r ϭ 230 000) and 2 pairs of light chains (M r 20 000 and 17 000). NMII has 3 paralogs, NMII-A, NMII-Bm and NMII-C, whose heavy chains are encoded by 3 different genes MYH9, MYH10, and MYH14, respectively, located on 3 different human chromosomes (22, 17, and 19). NMIIs are ubiquitously expressed but differ with respect to localization and expression levels in cells, although they may also overlap with each other. 5 The NMII protein is markedly asymmetric with a globular-shaped motor domain at one end containing the enzymatic activity that hydrolyzes MgATP to convert chemical energy into the mechanical translocation of actin filaments. The other end of the molecule is a long ␣-helical rod that dimerizes the 2 heavy chains and participates in the formation of bipolar filaments, composed of ϳ 28 molecules, 6 which are required for most NMII functions. As a motor prote...

Section: Discussionmentioning

confidence: 99%

Mouse models of MYH9-related disease: mutations in nonmuscle myosin II-A

Zhang

Conti

Malide

et al. 2012

160

187

“…Mice heterozygous for Myh9-null allele do not exhibit giant platelets and thrombocytopenia. 36,37 Evidence also indicates that different MYH9 mutations result in different consequences for the platelet phenotype. Patients with MYH9 mutations in the head domain, especially those at the R702 residue, have significantly larger platelets than those with mutations in the tail domain.…”

Section: Discussionmentioning

confidence: 99%

Differential expression of wild-type and mutant NMMHC-IIA polypeptides in blood cells suggests cell-specific regulation mechanisms in MYH9 disorders

Kunishima

Hamaguchi

Saito

2008

Self Cite

MYH9 disorders such as May-Hegglin anomaly are characterized by macrothrombocytopenia and cytoplasmic granulocyte inclusion bodies that result from mutations in MYH9, the gene for nonmuscle myosin heavy chain-IIA (NMMHC-IIA). We examined the expression of mutant NMMHC-IIA polypeptide in peripheral blood cells from patients with MYH9 5770delG and 5818delG mutations. A specific antibody to mutant NMMHC-IIA (NT629) was raised against the abnormal carboxyl-terminal residues generated by 5818delG. NT629 reacted to recombinant 5818delG NMMHC-IIA but not to wild-type NMMHC-IIA, and did not recognize any cellular components of normal peripheral blood cells. Immunofluorescence and immunoblotting revealed that mutant NMMHC-IIA was present and sequestrated only in inclusion bodies within neutrophils, diffusely distributed throughout lymphocyte cytoplasm, sparsely localized on a diffuse cytoplasmic background in monocytes, and uniformly distributed at diminished levels only in large platelets. Mutant NMMHC-IIA did not translocate to lamellipodia in surface activated platelets. Wild-type NMMHC-IIA was homogeneously distributed among megakaryocytes derived from the peripheral blood CD34 ؉ cells of patients, but coarse mutant NMMHC-IIA was heterogeneously scattered without abnormal aggregates in the cytoplasm. We show the differential expression of mutant NMMHC IntroductionMYH9 disorders are autosomal dominant macrothrombocytopenias characterized by giant platelets, thrombocytopenia, and Döhle body-like inclusion bodies in the cytoplasm of granulocytes. [1][2][3] These disorders are caused by heterozygous mutations in MYH9, which encodes nonmuscle myosin heavy chain-IIA (NMMHC-IIA), and include the May-Hegglin anomaly, Sebastian, Fechtner, and Epstein syndromes, Alport-like syndrome with macrothrombocytopenia, and nonsyndromic deafness (DFNA17). [4][5][6] An MYH9 mutation is always associated with macrothrombocytopenia and granulocyte inclusion bodies from birth, but it does not predict the development of Alport manifestations, including nephritis, deafness, and cataracts. A mutation of MYH9 alone does not seem to cause associated Alport manifestations, and unknown genetic and/or epigenetic factors might influence the phenotypic consequences of MYH9 mutations. [7][8][9][10] Although the molecular mechanisms of hematologic abnormalities are under investigation, those of the kidney, cochlea, and lens are completely unknown.We previously showed that NMMHC-IIA polypeptide accumulates in neutrophil cytoplasm and forms inclusion bodies in patients with MYH9 disorders. The nature of the cytoplasmic accumulation can be classified according to the number, size, and shape of accumulated NMMHC-IIA granules, into types I, II, and III. Type I comprises 1 or 2 large (0.5-2 m), intensely stained, oval-to spindle-shaped cytoplasmic NMMHC-IIA-positive granules. Type II comprises up to 20 circular to oval cytoplasmic spots (Յ 1 m). Type III appears as speckled staining. Furthermore, the pattern of localization correlates with the site of MYH9 ...

Section: Discussionmentioning

confidence: 99%

“…In humans, various mutations in the Myh9 gene that encodes the NMHC IIA cause autosomal dominant disease, whereas in mice the complete deficiency is embryonic lethal but heterozygous mice are nearly normal. 18,45 One hypothesis might be that the degree of inhibition of non-muscle myosin II is more strictly required in mice to observe any effect of this molecule on the enucleation of erythroblasts.Using human erythroblasts, we confirmed that the actin polymerization inhibitor cytochalasin D 5 and the Rac-specific inhibitor NSC23766 6 completely blocked enucleation. Tubulin/kinesin inhibitors blocked cell division of CFU-E but did not inhibit the enucleation, which suggests that the process of erythroblast enucleation is not entirely the same as that of cytokinesis.…”

mentioning

confidence: 99%

Enucleation of human erythroblasts involves non-muscle myosin IIB

Ubukawa

Guo

Takahashi

et al. 2012

Mammalian erythroblasts undergo enucleation, a process thought to be similar to cytokinesis. Although an assemblage of actin, non-muscle myosin II, and several other proteins is crucial for proper cytokinesis, the role of non-muscle myosin II in enucleation remains unclear. In this study, we investigated the effect of various celldivision inhibitors on cytokinesis and enucleation. For this purpose, we used human colony-forming unit-erythroid (CFU-E) and mature erythroblasts generated from purified CD34 ؉ cells as target cells for cytokinesis and enucleation assay, respectively. Here we show that the inhibition of myosin by blebbistatin, an inhibitor of non-muscle myosin II ATPase, blocks both cell division and enucleation, which suggests that non-muscle myosin II plays an essential role not only in cytokinesis but also in enucleation. When the function of non-muscle myosin heavy chain (NMHC) IIA or IIB was inhibited by an exogenous expression of myosin rod fragment, myosin IIA or IIB, each rod fragment blocked the proliferation of CFU-E but only the rod fragment for IIB inhibited the enucleation of mature erythroblasts. These data indicate that NMHC IIB among the isoforms is involved in the enucleation of human erythroblasts. IntroductionDuring erythropoiesis, stem cells undergo lineage specific commitment and generate erythroid progenitor cells through cellular division events including nuclear (mitosis) and cytoplasmic (cytokinesis) division. These progenitor cells consist of immature and mature erythroid progenitors, the burst-forming unit-erythroid (BFU-E) and the colony-forming unit-erythroid (CFU-E), respectively. The BFU-E can be considered as a progenitor of the CFU-E. Indeed, after 6 to 7 days in culture, cells generated from human BFU-E have all the functional characteristics of CFU-E 1 . After an additional 6 to 7 days in culture, human CFU-E proliferate and differentiate into mature erythroblasts. 1 Terminally differentiated erythroblasts in mammals expel their nuclei via a process termed enucleation, becoming reticulocytes and subsequently mature erythrocytes. The nucleus separates from the remainder of the cell and is phagocytosed by reticular cells such as macrophages (for a review, see Chasis et al 2 ).Enucleation of erythroblasts is thought to occur through a process similar to cytokinesis. Several general principles apply to cytokinesis. Firstly, the microtubule cytoskeleton plays an important role in both the choice and positioning of the division site. Once this site is chosen, the local assembly of the actomyosin contractile ring remodels the plasma membrane. Finally, membrane trafficking to, and membrane fusion at the division site result in the physical separation of the daughter cells, a process termed abscission (for reviews, see Barr et al 3 and Glotzer et al 4 ). Although modulation of the actomyosin cytoskeleton is crucial for proper cytokinesis, there is a paucity of information regarding how non-muscle myosin II contributes to enucleation.Several investigations have studied the mol...