Background: Three genes encode human nonmuscle myosin II (NM II) heavy chains, and the proteins have different intracellular roles and localizations. Results: NM II paralogs form bipolar filaments, but there are important differences in filament structure, enzymatic, and actin binding behavior. Conclusion: NM II filaments show diverse interactions with actin. Significance: NM II filaments are adapted to work in cytoskeletal networks.

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...

During vertebrate cytokinesis it is thought that contractile ring constriction is driven by nonmuscle myosin II (NM II) translocation of antiparallel actin filaments. Here we report in situ, in vitro, and in vivo observations that challenge this hypothesis. Graded knockdown of NM II in cultured COS-7 cells reveals that the amount of NM II limits ring constriction. Restoration of the constriction rate with motor-impaired NM II mutants shows that the ability of NM II to translocate actin is not required for cytokinesis. Blebbistatin inhibition of cytokinesis indicates the importance of myosin strongly binding to actin and exerting tension during cytokinesis. This role is substantiated by transient kinetic experiments showing that the load-dependent mechanochemical properties of mutant NM II support efficient tension maintenance despite the inability to translocate actin. Under loaded conditions, mutant NM II exhibits a prolonged actin attachment in which a single mechanoenzymatic cycle spans most of the time of cytokinesis. This prolonged attachment promotes simultaneous binding of NM II heads to actin, thereby increasing tension and resisting expansion of the ring. The detachment of mutant NM II heads from actin is enhanced by assisting loads, which prevent mutant NM II from hampering furrow ingression during cytokinesis. In the 3D context of mouse hearts, mutant NM II-B R709C that cannot translocate actin filaments can rescue multinucleation in NM II-B ablated cardiomyocytes. We propose that the major roles of NM II in vertebrate cell cytokinesis are to bind and cross-link actin filaments and to exert tension on actin during contractile ring constriction.cortical tension | myosin II kinetics | graded myosin knockdown | motor-impaired myosin | myosin-actin binding I n vertebrate cells, nonmuscle myosin II (NM II) and cytoplasmic actin are the two major contractile proteins mediating cytokinesis, the process by which one cell divides into two. The three NM II paralogs (II-A, II-B, and II-C) are composed of catalytically active heads, which bind to and translocate actin in an ATP-dependent manner, and bipolar filament-forming rods (1). Current concepts of vertebrate cytokinesis favor the notion of translocation of actin filaments by NM II motors to effect cell division (2-6). Translocation is linked to the hydrolysis of MgATP, resulting in conversion of chemical energy into mechanical movement. In cytokinesis, this movement was thought to be manifested by the sliding of actin filaments by bipolar filaments of NM II to form a narrow contractile ring in the cell's center that ultimately pinches closed to form two new cells.Previous experiments demonstrated that the three paralogs of vertebrate NM II differ in their heavy chains (NMHCs), which are the products of three different genes, but share the same two pairs of light chains (1). Each NM II has a unique enzymatic activity (rate of MgATP hydrolysis) and duty ratio (the fraction of time that the myosin molecule remains bound to actin during a contractile cycle). Pr...