Nonmuscle Myosin Heavy Chain IIA Research Articles

MYH9-related disease: May-Hegglin anomaly, Sebastian syndrome, Fechtner syndrome, and Epstein syndrome are not distinct entities but represent a variable expression of a single illness.

May-Hegglin anomaly, Sebastian syndrome, Fechtner syndrome, and Epstein syndrome are autosomal dominant macrothrombocytopenias distinguished by different combinations of clinical and laboratory signs, such as sensorineural hearing loss, cataract, nephritis, and polymorphonuclear Döhle-like bodies. Mutations in the MYH9 gene encoding for the nonmuscle myosin heavy chain IIA (NMMHC-IIA) have been identified in all these syndromes. To understand the role of the MYH9 mutations, we report the molecular defects in 12 new cases, which together with our previous works represent a cohort of 19 families. Since no genotype-phenotype correlation was established, we performed an accurate clinical and biochemical re-evaluation of patients. In addition to macrothrombocytopenia, an abnormal distribution of NMMHC-IIA within leukocytes was observed in all individuals, including those without Döhle-like bodies. Selective, high-tone hearing deficiency and cataract was diagnosed in 83% and 23%, respectively, of patients initially referred as having May-Hegglin anomaly or Sebastian syndrome. Kidney abnormalities, such as hematuria and proteinuria, affected not only patients referred as Fechtner syndrome and Epstein syndrome but also those referred as May-Hegglin anomaly and Sebastian syndrome. These findings allowed us to conclude that May-Hegglin anomaly, Sebastian syndrome, Fechtner syndrome, and Epstein syndrome are not distinct entities but rather a single disorder with a continuous clinical spectrum varying from mild macrothrombocytopenia with leukocyte inclusions to a severe form complicated by hearing loss, cataracts, and renal failure. For this new nosologic entity, we propose the term "MHY9-related disease," which better interprets the recent knowledge in this field and identifies all patients at risk of developing renal, hearing, or visual defects.

Induction of Nonmuscle Myosin Heavy Chain II-C by Butyrate in RAW 264.7 Mouse Macrophages

RAW 264.7 macrophages express nonmuscle myosin heavy chain II-A as the only significant nonmuscle myosin heavy chain isoform, with expression of nonmuscle myosin heavy chain II-B and II-C low or absent. Treatment of the cells with sodium butyrate, an inhibitor of histone deacetylase, led to the dose-dependent induction of nonmuscle myosin heavy chain II-C. Trichostatin A, another inhibitor of histone deacetylase, also induced nonmuscle myosin heavy chain II-C. Induction of nonmuscle myosin heavy chain II-C in response to these histone deacetylase inhibitors was attenuated by mithramycin, an inhibitor of Sp1 binding to GC-rich DNA sequences. Bacterial lipopolysaccharide alone had no effect on basal nonmuscle myosin heavy chain II-C expression, but attenuated butyrate-mediated induction of nonmuscle myosin heavy chain II-C. The effects of lipopolysaccharide were mimicked by the nitric oxide donors sodium nitroprusside and spermine NONOate, suggesting a role for nitric oxide in the lipopolysaccharide-mediated down-regulation of nonmuscle myosin heavy chain II-C induction. This was supported by experiments with the inducible nitric-oxide synthase inhibitor 1400W, which partially blocked the lipopolysaccharide-mediated attenuation of nonmuscle myosin heavy chain induction. 8-Bromo-cGMP had no effect on nonmuscle myosin heavy chain induction, consistent with a cGMP-independent mechanism for nitric oxide-mediated inhibition of nonmuscle myosin heavy chain II-C induction.

Myosin II in retinal pigmented epithelial cells: evidence for an association with membranous vesicles

The goal of this study was to further characterize and identify possible functions for a cytoplasmic myosin II protein which we have isolated from retinal pigmented epithelial (RPE) cells. The nucleotide and deduced amino acid sequences are highly identical to non-muscle myosin heavy chain II-A (NMMHC II-A). However, this RPE myosin displays characteristics that are atypical of other myosins, including an affinity for carbohydrate and a C-terminal sequence extension, suggesting it may have a specialized function. In this study, reverse transcriptase-PCR using isoform-specific primers demonstrated that the RPE myosin and conventional NMMHC II-A have overlapping but distinguishable tissue expression profiles. To gain clues to function, subcellular distribution was determined in motile RPE cells using indirect immunofluorescence. In addition to subtle differences in localization that appeared to further distinguish this molecule from NMMHC II-A, these studies revealed a colocalization with phagocytosed intracellular vesicles. In vitro experiments suggest that the association in situ was not simply coincidental, because isolated vesicles interacted with the protein in cosedimentation assays. Taken together, our observations suggest the RPE myosin exhibits characteristics different from conventional myosin II-A and may function in intracellular vesicle transport.

Cutting Edge: Association of the Motor Protein Nonmuscle Myosin Heavy Chain-IIA with the C Terminus of the Chemokine Receptor CXCR4 in T Lymphocytes

The binding of chemokines to their receptors guides lymphocyte migration. However, the precise mechanism that links the chemotactic signals with the energy and traction force generated by the actomyosin complex of the cell has not been elucidated. Using biochemical approaches and mass spectrometry analysis, we found an association between the C-termini of CXCR4 and CCR5 and the motor protein nonmuscle myosin H chain-IIA. Immunoprecipitation experiments revealed that this association also occurs between the endogenous molecules in T lymphocytes. As expected, myosin L chain was also associated with CXCR4. Confocal microscopy analysis showed that CXCR4 and motor protein nonmuscle myosin H chain-IIA colocalize at the leading edge of migrating T lymphocytes, together with filamentous actin and myosin L chain. These results provide the first evidence of a biochemical association between chemokine receptors and motor proteins, a mechanosignaling mechanism that may have a key role in lymphocyte migration.

Mutations in Human Nonmuscle Myosin IIA Found in Patients with May-Hegglin Anomaly and Fechtner Syndrome Result in Impaired Enzymatic Function

A family of autosomal-dominant diseases including May-Hegglin anomaly, Fechtner syndrome, Sebastian syndrome, Alport syndrome, and Epstein syndrome are commonly characterized by giant platelets and thrombocytopenia. In addition, there may be leukocyte inclusions, deafness, cataracts, and nephritis, depending on the syndrome. Mutations in the human nonmuscle myosin IIA heavy chain gene (MYH9) have been linked to these diseases. Two of the recently described mutations, N93K and R702C, are conserved in smooth and nonmuscle myosins from vertebrates and lie in the head domain of myosin. Interestingly, the two mutations lie within close proximity in the three-dimensional structure of myosin. These two mutations were engineered into a heavy meromyosin-like recombinant fragment of nonmuscle myosin IIA, which was expressed in baculovirus along with the appropriate light chains. The R702C mutant displays 25% of the maximal MgATPase activity of wild type heavy meromyosin and moves actin filaments at half the wild type rate. The effects of the N93K mutation are more dramatic. This heavy meromyosin has only 4% of the maximal MgATPase activity of wild type and does not translocate actin filaments in an in vitro motility assay. Biochemical characterization of the mutant is consistent with this mutant being unable to fully adopt the "on" conformation.

Cloning of the murine non-muscle myosin heavy chain IIA gene ortholog of human MYH9 responsible for May-Hegglin, Sebastian, Fechtner, and Epstein syndromes

Mutations in the non-muscle myosin heavy chain IIA gene ( MYH9) are responsible for May-Hegglin anomaly, Sebastian, Fechtner and Epstein syndromes. These ‘ MYH9-related’ diseases are inherited as an autosomal dominant trait and are characterized by a variable expressivity of clinical features, including macrothrombocytopenia, deafness, nephrites, cataract, and Döhle-like leukocyte inclusions. To gain information of the function of the non-muscle myosin heavy chain IIA protein (NMMHC-IIA), we have identified the murine orthologue Myh9 gene. The gene is localized in a region of chromosome 15 and encodes a predicted protein of 1960 amino acids. This protein shows a high homology to the human NMMHC-IIA with 98% identity. The Myh9 exon-intron junctions were deduced from a murine genomic clone that revealed a perfect conservation of the exon structure between the human and mouse gene. Myh9 is expressed in liver, kidney, lung, and spleen. A low level of transcripts was detected also in heart and brain while no expression was revealed in skeletal muscle and testis. In vertebrates, NMMHC-IIA shows a striking degree of homology to NMMHC-IIB, which is expressed at higher level in mouse brain and testis than in other tissues, confirming the hypothesis that the two non-muscle myosins have different functional roles within cells.

Expression of the nonmuscle myosin heavy chain IIA in the human kidney and screening for MYH9 mutations in Epstein and Fechtner syndromes.

Mutations in the MYH9 gene, which encodes the nonmuscle myosin heavy chain IIA, have been recently reported in three syndromes that share the association of macrothrombocytopenia (MTCP) and leukocyte inclusions: the May-Hegglin anomaly and Sebastian and Fechtner syndromes. Epstein syndrome, which associates inherited sensorineural deafness, glomerular nephritis, and MTCP without leukocyte inclusions, was shown to be genetically linked to the same locus at 22q12.3 to 13. The expression of MYH9 in the fetal and mature human kidney was studied, and the 40 coding exons of the gene were screened by single-strand conformation polymorphism in 12 families presenting with the association of MTCP and nephropathy. MYH9 is expressed in both fetal and mature kidney. During renal development, it is expressed in the late S-shaped body, mostly in its lower part, in the endothelial and the epithelial cell layers. Later, as well as in mature renal tissue, MYH9 is widely expressed in the kidney, mainly in the glomerulus and peritubular vessels. Within the glomerulus, MYH9 mRNA and protein are mostly expressed in the epithelial visceral cells. Four missense heterozygous mutations that are thought to be pathogenic were found in five families, including two families with Epstein syndrome. Three mutations were located in the coiled-coil rod domain of the protein, and one was in the motor domain. Two mutations (E1841K and D1424N) have been reported elsewhere in families with May-Hegglin anomaly. The two others (R1165L and S96L) are new mutations, although one of them affects a codon (R1165), found elsewhere to be mutated in Sebastian syndrome.

Nonmuscle Myosin Heavy Chain IIA Mutations Define a Spectrum of Autosomal Dominant Macrothrombocytopenias: May-Hegglin Anomaly and Fechtner, Sebastian, Epstein, and Alport-Like Syndromes

May-Hegglin anomaly (MHA) and Fechtner (FTNS) and Sebastian (SBS) syndromes are autosomal dominant platelet disorders that share macrothrombocytopenia and characteristic leukocyte inclusions. FTNS has the additional clinical features of nephritis, deafness, and cataracts. Previously, mutations in the nonmuscle myosin heavy chain 9 gene (MYH9), which encodes nonmuscle myosin heavy chain IIA (MYHIIA), were identified in all three disorders. The spectrum of mutations and the genotype-phenotype and structure-function relationships in a large cohort of affected individuals (n=27) has now been examined. Moreover, it is demonstrated that MYH9 mutations also result in two other FTNS-like macrothrombocytopenia syndromes: Epstein syndrome (EPS) and Alport syndrome with macrothrombocytopenia (APSM). In all five disorders, MYH9 mutations were identified in 20/27 (74%) affected individuals. Four mutations, R702C, D1424N, E1841K, and R1933X, were most frequent. R702C and R702H mutations were only associated with FTNS, EPS, or APSM, thus defining a region of MYHIIA critical in the combined pathogenesis of macrothrombocytopenia, nephritis, and deafness. The E1841K, D1424N, and R1933X coiled-coil domain mutations were common to both MHA and FTNS. Haplotype analysis using three novel microsatellite markers revealed that three E1841K carriers--one with MHA and two with FTNS--shared a common haplotype around the MYH9 gene, suggesting a common ancestor. The two new globular-head mutations, K371N and R702H, as well as the recently identified MYH9 mutation, R705H, which results in DFNA17, were modeled on the basis of X-ray crystallographic data. Altogether, our data suggest that MHA, SBS, FTNS, EPS, and APSM comprise a phenotypic spectrum of disorders, all caused by MYH9 mutations. On the basis of our genetic analyses, the name "MYHIIA syndrome" is proposed to encompass all of these disorders.

The Tip of the Coiled-coil Rod Determines the Filament Formation of Smooth Muscle and Nonmuscle Myosin

Myosin II self-assembles to form thick filaments that are attributed to its long coiled-coil tail domain. The present study has determined a region critical for filament formation of vertebrate smooth muscle and nonmuscle myosin II. A monoclonal antibody recognizing the 28 residues from the C-terminal end of the coiled-coil domain of smooth muscle myosin II completely inhibited filament formation, whereas other antibodies recognizing other parts of the coiled-coil did not. To determine the importance of this region in the filament assembly in vivo, green fluorescent protein (GFP)-tagged smooth muscle myosin was expressed in COS-7 cells, and the filamentous localization of the GFP signal was monitored by fluorescence microscopy. Wild type GFP-tagged smooth muscle myosin colocalized with F-actin during interphase and was also recruited into the contractile ring during cytokinesis. Myosin with the nonhelical tail piece deleted showed similar behavior, whereas deletion of the 28 residues at the C-terminal end of the coiled-coil domain abolished this localization. Deletion of the corresponding region of GFP-tagged nonmuscle myosin IIA also abolished this localization. We conclude that the C-terminal end of the coiled-coil domain, but not the nonhelical tail piece, of myosin II is critical for myosin filament formation both in vitro and in vivo.

Heterogeneity of bladder myocytes in vitro: modulation of myosin isoform expression

We studied the expression of myosin heavy chain isoforms differing at the N-terminal (SM-A, SM-B) and the C-terminal (SM1, SM2) regions and non-muscle myosin heavy chain II-A and II-B (NMMHC II-A and B) in newborn and adult rabbit bladder smooth muscle cells (SMCs) and in cultures of enzymatically dissociated neonatal detrusor. RT-PCR analyses revealed that 94.5 ± 3.27% of MHC transcripts of the adult bladder SMCs contained the 21-nucleotide insert (SM-B) compared with 83.8 ± 3.2% in the newborn bladder, with the remainder of the mRNA being non-inserted (SM-A). In 3, 7, and 10 days of primary culture (proliferating, confluent, and post-confluent, respectively) and up to 4 subculture passages, bladder myocytes expressed predominantly SM-A. Immunofluorescence microscopy revealed heterogeneity in cultured myocytes, i.e. SM-B positive cells coexisting with negatively stained cells. In adult bladder, the C-terminal isoforms SM1 and SM2 represented, 43.1 ± 4.3% and 56.89 ± 4.3% of the mRNA, respectively, while newborn bladders expressed 72.5 ± 7% SM1 and 27.5 ± 7% SM2. Upon culturing, cells predominantly expressed SM1 at both the mRNA and protein levels. NMMHC II-A was expressed by both adult and newborn bladders and in culture, whereas NMMHC II-B was expressed at low levels only in newborn bladders, but upregulated in culture. These data indicate that bladder myocytes in vitro undergo modulation with relative overexpression of SM-A and SM1 and upregulation of NMMHC II-B. Information on the mechanisms responsible for this modulation in vitro might provide an understanding of the nature of altered myosin isoform expression associated with smooth muscle dysfunction in certain bladder diseases.

Calcium-dependent Threonine Phosphorylation of Nonmuscle Myosin in Stimulated RBL-2H3 Mast Cells

Stimulation of RBL-2H3 m1 mast cells through the IgE receptor with antigen, or through a G protein-coupled receptor with carbachol, leads to the rapid appearance of phosphothreonine in nonmuscle myosin heavy chain II-A (NMHC-IIA). We demonstrate that this results from phosphorylation of Thr-1940 by calcium/calmodulin-dependent protein kinase II (CaM kinase II), activated by increased intracellular calcium. The phosphorylation site in rodent NMHC-IIA was localized to the carboxyl terminus of NMHC-IIA distal to the coiled-coil region, and identified as Thr-1940 by site-directed mutagenesis. A fusion protein containing the NMHC-IIA carboxyl terminus was phosphorylated by CaM kinase II in vitro, while mutation of Thr-1940 to Ala eliminated phosphorylation. In contrast to rodents, in humans Thr-1940 is replaced by Ala, and human NMHC-IIA fusion protein was not phosphorylated by CaM kinase II unless Ala-1940 was mutated to Thr. Similarly, co-transfected Ala --> Thr-1940 human NMHC-IIA was phosphorylated by activated CaM kinase II in HeLa cells, while wild type was not. In RBL-2H3 m1 cells, inhibition of CaM kinase II decreased Thr-1940 phosphorylation, and inhibited release of the secretory granule marker hexosaminidase in response to carbachol but not to antigen. These data indicate a role for CaM kinase stimulation and resultant threonine phosphorylation of NMHC-IIA in RBL-2H3 m1 cell activation.

Conditional expression of a truncated fragment of nonmuscle myosin II-A alters cell shape but not cytokinesis in HeLa cells.

A truncated fragment of the nonmuscle myosin II-A heavy chain (NMHC II-A) lacking amino acids 1-591, delta N592, was used to examine the cellular functions of this protein. Green fluorescent protein (GFP) was fused to the amino terminus of full-length human NMHC II-A, NMHC II-B, and delta N592 and the fusion proteins were stably expressed in HeLa cells by using a conditional expression system requiring absence of doxycycline. The HeLa cell line studied normally expressed only NMHC II-A and not NMHC II-B protein. Confocal microscopy indicated that the GFP fusion proteins of full-length NMHC II-A, II-B, and delta N592 were localized to stress fibers. However, in vitro assays showed that baculovirus-expressed delta N592 did not bind to actin, suggesting that delta N592 was localized to actin stress fibers through incorporation into endogenous myosin filaments. There was no evidence for the formation of heterodimers between the full-length endogenous nonmuscle myosin and truncated nonmuscle MHCs. Expression of delta N592, but not full-length NMHC II-A or NMHC II-B, induced cell rounding with rearrangement of actin filaments and disappearance of focal adhesions. These cells returned to their normal morphology when expression of delta N592 was repressed by addition of doxycycline. We also show that GFP-tagged full-length NMHC II-A or II-B, but not delta N592, were localized to the cytokinetic ring during mitosis, indicating that, in vertebrates, the amino-terminus part of mammalian nonmuscle myosin II may be necessary for localization to the cytokinetic ring.