Chain Of Non-muscle Myosin IIA Research Articles

A Novel Frameshift Mutation in a Case of Familial May-Hegglin Anomaly.

The May-Hegglin anomaly is a rare, autosomal dominant condition that is characterised by thrombocytopaenia, anisocytosis, and Dohlë-like inclusion bodies in leukocytes. The disorder generally results in a mild bleeding tendency. May-Hegglin anomaly results from defects in the MYH9 gene, which encodes the heavy chain of nonmuscle myosin IIA. The gene maps to chromosome 22q12.3-q13.2, comprises 40 exons, and produces a 5.8kb mRNA transcript.We present a case of familial May-Hegglin anomaly. This kindred was first identified, and published, in 1964; but no further analyses were carried out until recently.The proband, now aged 80 years, has a life long history of easy bruising and epistaxis, although the symptoms have improved with increasing age. Her sister (deceased) and daughter reported similar symptoms: the daughter requiring splenectomy for thrombocytopaenia at age 9 years. The proband has also suffered from deafness for in excess of 60 years.Recent investigations of the proband revealed a mild thrombocytopaenia, and the characteristic blood film of May -Hegglin anomaly. Platelet and neutrophil morphology were also consistent with the disorder. Karyotype analysis revealed good quality G banding throughout chromosome 22, and no gross alteration in the region 22q11.2., 22q13. Direct sequencing of the MYH9 gene identified a heterozygous deletion of a single base in a run of Gs at nucleotide positions 5818–5821 (codons 1940–1941). This frame shift mutation leads to the creation of a premature termination sequence at codon 1947, and the loss of the last fourteen amino acids. The deletion introduces a restriction site, and endonuclease digestion of a panel of normals failed to detect this change. Together with the fact that this is a conserved region across species (human, rat, rabbit, chicken), it is probable that this deletion is the causative mutation in this kindred.This novel mutation represents the second reported defect affecting the tail-piece domain in May-Hegglin anomaly. The protein's two heavy chains homodimerise in this region to form a tail, and multiple polypeptides associate through this tail domain to form bipolar filaments. Therefore, a mutation in this domain may exert its effect by the inhibition of myosin filament assembly.To date, approximately 20 different mutations have been described in the MYH9 gene giving rise to not only May-Hegglin anomaly, but also the similar disorders of Fechtner syndrome, Sebastian syndrome and Epstein syndrome. In fact some mutations have been identified in patients with different diagnoses. The proband presented here has a long standing diagnosis of May-Hegglin anomaly, but does exhibit one symptom (deafness) more usually associated with Fechtner syndrome. The genetic, phenotypic and clinical overlap in these disorders suggest that these actually represent allelic variations of a single genetic disorder, which should more correctly be termed ‘MYHIIA syndrome'.

Genetics, clinical and pathological features of glomerulonephrites associated with mutations of nonmuscle myosin IIA (Fechtner syndrome)

Background: Fechtner syndrome (FTNS), also known as Alport-like syndrome, is a rare inherited condition characterized by progressive nephritis, macrothrombocytopenia, Döhle-like leukocyte inclusions, deafness, and cataract. Although it recently was shown that FTNS derives from mutation of MYH9, the gene for the heavy chain of nonmuscle myosin IIA (NMMHC-IIA), its pathophysiological characteristics remain unknown. Methods: We studied a large FTNS family in which 10 components carried a missense mutation of MYH9 determining the D1424H substitution. Results: All affected subjects presented with macrothrombocytopenia and leukocyte Döhle-like bodies consisting of macroaggregates of NMMHC-IIA, but only two subjects had major renal problems characterized by proteinuria and renal failure. Electron microscopy showed focal and segmental effacement of podocytes and loss of the interpodocyte slit diaphragm. Immunohistochemistry showed apical localization of NMMHC-IIA in tubular epithelia and less podocyte staining in the two patients, whereas it was diffuse in normal epithelia. Three patients presented with stable microhematuria, and another five patients had no renal lesions, although they carried the same mutation of MYH9. Therefore, MYH9 mutation per se was responsible for platelet and leukocyte abnormalities, whereas additional predisposing conditions and/or environmental factors are necessary for nephropathy, cataract, and deafness. Looking at podocyte components conferring permselectivity properties to the kidney, we characterized the haplotype of podocin and found cosegregation of one specific allele in the two patients with nephrotic syndrome, suggesting a relationship between podocin features and proteinuria. Conclusion: Our study indicates a major role for the NMMHC-IIA abnormality in the pathogenesis of leukocyte, platelet, and kidney defects in FTNS. The basic feature in all cases is aggregation and compartmentation of NMMHC-IIA. However, proteinuria and podocyte lesions are the hallmark of nephropathy in patients who develop renal failure, and podocin may have some function in this setting. Am J Kidney Dis 41:95-104. © 2003 by the National Kidney Foundation, Inc.

Immunocytochemistry for the heavy chain of the non-muscle myosin IIA as a diagnostic tool for MYH9-related disorders.

May-Hegglin anomaly (MHA), Sebastian syndrome (SBS) and Fechtner syndrome (FTNS) are autosomal-dominant macrothrombocytopenias with Döhle-like leucocyte inclusions. These diseases are due to mutations of the MHY9 gene, encoding the heavy chain of non-muscle myosin IIA (NMMHC-A). We investigated the NMMHC-A localization in blood cells from eight MHA, SBS or FTNS patients with known MYH9 mutations. All the patients showed an altered localization of NMMHC-A in granulocytes and platelets, suggesting that Döhle-like bodies are due to the aggregation of NMMHC-A in the cytoplasm. Therefore, immunocytochemistry for NMMHC-A is a simple and sensitive method to detect pathological phenotypes of granulocytes and platelets in the diagnosis of MYH9-related disorders.

Specific Isoforms of Actin-binding Proteins on Distinct Populations of Golgi-derived Vesicles

Golgi membranes and Golgi-derived vesicles are associated with multiple cytoskeletal proteins and motors, the diversity and distribution of which have not yet been defined. Carrier vesicles were separated from Golgi membranes, using an in vitro budding assay, and different populations of vesicles were separated using sucrose density gradients. Three main populations of vesicles labeled with beta-COP, gamma-adaptin, or p200/myosin II were separated and analyzed for the presence of actin/actin-binding proteins. beta-Actin was bound to Golgi cisternae and to all populations of newly budded vesicles. Centractin was selectively associated with vesicles co-distributing with beta-COP-vesicles, while p200/myosin II (non-muscle myosin IIA) and non-muscle myosin IIB were found on different vesicle populations. Isoforms of the Tm5 tropomyosins were found on selected Golgi-derived vesicles, while other Tm isoforms did not colocalize with Tm5 indicating the association of specialized actin filaments with Golgi-derived vesicles. Golgi-derived vesicles were shown to bind to F-actin polymerized from cytosol with Jasplakinolide. Thus, newly budded, coated vesicles derived from Golgi membranes can bind to actin and are customized for differential interactions with microfilaments by the presence of selective arrays of actin-binding proteins.

Heparin rapidly and selectively regulates protein tyrosine phosphorylation in vascular smooth muscle cells.

Aberrant vascular smooth muscle cell (VSMC) hyperplasia is the hallmark of atherosclerosis and restenosis seen after vascular surgery. Heparin inhibits VSMC proliferation in animal models and in cell culture. To test our hypothesis that heparin mediates its antiproliferative effect by altering phosphorylation of key mitogenic signaling proteins in VSMC, we examined tyrosine phosphorylation of cellular proteins in quiescent VSMC stimulated with serum in the presence or absence of heparin. Western blot analysis with anti-phosphotyrosine antibodies shows that heparin specifically alters the tyrosine phosphorylation of only two proteins (42 kDa and 200 kDa). The 200 kDa protein (p200) is dephosphorylated within 2.5 min after heparin treatment with an IC50 that closely parallels the IC50 for growth inhibition. Studies using the tyrosine phosphatase inhibitor, sodium orthovanadate, indicate that heparin blocks p200 phosphorylation by inhibiting a kinase. Phosphorylation of p200 is not altered in heparin-resistant cells, supporting a role for p200 in mediating the antiproliferative effect of heparin. Purification and sequence analysis indicate that p200 exhibits very high homology to the heavy chain of nonmuscle myosin IIA. The 42 kDa protein, identified as mitogen activated protein kinase (MAPK), undergoes dephosphorylation within 15 min after heparin treatment, and this effect is also not seen in heparin-resistant cells. The identification of only two heparin-regulated tyrosine phosphoproteins suggests that they may be key mediators of the antiproliferative effect of heparin.

Myosin II is associated with Golgi membranes: identification of p200 as nonmuscle myosin II on Golgi-derived vesicles.

A variety of peripheral membrane proteins associate dynamically with Golgi membranes during the budding and trafficking of transport vesicles in eukaryotic cells. A monoclonal antibody (AD7) raised against Golgi membranes recognizes a peripheral membrane protein, p200, which associates with vesicles budding off the trans-Golgi network (TGN). Based on preliminary findings, a potential association between p200 and myosin on Golgi membranes was investigated. Immunofluorescence staining of cultured cells under a variety of fixation conditions was carried out using an antibody raised against chick brush border nonmuscle myosin II. We show that, in addition to being found in the cytoplasm or associated with stress fibres, nonmuscle myosin II is also specifically localized on Golgi membranes. Myosin II was also detected on Golgi membranes by immunoblotting and by immunogold labeling at the electron microscopy level where it was found to be concentrated on Golgi-derived vesicles. The association of myosin II with Golgi membranes is dynamic and was found to be enhanced following activation of G proteins. Myosin II staining of Golgi membranes was also disrupted by brefeldin A (BFA). Colocalization of the AD7 and myosin II antibodies at the light and electron microscopy levels led us to investigate the nature of the 200 kDa protein recognized by both antibodies. The 200 kDa protein immunoprecipiated by the AD7 antibody was isolated from MDCK cells and used for microsequencing. Amino acid sequence data enabled us to identify p200 as the heavy chain of nonmuscle myosin IIA. In addition, an extra protein (240 kDa) recognized by the AD7 antibody specifically in extracts of HeLa cells, was sequenced and identified as another actin-binding protein, filamin. These results show that nonmuscle myosin II is associated with Golgi membranes and that the vesicle-associated protein p200, is itself a heavy chain of myosin II.