Abundant expression of myosin heavy-chain IIB RNA in a subset of human masseter muscle fibres.
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Type IIB fast fibres are typically demonstrated in human skeletal muscle by histochemical staining for the ATPase activity of myosin heavy-chain (MyHC) isoforms. However, the monoclonal antibody specific for the mammalian IIB isoform does not detect MyHC IIB protein in man and MyHC IIX RNA is found in histochemically identified IIB fibres, suggesting that the IIB protein isoform may not be present in man; if this is not so, jaw-closing muscles, which express a diversity of isoforms, are likely candidates for their presence. ATPase histochemistry, immunohistochemistry polyacrylamide gel electrophoresis and in situ hybridization, which included a MyHC IIB-specific mRNA riboprobe, were used to compare the composition and RNA expression of MyHC isoforms in a human jaw-closing muscle, the masseter, an upper limb muscle, the triceps, an abdominal muscle, the external oblique, and a lower limb muscle, the gastrocnemius. The external oblique contained a mixture of histochemically defined type I, IIA and IIB fibres distributed in a mosaic pattern, while the triceps and gastrocnemius contained only type I and IIA fibres. Typical of limb muscle fibres, the MyHC I-specific mRNA probes hybridized with histochemically defined type I fibres, the IIA-specific probes with type IIA fibres and the IIX-specific probes with type IIB fibres. The MyHC IIB mRNA probe hybridized only with a few histochemically defined type I fibres in the sample from the external oblique; in addition to this IIB message, these fibres also expressed RNAs for MyHC I, IIA and IIX. MyHC IIB RNA was abundantly expressed in histochemical and immunohistochemical type IIA fibres of the masseter, together with transcripts for IIA and in some cases IIX. No MyHC IIB protein was detected in fibres and extracts of either the external oblique or masseter by immunohistochemistry, immunoblotting and electrophoresis. Thus, IIB RNA, but not protein, was found in the fibres of two different human skeletal muscles. It is believed this is the first report of the substantial expression of IIB mRNA in man as demonstrated in a subset of masseter fibres, but rarely in limb muscle, and in only a few fibres of the external oblique. These findings provide further evidence for the complexity of myosin gene expression, especially in jaw-closing muscles. (+info)
Nonmuscle myosin heavy chain IIA mutations define a spectrum of autosomal dominant macrothrombocytopenias: May-Hegglin anomaly and Fechtner, Sebastian, Epstein, and Alport-like syndromes.
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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. (+info)
Modulation of myosin A expression by a newly established tetracycline repressor-based inducible system in Toxoplasma gondii.
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We have developed a control system for regulating gene activation in Toxoplasma gondii. The elements of this system are derived from the Escherichia coli tetracycline resistance operon, which has been widely used to tightly control gene expression in eukaryotes. The tetracycline repressor (tetR) interferes with transcription initiation while the chimeric transactivator, composed of the tetR fused to the activating domain of VP16 transcriptional factor, allows tet-dependent transcription. Accordingly, tetracycline derivatives such as anhydrotetracycline, which we found to be well tolerated by T.gondii, can serve as effector molecules, allowing control of gene expression in a reversible manner. As a prerequisite to functionally express the tetR in T.gondii, we used a synthetic gene with change of codon frequency. Whereas no activation of transcription was achieved using the synthetic tetracycline-controlled transactivator, tTA2(s), the TetR(s )modulates parasite transcription over a range of approximately 15-fold as measured for several reporter genes. We show here that the tetR-dependent induction of the T.gondii myosin A transgene expression drastically down-regulates the level of endogenous MyoA. This myosin is under the control of a tight feedback mechanism, which occurs at the protein level. (+info)
Differential localization of non-muscle myosin II isoforms and phosphorylated regulatory light chains in human MRC-5 fibroblasts.
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We investigated the localization of non-muscle myosin II isoforms and mono- (at serine 19) and diphosphorylated (at serine 19 and threonine 18) regulatory light chains (RLCs) in motile and non-motile MRC-5 fibroblasts. In migrating cells, myosin IIA localized to the lamella and throughout the posterior region. Myosin IIB colocalized with myosin IIA to the posterior region except at the very end. Diphosphorylated RLCs were detected in the restricted region where myosin IIA was enriched. In non-motile cells, myosin IIA was enriched in peripheral stress fibers with diphosphorylated RLCs, but myosin IIB was not. Our results suggest that myosin IIA may be highly activated by diphosphorylation of RLCs and primarily involved in cell migration. (+info)
Evidence of a role for nonmuscle myosin II in herpes simplex virus type 1 egress.
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After cell entry, herpes simplex virus (HSV) particles are transported through the host cell cytoplasm to nuclear pores. Following replication, newly synthesized virus particles are transported back to the cell periphery via a complex pathway including a cytoplasmic phase involving some form of unenveloped particle. These various transport processes are likely to make use of one or more components of the cellular cytoskeletal systems and associated motor proteins. Here we report that the HSV type 1 (HSV-1) major tegument protein, VP22, interacts with the actin-associated motor protein nonmuscle myosin IIA (NMIIA). HSV-1 infection resulted in reorganization of NMIIA, inducing retraction of NMIIA from the cell periphery and condensation into a spoke-like distribution around the nucleus along with a second effect of accumulation in a perinuclear cluster. VP22 did not appear to colocalize with the reorganized cagelike distribution of NMIIA. However, VP22 has been previously reported to localize in a perinuclear vesicular pattern, and significant overlap was observed between this pattern and the perinuclear clusters of NMIIA. Inhibition of the ATPase activity of NMIIA with the myosin-specific inhibitor butanedione monoxime impaired the formation of the perinuclear vesicular VP22 accumulations and also the release of virus into the extracellular medium while having much less effect on the yield of cell-associated virus. Virus infection frequently results in the induction of highly extended processes emanating from the infected cell, and we observed that VP22-containing particles line up along NMIIA-containing filaments which run through these protrusions. (+info)
Toxoplasma gondii myosin A and its light chain: a fast, single-headed, plus-end-directed motor.
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Successful host cell invasion is a prerequisite for survival of the obligate intracellular apicomplexan parasites and establishment of infection. Toxoplasma gondii penetrates host cells by an active process involving its own actomyosin system and which is distinct from induced phagocytosis. Toxoplasma gondii myosin A (TgMyoA) is presumed to achieve power gliding motion and host cell penetration by the capping of apically released adhesins towards the rear of the parasite. We report here an extensive biochemical characterization of the functional TgMyoA motor complex. TgMyoA is anchored at the plasma membrane and binds a novel type of myosin light chain (TgMLC1). Despite some unusual features, the kinetic and mechanical properties of TgMyoA are unexpectedly similar to those of fast skeletal muscle myosins. Microneedle-laser trap and sliding velocity assays established that TgMyoA moves in unitary steps of 5.3 nm with a velocity of 5.2 microm/s towards the plus end of actin filaments. TgMyoA is the first fast, single-headed myosin and fulfils all the requirements for power parasite gliding. (+info)
Defective expression of GPIb/IX/V complex in platelets from patients with May-Hegglin anomaly and Sebastian syndrome.
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BACKGROUND AND OBJECTIVES: May-Hegglin anomaly (MHA) and Sebastian syndrome (SBS) are inherited macrothrombocytopenias with D hle-like bodies in leukocytes. MHA-SBS are due to mutations of the gene (MYH9) for the heavy chain of non-muscle myosin IIA (NMMHC-IIA), the only myosin II expressed in platelets. The bleeding tendency is often more severe than expected on the basis of platelet count, but no abnormality of platelet function has been identified. To characterize platelet abnormalities deriving from MYH9 mutations better, we studied surface glycoproteins (GPs) in platelets from MHA-SBS patients. DESIGN AND METHODS: Eight patients from 4 unrelated families were studied. Platelet surface GPs were studied by flow cytometry in both the whole platelet population and subpopulations of platelets identified according to their size. RESULTS: Flow cytometry identified a defect of the GPIb/IX/V complex in the whole platelet population in 7 of 8 patients. Moreover, in all patients the subpopulation of large platelets had defective expression of this complex. INTERPRETATION AND CONCLUSIONS: These findings indicate that MYH9 mutations may be responsible for reduced surface expression of GPIb/IX/V. This defect could contribute to the bleeding tendency of these patients. The identification of a GPIb/IX/V defect in MHA-SBS platelets raises the question of the differential diagnosis from heterozygous Bernard-Soulier syndrome. (+info)
Mutations in human nonmuscle myosin IIA found in patients with May-Hegglin anomaly and Fechtner syndrome result in impaired enzymatic function.
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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. (+info)