Myosin Va Gene Research Articles

Knockdown of microRNA‑143‑5p by STTM technology affects eumelanin and pheomelanin production in melanocytes.

MicroRNAs (miRNAs) serve various roles in the regulation of melanogenesis in mammalian melanocytes that contribute to the development of hair color. The manipulation of the melanocyte action is a new target for genetic improvement. Short tandem target mimic (STTM) is a potent approach for silencing miRNAs in plants and animals. To investigate the function of miR-143-5p in melanogenesis, STTM was used to block the expression of miR-143-5p (STTM-miR-143-5p). The molecular analysis and luciferase reporter assay identified myosin Va gene (MYO5A) as one of the miR-143-5p targets. STTM-miR-143-5p overexpression resulted in an increased expression of downstream melanogenesis genes including microphthalmia-associated transcription factor (MITF), tyrosinase family members [tyrosinase (TYR) and tyrosinase-related protein 1 (TYRP1)], melanophilin (MLPH), and Rab27a, thereby contributing to melanocyte pigmentation by promoting total alkali-soluble melanogenesis (ASM) and eumelanin (EM) contents; conversely, STTM-miR-143-5p overexpression resulted in decreased expression of the tyrosinase-related protein 2 (TYRP2)/dopachrome tautomerase (DCT), which is responsible for decreased pheomelanin (PM) content in mouse melanocytes. The results indicated that melanin production in melanocytes could be increased by manipulating miR-143-5p expression using STTM which resulted in ASM and EM production.

A role for myosin Va in cerebellar plasticity and motor learning: a possible mechanism underlying neurological disorder in myosin Va disease.

Mutations of the myosin Va gene cause the neurological diseases Griscelli syndrome type 1 and Elejalde syndrome in humans and dilute phenotypes in rodents. To understand the pathophysiological mechanisms underlying the neurological disorders in myosin Va diseases, we conducted an integrated analysis at the molecular, cellular, electrophysiological, and behavioral levels using the dilute-neurological (d-n) mouse mutant. These mice manifest an ataxic gait and clonic seizures during postnatal development, but the neurological disorders are ameliorated in adulthood. We found that smooth endoplasmic reticulum (SER) rarely extended into the dendritic spines of Purkinje cells (PCs) of young d-n mice, and there were few, if any, IP(3) receptors. Moreover, long-term depression (LTD) at parallel fiber-PC synapses was abolished, consistent with our previous observations in juvenile lethal dilute mutants. Young d-n mice exhibited severe impairment of cerebellum-dependent motor learning. In contrast, adult d-n mice showed restoration of motor learning and LTD, and these neurological changes were associated with accumulation of SER and IP(3) receptors in some PC spines and the expression of myosin Va proteins in the PCs. RNA interference-mediated repression of myosin Va caused a reduction in the number of IP(3) receptor-positive spines in cultured PCs. These findings indicate that myosin Va function is critical for subsequent processes in localization of SER and IP(3) receptors in PC spines, LTD, and motor learning. Interestingly, d-n mice had defects of motor coordination from young to adult ages, suggesting that the role of myosin Va in PC spines is not sufficient for motor coordination.

Involvement of Myosin Vb in Glutamate Receptor Trafficking

Myosin V motors mediate cargo transport; however, the identity of neuronal molecules transported by these proteins remains unknown. Here we show that myosin Vb is expressed in several neuronal populations and associates with the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-type glutamate receptor subunit GluR1. In developing hippocampal neurons, expression of the tail domain of myosin Vb, but not myosin Va, enhanced GluR1 accumulation in the soma and reduced its surface expression. These changes were accompanied by reduced GluR1 clustering and diminished frequency of excitatory but not inhibitory synaptic currents. Similar effects were observed upon expression of full-length myosin Vb lacking a C-terminal region required for binding to the small GTPase Rab11. In contrast, mutant myosin Vb did not change the localization of several other neurotransmitter receptors, including the glutamate receptor subunit NR1. These results reveal a novel mechanism for the transport of a specific glutamate receptor subunit in neurons mediated by a member of the myosin V family.

Missense mutations in the globular tail of myosin-Va in dilute mice partially impair binding of Slac2-a/melanophilin.

The well-known coat-color mutant mouse dilute exhibits a defect in melanosome transport, and although various mutations in the myosin-Va gene, which encodes an actin-based motor protein, have been identified in dilute mice, why missense mutations in the globular tail of myosin-Va, a putative cargo-binding site, cause the dilute phenotype (i.e. lighter coat color) has never been elucidated. In this study we discovered that missense mutations (I1510N, M1513K and D1519G) in the globular tail (GT) of myosin-Va partially impair the binding of Slac2-a/melanophilin, a linker protein between myosin-Va and Rab27A on the melanosome. The myosin-Va-GT-binding site in Slac2-a was mapped to the region (amino acids 147-240) adjacent to the N-terminal Rab27A-binding site, but it is distinct from the myosin-Va-exon-F-binding site (amino acids 320-406). The myosin-Va-GT.Slac2-a interaction was much weaker than the myosin-Va-exon-F.Slac2-a interaction. The missense mutations in the GT found in dilute mice abrogated only the myosin-Va-GT.Slac2-a interaction and had no effect on the myosin-Va-exon-F.Slac2-a interaction. We further showed that expression of green fluorescence protein-tagged Slac2-a lacking the myosin-Va-GT-binding site (DeltaGT), but not the wild-type Slac2-a, severely inhibits melanosome transport in melan-a cells, especially at the melanosome transfer step from microtubles to actin filaments (i.e. perinuclear aggregation of melanosomes). On the basis of our findings, we propose that myosin-Va interacts with Slac2-a.Rab27A complex on the melanosome via two distinct domains, both of which are essential for melanosome transport in melanocytes.

Assignment of the canine myosin Va gene (MYO5A) to chromosome 30q14 by fluorescence in situ hybridization and radiation hybrid mapping

Myosin Va belongs to the unconventional myosin family that has been identified in many non-muscle tissues of vertebrates and invertebrates. All known myosins bind actin and produce mechanical force through ATP hydrolysis. Myosin Va is only expressed in melanocytes and in cells of the neuronal system (Mercer et al., 1991). In melanocytes it is part of the melanosome transport complex (Wu et al., 1997). Mutations in the MYO5A gene cause the dilute phenotype in mice that is generally associated with severe neurological disorders (Mercer et al., 1991), the dilute-opisthotonus rat mutant (Futaki et al., 2000), and in human the rare Griscelli syndrome with neurological impairment (Pastural et al., 1997; Menasche et al., 2000). This autosomal recessive disorder results in a phenotype with pigmentary dilution of skin and hair, the presence of large clumps of pigment in hairshafts and an accumulation of melanosomes in melanocytes in addition to neurological defects. The MYO5A gene has been located on HSA 15q21→q22 and on the homologous mouse and rat chromosomes 8 and 9, respectively (Hasson et al., 1996; Ohuo et al., 1996). In dogs affected with black hair follicular dysplasia (BHFD) abnormal hair follicles showing clumps of melanosomes in the hair matrix and an abnormal shape of the hairshaft are also observed (Schmutz et al., 1998). Therefore, MYO5A might be a suitable candidate gene for this canine disorder. To facilitate future linkage studies we report here the assignment of the canine MYO5A gene to CFA30q14 by FISH and RH mapping.

Slac2-a/Melanophilin, the Missing Link between Rab27 and Myosin Va: IMPLICATIONS OF A TRIPARTITE PROTEIN COMPLEX FOR MELANOSOME TRANSPORT

Myosin Va is a member of the unconventional class V myosin family, and a mutation in the myosin Va gene causes pigment granule transport defects in human Griscelli syndrome and dilute mice. How myosin Va recognizes its cargo (i.e. melanosomes), however, has remained undetermined over the past decade. In this study, we discovered Slac2-a/melanophilin to be the "missing link" between myosin Va and GTP-Rab27A present in the melanosome. Deletion analysis and site-directed mutagenesis showed that the N-terminal Slp (synaptotagmin-like protein) homology domain of Slac2-a specifically binds Rab27A/B isoforms and that the C-terminal half directly binds the globular tail of myosin Va. The tripartite protein complex (Rab27A.Slac2-a.myosin Va) in melanoma cells was further confirmed by immunoprecipitation. The discovery that myosin Va indirectly recognizes its cargo through Slac2-a, a novel Rab27A/B effector, should shed light on molecular recognition of its specific cargo by class V myosin.

Identification of a novel myosin-Va mutation in an ataxic mutant rat, dilute-opisthotonus.

Mutations of the myosin-Va gene (Myo5a) cause diluted coat color in mice and are occasionally associated with severe neurological disorders. Dilute-opisthotonus (dop) is a spontaneous gene mutation in the rat, and phenotypes of the homozygote (dop/dop) are similar to those of the Myo5a-deficient mouse, suggesting that the mutation resides in the rat Myo5a gene. To elucidate the molecular basis of the dop mutation, we cloned the rat Myo5a cDNA from the wild type and the dop/dop. The wild-type rat Myo5a cDNA contained a 5487-bp ORF and showed higher homology with Myo5a of the other species than Myr6 (Myo5b) in the rat. A 141-bp in-frame deletion was detected in the head region in the dop cDNA. An intragenic rearrangement consisting of a 306-bp inversion associated with 17-bp and 217-bp deletions were identified in the Myo5a gene of the dop genome. This rearrangement involved a 141-bp exon, which was skipped in the dop transcript. The MyoVA protein expression was severely impaired in the dop/dop brain. This is the first report to define the dop mutation as the Myo5a gene abnormality in the rat.

Arg–Cys Substitution at Codon 1246 of the Human Myosin Va Gene is not Associated with Griscelli Syndrome

Myosin Va is an actin-associated motor protein involved in organelle transport such as melanosomes and neuron synaptic vesicles and has always been proposed as the candidate gene for the autosomal recessive Griscelli-Pruniéras syndrome, one of the silvery hair syndromes, which is a lethal disease combining immunodeficiency and neurologic and pigmentary abnormalities. Thus far, two mutations in the myosin Va gene have been described to be associated with this syndrome. One of these mutations was a homozygous mis-sense mutation causing an arginine to cysteine alteration at codon 1246. Because we also found this particular substitution after mutation analysis of a Griscelli patient, we checked its relevance in a control group of 124 unrelated healthy individuals and found it to be present, even in homozygous state, in normal unaffected individuals. It is clear that this arg1246cys substitution is a polymorphism occurring in the human population and not occurring in association with Griscelli syndrome. Distinguishing a polymorphism from a bona fide mutation is of utmost importance and has major ethical implications with regard to prenatal genetic counseling in affected families.

Correction: Griscelli disease maps to chromocome 15q and is associated with mutations in the Myosin-Va gene

Nature Genet. 16, 289–292 (1997). One of the two Myosin Va mutations described, the Arg→Cys alteration at codon 1,246 (C→T transition at nt 3,736), was found to be a relatively common polymorphism. Several healthy individuals are homozygous carriers of the Cys1246 allele. This fact was first pointedout to Nature Genetics by Jo Lambert, Jean Marie Naeyeart, Anne de Paepe, Rudy van Coster, Alina Ferster, Michele Song and Ludwine Messiaen of the University of Gent, Gent, Belgium.

Defective Myosin Genes in Mutant Mice and Human Diseases

ABSTRACTMyosins are highly divergent actin‐based molecular motors. In five of eight classes expressed in mammals, defects in genes have been identified in mutant mice and/or human diseases. A mutated myosin II‐7 gene is one of the causes of human familial hypertrophic cardiomyopathy (FHC). The defective myosin Va gene is responsible for Griscelli disease, which is characterized by partial albinism and immunodeficiency, while in its mouse homologue coat color dilution is seen with or without neurological defects. There are three classes of myosins, VI, VII and XV, that are essential in the inner ear function. In humans, mutations in the VIIa gene are associated with three deafness‐related diseases, Usher 1B/DFNB2/DFNA11, providing the first example of exhibition of recessive‐ and dominant‐inherited disorders by different mutations in a single myosin gene.There are variations in phenotype between human diseases and their mouse models, which appear to be explicable on the basis of differences in tissue expression patterns of the given myosin between mouse and man. In FHC and Usher 1BDFNB2DFNA11, a wide spectrum of clinical symptoms are observed. Evidence has accumulated suggesting that the more functionally important the mutation site of the molecule, the more serious and severe the symptoms, although involvement of additional factors such as modifier genes and genetic background can not be ruled out. Molecular genetic analyses of a variety of dilute alleles in mice have greatly facilitated our understanding of genotype‐phenotype correlations, including information about structurally and functionally important domains of the myosin Va protein and cell‐type‐specific functions of different isoforms produced by alternative splicing.

Griscelli disease maps to chromosome 15q21 and is associated with mutations in the myosin-Va gene.

Griscelli disease (OMIM 214450) is a rare autosomal recessive disorder characterized by pigmentary dilution, variable cellular immunodeficiency and onset of acute phases of uncontrolled lymphocyte and macrophage activation, leading to death in the absence of bone-marrow transplantation. The pigmentary dilution is characterized by a diffuse skin pigmentation, silvery hair, large clumps of pigments in the hair shafts (Fig. 1) and an accumulation of melanosomes in melanocytes, with abnormal transfer of the melanin granules to the keratinocytes. Immunological abnormalities are characterized by absent delayed-type cutaneous hypersensitivity and an impaired natural-killer cell function. A similar disorder has been described in the dilute lethal mouse--which, however, differs by the occurrence of a severe neurological disorder. The dilute locus encodes myosin-Va, a member of the unconventional myosin family. Myosins bind actin and produce mechanical force through ATP hydrolysis. Some members of this family are thought to participate in organelle-transport machinery. Because of the phenotype resulting in the dilute mouse and because of their potential role in intracellular transport, unconventional myosin-encoding genes were regarded as candidate genes for Griscelli disease. Here we report that the Griscelli disease locus co-localizes on chromosome 15q21 with the myosin-Va gene, MYO5a, and that mutations of this gene occur in two patients with the disease. Griscelli disease is therefore a human equivalent of dilute expression in the mouse.