RESUMEN
BACKGROUND: Myo1e is a nonmuscle motor protein enriched in podocytes. Mutations in MYO1E are associated with steroid-resistant nephrotic syndrome (SRNS). Most of the MYO1E variants identified by genomic sequencing have not been functionally characterized. Here, we set out to analyze two mutations in the Myo1e motor domain, T119I and D388H, which were selected on the basis of protein sequence conservation. METHODS: EGFP-tagged human Myo1e constructs were delivered into the Myo1e-KO mouse podocyte-derived cells via adenoviral infection to analyze Myo1e protein stability, Myo1e localization, and clathrin-dependent endocytosis, which is known to involve Myo1e activity. Furthermore, truncated Myo1e constructs were expressed using the baculovirus expression system and used to measure Myo1e ATPase and motor activity in vitro. RESULTS: Both mutants were expressed as full-length proteins in the Myo1e-KO cells. However, unlike wild-type (WT) Myo1e, the T119I variant was not enriched at the cell junctions or clathrin-coated vesicles (CCVs). In contrast, D388H variant localization was similar to that of WT. The rate of dissociation of the D388H variant from cell-cell junctions and CCVs was decreased, suggesting this mutation affects Myo1e interactions with binding partners. ATPase activity and ability to translocate actin filaments were drastically reduced for the D388H mutant, supporting findings from cell-based experiments. CONCLUSIONS: T119I and D388H mutations are deleterious to Myo1e functions. The experimental approaches used in this study can be applied to future characterization of novel MYO1E variants associated with SRNS.
Asunto(s)
Miosina Tipo I , Síndrome Nefrótico , Animales , Humanos , Ratones , Mutación , Miosina Tipo I/genética , Miosina Tipo I/metabolismo , Síndrome Nefrótico/genética , EsteroidesRESUMEN
In both unicellular and multicellular organisms, long-tailed class I myosins function in clathrin-mediated endocytosis. Myosin 1e (Myo1e) in vertebrates and Myo1 in fission yeast have similar domain organization, yet whether these proteins or their individual protein domains are functionally interchangeable remains unknown. In an effort to assess functional conservation of class I myosins, we tested whether human Myo1e could replace Myo1 in fission yeast Schizosaccharomyces pombe and found that it was unable to substitute for yeast Myo1. To determine if any individual protein domain is responsible for the inability of Myo1e to function in yeast, we created human-yeast myosin-I chimeras. By functionally testing these chimeric myosins in vivo, we concluded that the Myo1e motor domain is unable to function in yeast, even when combined with the yeast Myo1 tail and a full complement of yeast regulatory light chains. Conversely, the Myo1e tail, when attached to the yeast Myo1 motor domain, supports localization to endocytic actin patches and partially rescues the endocytosis defect in myo1Δ cells. Further dissection showed that both the TH1 and TH2-SH3 domains in the human Myo1e tail are required for localization and function of chimeric myosin-I at endocytic sites. Overall, this study provides insights into the role of individual myosin-I domains, expands the utility of fission yeast as a simple model system to study the effects of disease-associated MYO1E mutations, and supports a model of co-evolution between a myosin motor and its actin track.
Asunto(s)
Endocitosis/fisiología , Cadenas Pesadas de Miosina/metabolismo , Miosina Tipo I/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Actinas/metabolismo , Humanos , Dominios Proteicos/fisiologíaRESUMEN
Several missense mutations in the Z-band protein, myotilin, have been implicated in human muscle diseases such as myofibrillar myopathy, spheroid body myopathy, and distal myopathy. Recently, we have reported the cloning of chicken myotilin cDNA. In this study, we have investigated the expression of myotilin in cross-striated muscles from developing chicken by qRT-PCR and in situ hybridizations. In situ hybridization of embryonic stages shows myotilin gene expression in heart, somites, neural tissue, eyes and otocysts. RT-PCR and qRT-PCR data, together with in situ hybridization results point to a biphasic transcriptional pattern for MYOT gene during early heart development with maximum expression level in the adult. In skeletal muscle, the expression level starts decreasing after embryonic day 20 and declines in the adult skeletal muscles. Western blot assays of myotilin in adult skeletal muscle reveal a decrease in myotilin protein compared with levels in embryonic skeletal muscle. Our results suggest that MYOT gene may undergo transcriptional activation and repression that varies between tissues in developing chicken. We believe this is the first report of the developmental regulation on myotilin expression in non-mammalian species.
Asunto(s)
Conectina/metabolismo , Corazón/embriología , Músculo Esquelético/metabolismo , Miocitos Cardíacos/metabolismo , Animales , Embrión de Pollo , Pollos , Conectina/genética , Regulación del Desarrollo de la Expresión Génica , Músculo Esquelético/embriología , ARN Mensajero/metabolismo , Factores de Tiempo , Transcripción Genética , Activación TranscripcionalRESUMEN
Glomerular visceral epithelial cells (podocytes) play a key role in maintaining selective protein filtration in the kidney. Podocytes have a complex cell shape characterized by the presence of numerous actin-rich processes, which cover the surface of glomerular capillaries and are connected by specialized cell-cell adhesion complexes (slit diaphragms). Human genetic studies and experiments in knockout mouse models show that actin filaments and actin-associated proteins are indispensable for the maintenance of podocyte shape, slit diaphragm integrity, and normal glomerular filtration. The ability to examine cytoskeletal protein organization and dynamics in podocytes and to test the effects of disease-associated mutations on protein localization provides valuable information for researchers aiming to dissect the molecular mechanisms of podocyte dysfunction. We describe how adenovirus-mediated transduction of cultured podocytes with DNA constructs can be used to reliably introduce fluorescently tagged cytoskeletal markers for live cell imaging with high efficiency and low toxicity. This technique can be used to study the dynamic reorganization of the podocyte cytoskeleton and to test the effects of novel mutations on podocyte cytoskeletal dynamics.
Asunto(s)
Adenoviridae/genética , Citoesqueleto/metabolismo , Vectores Genéticos/metabolismo , Imagen Molecular/métodos , Podocitos/citología , Animales , Biomarcadores/metabolismo , Western Blotting , Diferenciación Celular , Extractos Celulares , Supervivencia Celular , Colorantes Fluorescentes/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Ratones , Miosinas/metabolismo , Podocitos/virología , Ratas , Proteínas Recombinantes/metabolismo , Transducción GenéticaRESUMEN
Although the growth of experimental oral cancers can be inhibited by infection with the herpes simplex virus type 1 (HSV-1), the effect is incomplete. To define factors that might limit the effectiveness of the virus, we examined the roles of the innate immune system and the replication status of the tumor cells. AT-84 tumors were induced in strains of mice that had specific immune defects and were treated with the virus. Explanted tumors and tumor cells in culture were also infected. No differences in viral replication or in the effect of virus on the tumor were seen between mice with a lack of T or B lymphocytes, natural killer cells, phagocytic spleen cells, or complement. The virus did not replicate significantly more in tumors that were maintained as explants. Immediately after recovery of cells from a tumor the proportion of cells in the S phase was around 18%, and replication of virus in those cells was very limited. After 3 weeks in culture, the proportion in S had increased to 50% and both the recovery of virus from the cells and the toxic effect of the virus on the cells had increased significantly. The innate immune system thus seemed to have a minimal effect on replication of HSV-1 when used as an oncolytic virus for oral cancers in mice. Instead, the fraction of cells in the S phase was important. Because human oral cancers, like mouse tumors, have a low fraction of cells in the S phase, it is likely that the in vivo use of HSV-1 as cancer therapy will be limited by the replication of the virus.