RESUMEN
Palladin (PALLD) belongs to the PALLD/myopalladin (MYPN)/myotilin family of actin-associated immunoglobulin-containing proteins in the sarcomeric Z-line. PALLD is ubiquitously expressed in several isoforms, and its longest 200 kDa isoform, predominantly expressed in striated muscle, shows high structural homology to MYPN. MYPN gene mutations are associated with human cardiomyopathies, whereas the role of PALLD in the heart has remained unknown, partly due to embryonic lethality of PALLD knockout mice. In a yeast two-hybrid screening, CARP/Ankrd1 and FHOD1 were identified as novel interaction partners of PALLD's N-terminal region. To study the role of PALLD in the heart, we generated conditional (cPKO) and inducible (cPKOi) cardiomyocyte-specific PALLD knockout mice. While cPKO mice exhibited no pathological phenotype, ablation of PALLD in adult cPKOi mice caused progressive cardiac dilation and systolic dysfunction, associated with reduced cardiomyocyte contractility, intercalated disc abnormalities, and fibrosis, demonstrating that PALLD is essential for normal cardiac function. Double cPKO and MYPN knockout (MKO) mice exhibited a similar phenotype as MKO mice, suggesting that MYPN does not compensate for the loss of PALLD in cPKO mice. Altered transcript levels of MYPN and PALLD isoforms were found in myocardial tissue from human dilated and ischemic cardiomyopathy patients, whereas their protein expression levels were unaltered.
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Cardiomiopatías , Cardiomiopatía Dilatada , Proteínas del Citoesqueleto , Animales , Humanos , Ratones , Cardiomiopatías/genética , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Ratones Noqueados , Proteínas Musculares/metabolismo , Miocardio/metabolismo , Isoformas de Proteínas/genéticaRESUMEN
CD73, a cell-surface N-linked glycoprotein that produces extracellular adenosine, is a novel target for cancer immunotherapy. Although anti-CD73 antibodies have entered clinical development, CD73 has both protumor and antitumor functions, depending on the target cell and tumor type. The aim of this study was to characterize CD73 regulation in human hepatocellular carcinoma (HCC). We examined CD73 expression, localization, and activity using molecular, biochemical, and cellular analyses on primary HCC surgical specimens, coupled with mechanistic studies in HCC cells. We analyzed CD73 glycan signatures and global alterations in transcripts encoding other N-linked glycoproteins by using mass spectrometry glycomics and RNA sequencing (RNAseq), respectively. CD73 was expressed on tumor hepatocytes where it exhibited abnormal N-linked glycosylation, independent of HCC etiology, tumor stage, or fibrosis presence. Aberrant glycosylation of tumor-associated CD73 resulted in a 3-fold decrease in 5'-nucleotidase activity (P < 0.0001). Biochemically, tumor-associated CD73 was deficient in hybrid and complex glycans specifically on residues N311 and N333 located in the C-terminal catalytic domain. Blocking N311/N333 glycosylation by site-directed mutagenesis produced CD73 with significantly decreased 5'-nucleotidase activity in vitro, similar to the primary tumors. Glycosylation-deficient CD73 partially colocalized with the Golgi structural protein GM130, which was strongly induced in HCC tumors. RNAseq analysis further revealed that N-linked glycoprotein-encoding genes represented the largest category of differentially expressed genes between HCC tumor and adjacent tissue. Conclusion: We provide the first detailed characterization of CD73 glycosylation in normal and tumor tissue, revealing a novel mechanism that leads to the functional suppression of CD73 in human HCC tumor cells. The present findings have translational implications for therapeutic candidate antibodies targeting cell-surface CD73 in solid tumors and small-molecule adenosine receptor agonists that are in clinical development for HCC.
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Background Podocyte loss and effacement of interdigitating podocyte foot processes are the major cause of a leaky filtration barrier and ESRD. Because the complex three-dimensional morphology of podocytes depends on the actin cytoskeleton, we studied the role in podocytes of the actin bundling protein palladin, which is highly expressed therein.Methods We knocked down palladin in cultured podocytes by siRNA transfection or in zebrafish embryos by morpholino injection and studied the effects by immunofluorescence and live imaging. We also investigated kidneys of mice with podocyte-specific knockout of palladin (PodoPalld-/- mice) by immunofluorescence and ultrastructural analysis and kidney biopsy specimens from patients by immunostaining for palladin.Results Compared with control-treated podocytes, palladin-knockdown podocytes had reduced actin filament staining, smaller focal adhesions, and downregulation of the podocyte-specific proteins synaptopodin and α-actinin-4. Furthermore, palladin-knockdown podocytes were more susceptible to disruption of the actin cytoskeleton with cytochalasin D, latrunculin A, or jasplakinolide and showed altered migration dynamics. In zebrafish embryos, palladin knockdown compromised the morphology and dynamics of epithelial cells at an early developmental stage. Compared with PodoPalld+/+ controls, PodoPalld-/- mice developed glomeruli with a disturbed morphology, an enlarged subpodocyte space, mild effacement, and significantly reduced expression of nephrin and vinculin. Furthermore, nephrotoxic serum injection led to significantly higher levels of proteinuria in PodoPalld-/- mice than in controls. Kidney biopsy specimens from patients with diabetic nephropathy and FSGS showed downregulation of palladin in podocytes as well.Conclusions Palladin has an important role in podocyte function in vitro and in vivo.
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Actinas/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Podocitos/metabolismo , Animales , Citoesqueleto , Femenino , Adhesiones Focales , Expresión Génica , Silenciador del Gen , Humanos , Glomérulos Renales/patología , Masculino , Ratones Noqueados , Proteínas de Microfilamentos/metabolismo , Morfolinos/farmacología , Podocitos/patología , ARN Mensajero/metabolismo , Vinculina/genética , Vinculina/metabolismo , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Palladin is an important component of motile actin-rich structures and nucleates branched actin filament arrays in vitro Here we examine the role of palladin during Listeria monocytogenes infections in order to tease out novel functions of palladin. We show that palladin is co-opted by L. monocytogenes during its cellular entry and intracellular motility. Depletion of palladin resulted in shorter and misshapen comet tails, and when actin- or VASP-binding mutants of palladin were overexpressed in cells, comet tails disintegrated or became thinner. Comet tail thinning resulted in parallel actin bundles within the structures. To determine whether palladin could compensate for the Arp2/3 complex, we overexpressed palladin in cells treated with the Arp2/3 inhibitor CK-666. In treated cells, bacterial motility could be initiated and maintained when levels of palladin were increased. To confirm these findings, we utilized a cell line depleted of multiple Arp2/3 complex subunits. Within these cells, L. monocytogenes failed to generate comet tails. When palladin was overexpressed in this Arp2/3 functionally null cell line, the ability of L. monocytogenes to generate comet tails was restored. Using purified protein components, we demonstrate that L. monocytogenes actin clouds and comet tails can be generated (in a cell-free system) by palladin in the absence of the Arp2/3 complex. Collectively, our results demonstrate that palladin can functionally replace the Arp2/3 complex during bacterial actin-based motility.IMPORTANCE Structures containing branched actin filaments require the Arp2/3 complex. One of the most commonly used systems to study intracellular movement generated by Arp2/3-based actin motility exploits actin-rich comet tails made by Listeria Using these infections together with live imaging and cell-free protein reconstitution experiments, we show that another protein, palladin, can be used in place of Arp2/3 to form actin-rich structures. Additionally, we show that palladin is needed for the structural integrity of comet tails as its depletion or mutation of critical regions causes dramatic changes to comet tail organization. These findings are the first to identify a protein that can functionally replace the Arp2/3 complex and have implications for all actin-based structures thought to exclusively use that complex.
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Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Endocitosis , Interacciones Huésped-Patógeno , Listeria monocytogenes/fisiología , Locomoción , Fosfoproteínas/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/antagonistas & inhibidores , Animales , Línea Celular , Humanos , Indoles/metabolismoRESUMEN
BACKGROUND: Cancer-associated fibroblasts (CAFs) support tumour progression and invasion, and they secrete abundant extracellular matrix (ECM) that may shield tumour cells from immune checkpoint or kinase inhibitors. Targeting CAFs using drugs that revert their differentiation, or inhibit their tumour-supportive functions, has been considered as an anti-cancer strategy. METHODS: We have used human and murine cell culture models, atomic force microscopy (AFM), microarray analyses, CAF/tumour cell spheroid co-cultures and transgenic fibroblast reporter mice to study how targeting HDACs using small molecule inhibitors or siRNAs re-directs CAF differentiation and function in vitro and in vivo. RESULTS: From a small molecule screen, we identified Scriptaid, a selective inhibitor of HDACs 1/3/8, as a repressor of TGFß-mediated CAF differentiation. Scriptaid inhibits ECM secretion, reduces cellular contraction and stiffness, and impairs collective cell invasion in CAF/tumour cell spheroid co-cultures. Scriptaid also reduces CAF abundance and delays tumour growth in vivo. CONCLUSIONS: Scriptaid is a well-tolerated and effective HDACi that reverses many of the functional and phenotypic properties of CAFs. Impeding or reversing CAF activation/function by altering the cellular epigenetic regulatory machinery could control tumour growth and invasion, and be beneficial in combination with additional therapies that target cancer cells or immune cells directly.
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Fibroblastos Asociados al Cáncer/efectos de los fármacos , Inhibidores de Histona Desacetilasas/administración & dosificación , Hidroxilaminas/administración & dosificación , Neoplasias/tratamiento farmacológico , Quinolinas/administración & dosificación , Factor de Crecimiento Transformador beta/genética , Animales , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/ultraestructura , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Técnicas de Cocultivo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/ultraestructura , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/ultraestructura , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Ratones , Análisis por Micromatrices , Microscopía de Fuerza Atómica , Neoplasias/patología , Neoplasias/ultraestructura , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cells organize actin filaments into higher-order structures by regulating the composition, distribution and concentration of actin crosslinkers. Palladin is an actin crosslinker found in the lamellar actin network and stress fibers, which are critical for mechanosensing of the environment. Palladin also serves as a molecular scaffold for α-actinin, another key actin crosslinker. By virtue of its close interactions with actomyosin structures in the cell, palladin may play an important role in cell mechanics. However, the role of palladin in cellular force generation and mechanosensing has not been studied. Here, we investigate the role of palladin in regulating the plasticity of the actin cytoskeleton and cellular force generation in response to alterations in substrate stiffness. Traction force microscopy revealed that tumor-associated fibroblasts generate larger forces on substrates of increased stiffness. Contrary to expectations, knocking down palladin increased the forces generated by cells and inhibited their ability to sense substrate stiffness for very stiff gels. This was accompanied by significant differences in actin organization, adhesion dynamics and altered myosin organization in palladin knock-down cells. Our results suggest that actin crosslinkers such as palladin and myosin motors coordinate for optimal cell function and to prevent aberrant behavior as in cancer metastasis.
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Fibroblastos Asociados al Cáncer/fisiología , Proteínas del Citoesqueleto/fisiología , Fosfoproteínas/fisiología , Actinas/metabolismo , Fibroblastos Asociados al Cáncer/efectos de los fármacos , Adhesión Celular , Células Cultivadas , Adhesiones Focales/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Mecanotransducción CelularRESUMEN
The stroma surrounding solid tumors contributes in complex ways to tumor progression. Cancer-associated fibroblasts (CAFs) are the predominant cell type in the tumor stroma. Previous studies have shown that the actin-binding protein palladin is highly expressed in the stroma of pancreas tumors, but the interpretation of these results is complicated by the fact that palladin exists as multiple isoforms. In the current study, the expression and localization of palladin isoform 4 was examined in normal specimens and adenocarcinomas of human pancreas, lung, colon, and stomach samples. Immunohistochemistry with isoform-selective antibodies revealed that expression of palladin isoform 4 was higher in adenocarcinomas versus normal tissues, and highest in CAFs. Immunohistochemistry staining revealed that palladin was present in both the cytoplasm and the nucleus of CAFs, and this was confirmed using immunofluorescence staining and subcellular fractionation of a pancreatic CAF cell line. To investigate the functional significance of nuclear palladin, RNA Seq analysis of palladin knockdown CAFs versus control CAFs was performed, and the results showed that palladin regulates the expression of genes involved in the biosynthesis and assembly of collagen, and organization of the extracellular matrix. These results suggested that palladin isoform 4 may play a conserved role in establishing the phenotype of CAFs in multiple tumor types.
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Adenocarcinoma/metabolismo , Proteínas del Citoesqueleto/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Pancreáticas/metabolismo , Fosfoproteínas/metabolismo , Microambiente Tumoral , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Humanos , Inmunohistoquímica , Fosfoproteínas/química , Fosfoproteínas/genética , Isoformas de Proteínas/metabolismo , Células Tumorales CultivadasRESUMEN
Recovery from acute kidney injury involving tubular epithelial cells requires proliferation and migration of healthy cells to the area of injury. In this study, we show that palladin, a previously characterized cytoskeletal protein, is upregulated in injured tubules and suggest that one of its functions during repair is to facilitate migration of remaining cells to the affected site. In a mouse model of anti-neutrophilic cytoplasmic antibody involving both tubular and glomerular disease, palladin is upregulated in injured tubular cells, crescents and capillary cells with angiitis. In human biopsies of kidneys from patients with other kidney diseases, palladin is also upregulated in crescents and injured tubules. In LLC-PK1 cells, a porcine proximal tubule cell line, stress induced by transforming growth factor-ß1 (TGF-ß1) leads to palladin upregulation. Knockdown of palladin in LLC-PK1 does not disrupt cell morphology but does lead to a defect in cell migration. Furthermore, TGF-ß1 induced increase in the 75 kDa palladin isoform occurs in both the nucleus and the cytoplasm. These data suggest that palladin expression is induced in injured cells and contributes to proper migration of cells in proximal tubules, possibly by regulation of gene expression as part of the healing process after acute injury.
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Proteínas del Citoesqueleto/metabolismo , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/patología , Animales , Línea Celular , Movimiento Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas del Citoesqueleto/antagonistas & inhibidores , Proteínas del Citoesqueleto/genética , Células Epiteliales/citología , Células Epiteliales/metabolismo , Humanos , Inmunohistoquímica , Riñón/patología , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Ratones , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Porcinos , Factor de Crecimiento Transformador beta1/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Sertoli cells undergo terminal differentiation at puberty to support all phases of germ cell development, which occurs in the mouse beginning in the second week of life. By â¼18 days postpartum (dpp), nearly all Sertoli cells have ceased proliferation. This terminal differentiation is accompanied by the development of unique and regionally concentrated filamentous actin (F-actin) structures at the basal and apical aspects of the seminiferous epithelium, and this reorganization is likely to involve the action of actin-binding proteins. Palladin (PALLD) is a widely expressed F-actin-binding and bundling protein recently shown to regulate these structures, yet it is predominantly nuclear in Sertoli cells at puberty. We found that PALLD localized within nuclei of primary Sertoli cells grown in serum-free media but relocalized to the cytoplasm upon serum stimulation. We utilized this system with in vivo relevance to Sertoli cell development to investigate mechanisms regulating nuclear localization of this F-actin-binding protein. Our results indicate that PALLD can be shuttled from the nucleus to the cytoplasm, and that this relocalization occurred following depolymerization of the F-actin cytoskeleton in response to cAMP signaling. Nuclear localization was reduced in Hpg-mutant testes, suggesting the involvement of gonadotropin signaling. We found that PALLD nuclear localization was unaffected in testis tissues from LH receptor and androgen receptor-mutant mice. However, PALLD nuclear localization was reduced in the testes of FSH receptor-mutant mice, suggesting that FSH signaling during Sertoli cell maturation regulates this subcellular localization.
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Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Fosfoproteínas/metabolismo , Células de Sertoli/metabolismo , Transporte Activo de Núcleo Celular , Animales , Células Cultivadas , AMP Cíclico/metabolismo , Citoplasma/metabolismo , Hormona Folículo Estimulante/metabolismo , Hormona Liberadora de Gonadotropina/genética , Hormona Liberadora de Gonadotropina/metabolismo , Humanos , Carioferinas/metabolismo , Hormona Luteinizante/metabolismo , Masculino , Ratones , Ratones Noqueados , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de HFE/genética , Receptores de HFE/metabolismo , Receptores de HL/genética , Receptores de HL/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Proteína Exportina 1RESUMEN
Here, we report the NMR structure of the actin-binding domain contained in the cell adhesion protein palladin. Previously, we demonstrated that one of the immunoglobulin domains of palladin (Ig3) is both necessary and sufficient for direct filamentous actin binding in vitro. In this study, we identify two basic patches on opposite faces of Ig3 that are critical for actin binding and cross-linking. Sedimentation equilibrium assays indicate that the Ig3 domain of palladin does not self-associate. These combined data are consistent with an actin cross-linking mechanism that involves concurrent attachment of two actin filaments by a single palladin molecule by an electrostatic mechanism. Palladin mutations that disrupt actin binding show altered cellular distributions and morphology of actin in cells, revealing a functional requirement for the interaction between palladin and actin in vivo.
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Actinas/metabolismo , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/fisiología , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosfoproteínas/fisiología , Dominios y Motivos de Interacción de Proteínas , Actinas/química , Sustitución de Aminoácidos/fisiología , Animales , Células COS , Chlorocebus aethiops , Proteínas del Citoesqueleto/genética , Inmunoglobulinas/química , Ratones , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Fosfoproteínas/genética , Unión Proteica/genética , Dominios y Motivos de Interacción de Proteínas/genética , Mapeo de Interacción de Proteínas , Transporte de Proteínas/genética , Conejos , TransfecciónRESUMEN
The interaction between α-actinin and palladin, two actin-cross-linking proteins, is essential for proper bidirectional targeting of these proteins. As a first step toward understanding the role of this complex in organizing cytoskeletal actin, we have characterized binding interactions between the EF-hand domain of α-actinin (Act-EF34) and peptides derived from palladin and generated an NMR-derived structural model for the Act-EF34/palladin peptide complex. The critical binding site residues are similar to an α-actinin binding motif previously suggested for the complex between Act-EF34 and titin Z-repeats. The structure-based model of the Act-EF34/palladin peptide complex expands our understanding of binding specificity between the scaffold protein α-actinin and various ligands, which appears to require an α-helical motif containing four hydrophobic residues, common to many α-actinin ligands. We also provide evidence that the Family X mutation in palladin, associated with a highly penetrant form of pancreatic cancer, does not interfere with α-actinin binding.
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Actinina/química , Actinina/metabolismo , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Sitios de Unión , Dicroismo Circular , Cristalografía por Rayos X , Motivos EF Hand , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
PURPOSE: To compare the incidence of pancreatic abnormalities detected by MR imaging between high-risk patients and control patients. MATERIALS AND METHODS: Forty-one consecutive patients who had two or more first-degree relatives with pancreatic cancer and who were asymptomatic with no clinical evidence of pancreatic cancer were prospectively included in this study. A control group was obtained by reviewing consecutive patients undergoing 3 Tesla (T) MRI examinations for nonpancreatic indications. On MR imaging, the presence of pancreatic abnormalities were evaluated in consensus by two radiologists who were blinded to clinical history. Pancreatic abnormalities were categorized as developmental abnormalities, mass-type lesions, inflammatory disease, and others. RESULTS: Overall, the incidence of pancreatic abnormalities was greater in the high-risk group than in the control group, but not statistically significant (P = 0.244). In the high-risk group, a total of 16 patients (39%) were diagnosed with pancreatic abnormalities, whereas in the control group, 11 patients (25%) were diagnosed with pancreatic abnormalities. Regarding mass-type lesions, there was a significant difference in incidence between the high-risk group, with a total of seven patients (17%), and the control group, with one patient (2%) (P = 0.028). There were no cases of imaging diagnosis of pancreatic cancer or tissue evaluation by surgical pathology in either group. CONCLUSION: Our prospective pilot study demonstrated a higher incidence of mass-type lesions in patients at increased risk for pancreatic cancer.
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Imagen por Resonancia Magnética/métodos , Páncreas/anomalías , Enfermedades Pancreáticas/patología , Adulto , Anciano , Estudios de Casos y Controles , Femenino , Humanos , Incidencia , Inflamación , Masculino , Persona de Mediana Edad , Enfermedades Pancreáticas/epidemiología , Proyectos Piloto , Estudios Prospectivos , RiesgoRESUMEN
Palladin, an actin associated protein, plays a significant role in regulating cell adhesion and cell motility. Palladin is important for development, as knockdown in mice is embryonic lethal, yet its role in the development of the vasculature is unknown. We have shown that palladin is essential for the expression of smooth muscle cells (SMC) marker genes and force development in response to agonist stimulation in palladin deficient SMCs. The goal of the study was to determine the molecular mechanisms underlying palladin's ability to regulate the expression of SMC marker genes. Results showed that palladin expression was rapidly induced in an A404 cell line upon retinoic acid (RA) induced differentiation. Suppression of palladin expression with siRNAs inhibited the expression of RA induced SMC differentiation genes, SM α-actin (SMA) and SM22, whereas over-expression of palladin induced SMC gene expression. Chromatin immunoprecipitation assays provided evidence that palladin bound to SMC genes, whereas co-immunoprecipitation assays also showed binding of palladin to myocardin related transcription factors (MRTFs). Endogenous palladin was imaged in the nucleus, increased with leptomycin treatment and the carboxyl-termini of palladin co-localized with MRTFs in the nucleus. Results support a model wherein palladin contributes to SMC differentiation through regulation of CArG-SRF-MRTF dependent transcription of SMC marker genes and as previously published, also through actin dynamics. Finally, in E11.5 palladin null mouse embryos, the expression of SMA and SM22 mRNA and protein is decreased in the vessel wall. Taken together, our findings suggest that palladin plays a key role in the differentiation of SMCs in the developing vasculature.
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Actinas/metabolismo , Diferenciación Celular , Proteínas del Citoesqueleto/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/citología , Fosfoproteínas/metabolismo , Actinas/genética , Animales , Línea Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas del Citoesqueleto/genética , Regulación de la Expresión Génica , Ratones , Ratones Noqueados , Músculo Liso Vascular/citología , Músculo Liso Vascular/embriología , Miocitos del Músculo Liso/metabolismo , Fosfoproteínas/genética , Unión Proteica , Transporte de Proteínas , RatasRESUMEN
Pancreatic ductal adenocarcinoma (PDA) is a lethal disease with a characteristic pattern of early metastasis, which is driving a search for biomarkers that can be used to detect the cancer at an early stage. Recently, the actin-associated protein palladin was identified as a candidate biomarker when it was shown that palladin is mutated in a rare inherited form of PDA, and overexpressed in many sporadic pancreas tumors and premalignant precursors. In this study, we analyzed the expression of palladin isoforms in murine and human PDA and explored palladin's potential use in diagnosing PDA. We performed immunohistochemistry and immunoblot analyses on patient samples and tumor-derived cells using an isoform-selective monoclonal antibody and a pan-palladin polyclonal antibody. Immunoblot and real-time quantitative reverse transcription-PCR were used to quantify palladin mRNA levels in human samples. We show that there are two major palladin isoforms expressed in pancreas: 65 and 85-90 kDa. The 65 kDa isoform is expressed in both normal and neoplastic ductal epithelial cells. The 85-90 kDa palladin isoform is highly overexpressed in tumor-associated fibroblasts (TAFs) in both primary and metastatic tumors compared to normal pancreas, in samples obtained from either human patients or genetically engineered mice. In tumor-derived cultured cells, expression of palladin isoforms follows cell-type specific patterns, with the 85-90 kDa isoform in TAFs, and the 65 kDa isoform predominating in normal and neoplastic epithelial cells. These results suggest that upregulation of 85-90 kDa palladin isoform may play a role in the establishment of the TAF phenotype, and thus in the formation of a desmoplastic tumor microenvironment. Thus, palladin may have a potential use in the early diagnosis of PDA and may have much broader significance in understanding metastatic behavior.
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Carcinoma Ductal Pancreático/diagnóstico , Proteínas del Citoesqueleto/genética , Fosfoproteínas/genética , Animales , Biomarcadores de Tumor , Proteínas del Citoesqueleto/análisis , Fibroblastos/patología , Humanos , Inmunoensayo , Ratones , Mutación , Proteínas de Neoplasias , Fosfoproteínas/análisis , Isoformas de Proteínas , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia ArribaRESUMEN
Immunoglobulin domains are found in a wide variety of functionally diverse transmembrane proteins, and also in a smaller number of cytoplasmic proteins. Members of this latter group are usually associated with the actin cytoskeleton, and most of them bind directly to either actin or myosin, or both. Recently, studies of inherited human disorders have identified disease-causing mutations in five cytoplasmic Ig-domain proteins: myosin-binding protein C, titin, myotilin, palladin, and myopalladin. Together with results obtained from cultured cells and mouse models, these clinical studies have yielded novel insights into the unexpected roles of Ig domain proteins in mechanotransduction and signaling to the nucleus. An emerging theme in this field is that cytoskeleton-associated Ig domain proteins are more than structural elements of the cell, and may have evolved to fill different needs in different cellular compartments. Cell Motil. Cytoskeleton 2009. (c) 2009 Wiley-Liss, Inc.
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Citoplasma/metabolismo , Citoesqueleto/metabolismo , Proteínas Portadoras/metabolismo , Conectina , Proteínas del Citoesqueleto/metabolismo , Humanos , Proteínas de Microfilamentos , Modelos Biológicos , Proteínas Musculares/metabolismo , Fosfoproteínas/metabolismo , Proteínas Quinasas/metabolismoRESUMEN
Palladin, a cytoskeletal protein with essential functions for stress fiber formation, is found in developing and mature tissues, including the kidney. To define its role in the kidney, we measured its expression in mouse kidney and found it co-localized with F-actin in smooth muscle cells of renal arterial vessels, mesangial cells, and podocytes but not in tubular epithelium. Using immunoelectron microscopy, we confirmed that palladin was present in podocytes. In cultured mouse podocytes, palladin co-localized with F-actin in dense regions of stress fibers, focal adhesions, cell-cell contacts and motile cell margins. Transfection with the N-terminal half of palladin targeted it to F-actin-containing structures in podocytes while the C-terminal half accumulated in the nucleus, a result also found for endogenous palladin in cultured cells after leptomycin B was used to block nuclear export. Green fluorescent protein (GFP)-tagged palladin was found in dynamic ring-like F-actin structures and ruffles in cultured podocytes after stimulation with epidermal growth factor. Inhibition of palladin expression by transfection of an antisense construct reduced the formation of ring-like structures. Photo-bleaching analysis showed that GFP-palladin turned over with a half-time of 10 s in focal adhesions and dense regions of stress fibers, suggesting that palladin is a dynamic scaffolding protein. Our study shows that palladin is expressed in podocytes and plays an important role in actin dynamics.
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Actinas/análisis , Proteínas del Citoesqueleto/fisiología , Fosfoproteínas/fisiología , Podocitos/química , Actinas/metabolismo , Animales , Células Cultivadas , Proteínas del Citoesqueleto/análisis , Proteínas del Citoesqueleto/metabolismo , Adhesiones Focales/química , Cinética , Ratones , Fosfoproteínas/análisis , Fosfoproteínas/metabolismo , Fibras de Estrés/químicaRESUMEN
Palladin is a widely expressed protein found in stress fibers, focal adhesions, growth cones, Z-discs, and other actin-based subcellular structures. It belongs to a small gene family that includes the Z-disc proteins myopalladin and myotilin, all of which share similar Ig-like domains. Recent advances have shown that palladin shares with myotilin the ability to bind directly to F-actin, and to crosslink actin filaments into bundles, in vitro. Studies in a variety of cultured cells suggest that the actin-organizing activity of palladin plays a central role in promoting cell motility. Correlative evidence also supports this hypothesis, as palladin levels are typically up-regulated in cells that are actively migrating: in developing vertebrate embryos, in cells along a wound edge, and in metastatic cancer cells. Recently, a mutation in the human palladin gene was implicated in an unusually penetrant form of inherited pancreatic cancer, which has stimulated new ideas about the role of palladin in invasive cancer.
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Citoesqueleto de Actina/ultraestructura , Movimiento Celular/fisiología , Proteínas de Microfilamentos/fisiología , Citoesqueleto de Actina/fisiología , Animales , Moléculas de Adhesión Celular/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Humanos , Proteínas de Microfilamentos/genética , Modelos Biológicos , Mutación , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Cicatrización de Heridas/fisiologíaRESUMEN
Palladin is a recently described phosphoprotein that plays an important role in cell adhesion and motility. Previous studies have shown that palladin overexpression results in profound changes in actin organization in cultured cells. Palladin binds to the actin-associated proteins alpha-actinin, vasodilator-stimulated phosphoprotein, profilin, Eps8, and ezrin, suggesting that it may affect actin organization indirectly. To determine its molecular function in generating actin arrays, we purified palladin and asked if it is also capable of binding to F-actin directly. In co-sedimentation and differential sedimentation assays, palladin was found to both bind and cross-link actin filaments. This bundling activity was confirmed by fluorescence and electron microscopy. Palladin fragments were then purified and used to determine the sequences necessary to bind and bundle F-actin. The Ig3 domain of palladin bound to F-actin, and a palladin fragment containing Ig3, Ig4, and the region linking these domains was identified as a fragment that was able to bundle F-actin. Because palladin has multiple Ig domains, and only one of them binds to F-actin, this suggests that different Ig domains may be specialized for distinct biological functions. In addition, our results suggest a potential role for palladin in generating specialized, actin-based cell morphologies via both direct actin cross-linking activity and indirect scaffolding activity.
Asunto(s)
Citoesqueleto de Actina/química , Proteínas del Citoesqueleto/química , Fosfoproteínas/química , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestructura , Animales , Adhesión Celular/fisiología , Movimiento Celular/fisiología , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/aislamiento & purificación , Proteínas del Citoesqueleto/metabolismo , Humanos , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Microscopía Electrónica de Transmisión , Fosfoproteínas/genética , Fosfoproteínas/aislamiento & purificación , Fosfoproteínas/metabolismo , Unión Proteica/fisiología , Estructura Terciaria de Proteína/fisiología , Conejos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Espectrometría de FluorescenciaRESUMEN
Force generated in contractile actin filament bundles (stress fibers-SFs) is transmitted to the extracellular matrix (ECM) via linker proteins and transmembrane integrins at focal adhesions (FAs). Though it has long been known that actin is rapidly exchanged in FAs, the connection between SFs and FAs has not been studied in detail. We introduced fiduciary marks on SFs by expressing GFP-palladin or GFP-alpha-actinin-1, which are both FA and dense body proteins, and by pattern bleaching of GFP-actin. Following fiduciary marks on SFs over time by time-lapse fluorescence microscopy, we detected assembly of SFs at FAs in stationary cells resulting in movement of SFs away from FAs with a velocity of 0.2-0.4 microm/min. Visualization of FAs in GFP-palladin/DsRed-paxillin double transfected cells showed that SF elongation was not accompanied by a change in FA length. SF elongation at FAs depended on actin polymerization and force as demonstrated by inhibitors of actin polymerization (cytochalasin D, jasplakinolide) and inhibitors of myosin-dependent contraction (blebbistatin, Y-27632), respectively. Our finding of SF assembly at FAs has important implications for SF formation, force transmission, and tension distribution within the actin cytoskeletal network of stationary cells.
Asunto(s)
Actinas/metabolismo , Movimiento Celular , Adhesiones Focales , Fibras de Estrés/metabolismo , Actinina/análisis , Actinina/genética , Actinas/antagonistas & inhibidores , Animales , Citocalasinas/farmacología , Proteínas del Citoesqueleto/análisis , Proteínas del Citoesqueleto/genética , Citoesqueleto , Depsipéptidos/farmacología , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Ratones , Células 3T3 NIH , Fosfoproteínas/análisis , Fosfoproteínas/genética , Fibras de Estrés/químicaRESUMEN
Cell morphology may be an important stimulus during differentiation of human adipose-derived adult stem (hADAS) cells, but there are limited studies that have investigated the role of the cytoskeleton or associated proteins in hADAS cells undergoing differentiation. Palladin is an actin-associated protein that plays an integral role in focal adhesion and cytoskeleton organization. In this study we show that palladin was expressed by hADAS cells and was modulated during osteogenic differentiation and in response to cyclic tensile strain. Human ADAS cells expressed the 90- and 140-kDa palladin isoforms and upregulated expression of both isoforms after culture in conditions that promoted osteogenesis. Palladin mRNA expression levels were also increased in hADAS cells subjected to cyclic tensile strain. Knockdown of the palladin gene during osteogenesis resulted in decreased actin stress fibers and decreased protein levels of Eps8, an epidermal growth factor receptor tyrosine kinase that colocalizes with actin. Silencing the palladin gene, however, did not affect hADAS cells' commitment down the osteogenic lineage.