RESUMEN
BACKGROUND: The expression of aquaporin 4 (AQP4) and intermediate filament (IF) proteins is altered in malignant glioblastoma (GBM), yet the expression of the major IF-based cytolinker, plectin (PLEC), and its contribution to GBM migration and invasiveness, are unknown. Here, we assessed the contribution of plectin in affecting the distribution of plasmalemmal AQP4 aggregates, migratory properties, and regulation of cell volume in astrocytes. METHODS: In human GBM, the expression of glial fibrillary acidic protein (GFAP), AQP4 and PLEC transcripts was analyzed using publicly available datasets, and the colocalization of PLEC with AQP4 and with GFAP was determined by immunohistochemistry. We performed experiments on wild-type and plectin-deficient primary and immortalized mouse astrocytes, human astrocytes and permanent cell lines (U-251 MG and T98G) derived from a human malignant GBM. The expression of plectin isoforms in mouse astrocytes was assessed by quantitative real-time PCR. Transfection, immunolabeling and confocal microscopy were used to assess plectin-induced alterations in the distribution of the cytoskeleton, the influence of plectin and its isoforms on the abundance and size of plasmalemmal AQP4 aggregates, and the presence of plectin at the plasma membrane. The release of plectin from cells was measured by ELISA. The migration and dynamics of cell volume regulation of immortalized astrocytes were assessed by the wound-healing assay and calcein labeling, respectively. RESULTS: A positive correlation was found between plectin and AQP4 at the level of gene expression and protein localization in tumorous brain samples. Deficiency of plectin led to a decrease in the abundance and size of plasmalemmal AQP4 aggregates and altered distribution and bundling of the cytoskeleton. Astrocytes predominantly expressed P1c, P1e, and P1g plectin isoforms. The predominant plectin isoform associated with plasmalemmal AQP4 aggregates was P1c, which also affected the mobility of astrocytes most prominently. In the absence of plectin, the collective migration of astrocytes was impaired and the dynamics of cytoplasmic volume changes in peripheral cell regions decreased. Plectin's abundance on the plasma membrane surface and its release from cells were increased in the GBM cell lines. CONCLUSIONS: Plectin affects cellular properties that contribute to the pathology of GBM. The observed increase in both cell surface and released plectin levels represents a potential biomarker and therapeutic target in the diagnostics and treatment of GBMs.
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Glioblastoma , Animales , Humanos , Ratones , Acuaporina 4 , Astrocitos , Biomarcadores , Plectina , Isoformas de ProteínasRESUMEN
The body size traits are major traits in livestock, which intuitively displays the development of the animal's bones and muscles. This study used PCR amplification, Sanger sequencing, KASPar genotyping, and quantitative real-time reverse transcription PCR (qRT-PCR) to analyze the Single-nucleotide polymorphism and expression characteristics of Argonaute RISC catalytic component 2 (AGO2) and Plectin (PLEC) genes in Hu sheep. Two intron mutations were found in Hu sheep, which were AGO2 g.51700 A > C and PLEC g.23157 C > T, respectively. Through association analysis of two mutation sites and body size traits, it was found that AGO2 g.51700 A > C mainly affects the chest and cannon circumference of Hu sheep of while PLEC g.23157 C mainly affects body height and body length. The combined genotypes of AGO2 and PLEC genes with body size traits showed SNPs at the AGO2 g.51700 A > C and PLEC g.23157 C > T loci significantly improved the body size traits of Hu sheep. In addition, the AGO2 gene has the highest expression levels in the heart, rumen, and tail fat, and the PLEC gene is highly expressed in the heart. These two loci can provide new research ideas for improving the body size traits of Hu sheep.
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Plectina , Polimorfismo de Nucleótido Simple , Ovinos/genética , Animales , Plectina/genética , Tamaño Corporal/genética , Polimorfismo de Nucleótido Simple/genética , Genotipo , FenotipoRESUMEN
Congenital insensitivity to pain is a rare human condition in which affected individuals do not experience pain throughout their lives. This study aimed to identify the molecular etiology of congenital insensitivity to pain in two Thai patients. Clinical, radiographic, histopathologic, immunohistochemical, and molecular studies were performed. Patients were found to have congenital insensitivity to pain, self-mutilation, acro-osteolysis, cornea scars, reduced temperature sensation, tooth agenesis, root maldevelopment, and underdeveloped maxilla and mandible. The skin biopsies revealed fewer axons, decreased vimentin expression, and absent neurofilament expression, indicating lack of dermal nerves. Whole exome and Sanger sequencing identified a rare homozygous variant c.4039C>T; p.Arg1347Cys in the plakin domain of Plec, a cytolinker protein. This p.Arg1347Cys variant is in the spectrin repeat 9 region of the plakin domain, a region not previously found to harbor pathogenic missense variants in other plectinopathies. The substitution with a cysteine is expected to decrease the stability of the spectrin repeat 9 unit of the plakin domain. Whole mount in situ hybridization and an immunohistochemical study suggested that Plec is important for the development of maxilla and mandible, cornea, and distal phalanges. Additionally, the presence of dental anomalies in these patients further supports the potential involvement of Plec in tooth development. This is the first report showing the association between the Plec variant and congenital insensitivity to pain in humans.
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Homocigoto , Insensibilidad Congénita al Dolor , Plectina , Humanos , Masculino , Plectina/genética , Plectina/metabolismo , Femenino , Insensibilidad Congénita al Dolor/genética , Niño , Linaje , Mutación Missense , Secuenciación del ExomaRESUMEN
Objective: To explore the relationship between the expression of plectin and the migration of hepatocellular carcinoma (HCC) cells and to elucidate the molecular mechanisms by which plectin expression affects the migration of HCC cells. Methods: First of all, Western blot was performed to determine the expression of plectin in normal hepatocytes and HCC cells. Secondly, a plectin-downregulated HCC cell strain was established and the control group (shNC group) and shPLEC group were set up. Each group was divided into a vehicle control group (shNC+DMSO group or shPLEC+DMSO group) and a F-actin cytoskeleton polymerization inducer Jasplakinolide group (shNC+Jasp group or shPLEC+Jasp group). Western blot was performed to determine the expression of plectin and epithelial-mesenchymal transition (EMT)-related proteins, including N-cadherin, vimentin, and E-cadherin. HCC cell migration was evaluated by Transwell assay. KEGG (Kyoto Encyclopedia of Genes and Genomes) was used to analyze the signaling pathways related to plectin gene. The polymerization of F-actin was analyzed by immunofluorescence assay. Results: Compared with the normal hepatocytes, HCC cells showed high expression of plectin. Compared with those in the shNC group, the expression of plectin in the shPLEC group was decreased (P<0.05), the migration ability of HCC cells was weakened (P<0.05), and the EMT process was inhibited (with the expression of N-cadherin and vimentin being decreased and the expression of E-cadherin being increased) (P<0.05). KEGG analysis showed that the regulation of cytoskeletal F-actin was most closely associated with plectin and cytoskeletal F-actin depolymerized in the shPLEC group. After treatment with Jasplakinolide, an inducer of F-actin cytoskeleton polymerization, the migration ability of HCC cells in the shPLEC+Jasp group was enhanced compared with that of shPLEC+DMSO group (P<0.05) and the EMT process was restored (with the expression of N-cadherin and vimentin being increased and the expression of E-cadherin being decreased) (P<0.05). In addition, the polymerization of cytoskeletal F-actin in HCC cells was also restored. Conclusion: Plectin is highly expressed in HCC cells. Plectin promotes the migration and the EMT of HCC cells through inducing F-actin polymerization.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Plectina , Humanos , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/patología , Actinas/metabolismo , Cadherinas/metabolismo , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Movimiento Celular , Dimetilsulfóxido , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/metabolismo , Plectina/genética , Plectina/metabolismo , Polimerizacion , Vimentina/metabolismoRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and lethal malignancy with extremely poor patient survival rates. A key reason for the poor prognosis is the lack of effective diagnostic tools to detect the disease at curable, premetastatic stages. Tumor surgical resection is PDAC's first-line treatment, however distinguishing between cancerous and healthy tissue with current imaging tools remains a challenge. In this work, we report a DOTA-based fluorescent probe targeting plectin-1 for imaging PDAC with high specificity. To enable heterogeneous functionalization of the DOTA-core with multiple targeting peptide units and the fluorophore, a novel, fully clickable synthetic route that proceeds in one pot was developed. Extensive validation of the probe set the stage for PDAC detection in mice and human tissue. Altogether, these findings may pave the way for improved clinical understanding and early detection of PDAC progression as well as more accurate resection criteria.
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Medios de Contraste , Compuestos Heterocíclicos con 1 Anillo , Neoplasias Pancreáticas , Plectina , Humanos , Neoplasias Pancreáticas/diagnóstico por imagen , Neoplasias Pancreáticas/patología , Plectina/metabolismo , Animales , Medios de Contraste/química , Ratones , Compuestos Heterocíclicos con 1 Anillo/química , Colorantes Fluorescentes/química , Colorantes Fluorescentes/síntesis química , Carcinoma Ductal Pancreático/diagnóstico por imagen , Carcinoma Ductal Pancreático/patología , Imagen ÓpticaRESUMEN
In muscle cells subjected to mechanical stimulation, LINC complex and cytoskeletal proteins are basic to preserve cellular architecture and maintain nuclei orientation and positioning. In this context, the role of lamin A/C remains mostly elusive. This study demonstrates that in human myoblasts subjected to mechanical stretching, lamin A/C recruits desmin and plectin to the nuclear periphery, allowing a proper spatial orientation of the nuclei. Interestingly, in Emery-Dreifuss Muscular Dystrophy (EDMD2) myoblasts exposed to mechanical stretching, the recruitment of desmin and plectin to the nucleus and nuclear orientation were impaired, suggesting that a functional lamin A/C is crucial for the response to mechanical strain. While describing a new mechanism of action headed by lamin A/C, these findings show a structural alteration that could be involved in the onset of the muscle defects observed in muscular laminopathies.