RESUMEN
The primary cilium is required for Sonic hedgehog (Shh) signaling in vertebrates. In contrast to mutants affecting ciliary assembly, mutations in the intraflagellar transport complex A (IFT-A) paradoxically cause increased Shh signaling. We previously showed that the IFT-A complex, in addition to its canonical role in retrograde IFT, binds to the tubby-like protein, Tulp3, and recruits it to cilia. Here, we describe a conserved vertebrate G-protein-coupled receptor, Gpr161, which localizes to primary cilia in a Tulp3/IFT-A-dependent manner. Complete loss of Gpr161 in mouse causes midgestation lethality and increased Shh signaling in the neural tube, phenocopying Tulp3/IFT-A mutants. Constitutive Gpr161 activity increases cAMP levels and represses Shh signaling by determining the processing of Gli3 to its repressor form. Conversely, Shh signaling directs Gpr161 to be internalized from cilia, preventing its activity. Thus, Gpr161 defines a morphogenetic pathway coupling protein kinase A activation to Shh signaling during neural tube development.
Asunto(s)
Cilios/metabolismo , Embrión de Mamíferos/metabolismo , Tubo Neural/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular , Péptidos y Proteínas de Señalización Intracelular , Ratones , Datos de Secuencia Molecular , Filogenia , Proteínas/metabolismo , Receptores Acoplados a Proteínas G/química , Alineación de SecuenciaRESUMEN
Cancer immunotherapy has transformed the clinical approach to patients with malignancies, as profound benefits can be seen in a subset of patients. To identify this subset, biomarker analyses increasingly focus on phenotypic and functional evaluation of the tumor microenvironment to determine if density, spatial distribution, and cellular composition of immune cell infiltrates can provide prognostic and/or predictive information. Attempts have been made to develop standardized methods to evaluate immune infiltrates in the routine assessment of certain tumor types; however, broad adoption of this approach in clinical decision-making is still missing. We developed approaches to categorize solid tumors into 'desert', 'excluded', and 'inflamed' types according to the spatial distribution of CD8+ immune effector cells to determine the prognostic and/or predictive implications of such labels. To overcome the limitations of this subjective approach, we incrementally developed four automated analysis pipelines of increasing granularity and complexity for density and pattern assessment of immune effector cells. We show that categorization based on 'manual' observation is predictive for clinical benefit from anti-programmed death ligand 1 therapy in two large cohorts of patients with non-small cell lung cancer or triple-negative breast cancer. For the automated analysis we demonstrate that a combined approach outperforms individual pipelines and successfully relates spatial features to pathologist-based readouts and the patient's response to therapy. Our findings suggest that tumor immunophenotype generated by automated analysis pipelines should be evaluated further as potential predictive biomarkers for cancer immunotherapy. © 2024 The Pathological Society of Great Britain and Ireland.
Asunto(s)
Automatización , Antígeno B7-H1 , Biomarcadores de Tumor , Carcinoma de Pulmón de Células no Pequeñas , Inmunofenotipificación , Neoplasias de la Mama Triple Negativas , Humanos , Inmunoterapia , Antígeno B7-H1/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Neoplasias/patología , Inmunofenotipificación/métodos , Terapia Molecular Dirigida , Automatización/métodos , Estudios de Cohortes , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/inmunología , Neoplasias de la Mama Triple Negativas/patología , Biomarcadores de Tumor/análisis , Resultado del TratamientoRESUMEN
The small intestine epithelium renews every 2 to 5 days, making it one of the most regenerative mammalian tissues. Genetic inducible fate mapping studies have identified two principal epithelial stem cell pools in this tissue. One pool consists of columnar Lgr5-expressing cells that cycle rapidly and are present predominantly at the crypt base. The other pool consists of Bmi1-expressing cells that largely reside above the crypt base. However, the relative functions of these two pools and their interrelationship are not understood. Here we specifically ablated Lgr5-expressing cells in mice using a human diphtheria toxin receptor (DTR) gene knocked into the Lgr5 locus. We found that complete loss of the Lgr5-expressing cells did not perturb homeostasis of the epithelium, indicating that other cell types can compensate for the elimination of this population. After ablation of Lgr5-expressing cells, progeny production by Bmi1-expressing cells increased, indicating that Bmi1-expressing stem cells compensate for the loss of Lgr5-expressing cells. Indeed, lineage tracing showed that Bmi1-expressing cells gave rise to Lgr5-expressing cells, pointing to a hierarchy of stem cells in the intestinal epithelium. Our results demonstrate that Lgr5-expressing cells are dispensable for normal intestinal homeostasis, and that in the absence of these cells, Bmi1-expressing cells can serve as an alternative stem cell pool. These data provide the first experimental evidence for the interrelationship between these populations. The Bmi1-expressing stem cells may represent both a reserve stem cell pool in case of injury to the small intestine epithelium and a source for replenishment of the Lgr5-expressing cells under non-pathological conditions.
Asunto(s)
Intestino Delgado/citología , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Represoras/metabolismo , Células Madre/citología , Animales , Linaje de la Célula , Células Epiteliales/citología , Células Epiteliales/metabolismo , Femenino , Factor de Crecimiento Similar a EGF de Unión a Heparina , Homeostasis , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Intestino Delgado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Complejo Represivo Polycomb 1 , Receptores Acoplados a Proteínas G/deficiencia , Receptores Acoplados a Proteínas G/genética , Regeneración , Células Madre/metabolismoRESUMEN
Mammalian hosts are colonized with commensal microbes in various mucosal and epithelial tissues, including the intestinal tract. In mice, the presence of segmented filamentous bacteria (SFB) promotes Th17 differentiation and the development of autoimmune disease. Here, we demonstrate that the IL-23 pathway dynamically regulates the abundance of SFB as well as mucosal barrier function in the adult animal. Genetic or pharmacological inactivation of the pathway selectively perturbs the abundance of a small group of commensals, including SFB, and results in an impaired mucosal barrier. Defective barrier function leads to systemic dissemination of microbial products, provoking induction of the IL-23 pathway with dual consequences: IL-23 drives IL-22 production to reinforce mucosal barrier function and elicit antimicrobial activities, and it also drives the differentiation of Th17 cells in an attempt to combat escaped microbes in the lamina propria and in distal tissues. Thus, barrier defects generate a systemic environment that facilitates Th17 development.
Asunto(s)
Interleucinas/inmunología , Mucosa Intestinal/inmunología , Microbiota/inmunología , Receptores de Interleucina/inmunología , Células Th17/inmunología , Animales , Diferenciación Celular/inmunología , Interleucinas/genética , Mucosa Intestinal/microbiología , Ratones , Ratones Noqueados , Receptores de Interleucina/genética , Interleucina-22RESUMEN
Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. Limited treatment options have only marginally impacted patient survival over the past decades. The phophatidylinositol 3-kinase (PI3K) pathway, frequently altered in GBM, represents a potential target for the treatment of this glioma. 5-(6,6-Dimethyl-4-morpholino-8,9-dihydro-6H-[1,4]oxazino[4,3-e]purin-2-yl)pyrimidin-2-amine (GDC-0084) is a PI3K inhibitor that was specifically optimized to cross the blood-brain barrier. The goals of our studies were to characterize the brain distribution, pharmacodynamic (PD) effect, and efficacy of GDC-0084 in orthotopic xenograft models of GBM. GDC-0084 was tested in vitro to assess its sensitivity to the efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) and in vivo in mice to evaluate its effects on the PI3K pathway in intact brain. Mice bearing U87 or GS2 intracranial tumors were treated with GDC-0084 to assess its brain distribution by matrix-assisted laser desorption ionization (MALDI) imaging and measure its PD effects and efficacy in GBM orthotopic models. Studies in transfected cells indicated that GDC-0084 was not a substrate of P-gp or BCRP. GDC-0084 markedly inhibited the PI3K pathway in mouse brain, causing up to 90% suppression of the pAkt signal. MALDI imaging showed GDC-0084 distributed evenly in brain and intracranial U87 and GS2 tumors. GDC-0084 achieved significant tumor growth inhibition of 70% and 40% against the U87 and GS2 orthotopic models, respectively. GDC-0084 distribution throughout the brain and intracranial tumors led to potent inhibition of the PI3K pathway. Its efficacy in orthotopic models of GBM suggests that it could be effective in the treatment of GBM. GDC-0084 is currently in phase I clinical trials.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Encéfalo/metabolismo , Glioblastoma/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Animales , Barrera Hematoencefálica/metabolismo , Encéfalo/efectos de los fármacos , Neoplasias Encefálicas/tratamiento farmacológico , Línea Celular , Línea Celular Tumoral , Perros , Femenino , Glioblastoma/tratamiento farmacológico , Glioma/tratamiento farmacológico , Glioma/metabolismo , Humanos , Indazoles/metabolismo , Indazoles/farmacología , Células de Riñón Canino Madin Darby , Ratones , Ratones Desnudos , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
BACKGROUND: Protein secretion to the periplasm of Escherichia coli offers an attractive route for producing heterologous proteins including antibodies. In this approach, a signal peptide is fused to the N-terminus of the heterologous protein. The signal peptide mediates translocation of the heterologous protein from the cytoplasm to the periplasm and is cleaved during the translocation process. It was previously shown that optimization of the translation initiation region (TIR) which overlaps with the nucleotide sequence of the signal sequence improves the production of heterologous proteins. Despite the progress, there is still room to improve yields using secretion as a means to produce protein complexes such as full-length monoclonal antibodies (mAbs). RESULTS: In this study we identified the inefficient secretion of heavy chain as the limitation for full-length mAb accumulation in the periplasm. To improve heavy chain secretion we investigated the effects of various signal peptides at controlled TIR strengths. The signal peptide of disulfide oxidoreductase (DsbA) mediated more efficient secretion of heavy chain than the other signal peptides tested. Mutagenesis studies demonstrated that at controlled translational levels, hydrophobicity of the hydrophobic core (H-region) of the signal peptide is a critical factor for heavy chain secretion and full-length mAb accumulation in the periplasm. Increasing the hydrophobicity of a signal peptide enhanced heavy chain secretion and periplasmic levels of assembled full-length mAbs, while decreasing the hydrophobicity had the opposite effect. CONCLUSIONS: This study demonstrates that under similar translational strengths, the hydrophobicity of the signal peptide plays an important role in heavy chain secretion. Increasing the hydrophobicity of the H-region and controlling TIR strengths can serve as an approach to improve heavy chain secretion and full-length mAb production in E. coli.
Asunto(s)
Anticuerpos/metabolismo , Ingeniería de Proteínas/métodos , Señales de Clasificación de Proteína , Secuencia de Aminoácidos , Anticuerpos Monoclonales/metabolismo , Escherichia coli/metabolismo , Escherichia coli/ultraestructura , Interacciones Hidrofóbicas e Hidrofílicas , Cadenas Pesadas de Inmunoglobulina/metabolismo , Cuerpos de Inclusión/metabolismo , Cuerpos de Inclusión/ultraestructura , Datos de Secuencia Molecular , Iniciación de la Cadena Peptídica Traduccional , Periplasma/metabolismoRESUMEN
Depletion of the central metabolite NAD in cells results in broad metabolic defects leading to cell death and is a proposed novel therapeutic strategy in oncology. There is, however, a limited understanding of the underlying mechanisms that connect disruption of this central metabolite with cell death. Here we utilize GNE-617, a small molecule inhibitor of NAMPT, a rate-limiting enzyme required for NAD generation, to probe the pathways leading to cell death following NAD depletion. In all cell lines examined, NAD was rapidly depleted (average t½ of 8.1 h) following NAMPT inhibition. Concurrent with NAD depletion, there was a decrease in both cell proliferation and motility, which we attribute to reduced activity of NAD-dependent deacetylases because cells fail to deacetylate α-tubulin-K40 and histone H3-K9. Following depletion of NAD by >95%, cells lose the ability to regenerate ATP. Cell lines with a slower rate of ATP depletion (average t½ of 45 h) activate caspase-3 and show evidence of apoptosis and autophagy, whereas cell lines with rapid depletion ATP (average t½ of 32 h) do not activate caspase-3 or show signs of apoptosis or autophagy. However, the predominant form of cell death in all lines is oncosis, which is driven by the loss of plasma membrane homeostasis once ATP levels are depleted by >20-fold. Thus, our work illustrates the sequence of events that occurs in cells following depletion of a key metabolite and reveals that cell death caused by a loss of NAD is primarily driven by the inability of cells to regenerate ATP.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Compuestos Heterocíclicos con 2 Anillos/farmacología , NAD/metabolismo , Sulfonas/farmacología , Acetilación/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Western Blotting , Caspasa 3/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células HCT116 , Compuestos Heterocíclicos con 2 Anillos/química , Histonas/metabolismo , Humanos , Microscopía Electrónica de Transmisión , Estructura Molecular , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Nicotinamida Fosforribosiltransferasa/metabolismo , Sulfonas/química , Factores de Tiempo , Tubulina (Proteína)/metabolismoRESUMEN
Complement is an important component of the innate and adaptive immune response, yet complement split products generated through activation of each of the three complement pathways (classical, alternative, and lectin) can cause inflammation and tissue destruction. Previous studies have shown that complement activation through the alternative, but not classical, pathway is required to initiate antibody-induced arthritis in mice, but it is unclear if the alternative pathway (AP) plays a role in established disease. Previously, we have shown that human complement receptor of the immunoglobulin superfamily (CRIg) is a selective inhibitor of the AP of complement. Here, we present the crystal structure of murine CRIg and, using mutants, provide evidence that the structural requirements for inhibition of the AP are conserved in human and mouse. A soluble form of CRIg reversed inflammation and bone loss in two experimental models of arthritis by inhibiting the AP of complement in the joint. Our data indicate that the AP of complement is not only required for disease induction, but also disease progression. The extracellular domain of CRIg thus provides a novel tool to study the effects of inhibiting the AP of complement in established disease and constitutes a promising therapeutic with selectivity for a single complement pathway.
Asunto(s)
Artritis Experimental/tratamiento farmacológico , Resorción Ósea/tratamiento farmacológico , Modelos Moleculares , Receptores de Complemento/genética , Animales , Artritis Experimental/complicaciones , Resorción Ósea/etiología , Inactivadores del Complemento , Cristalización , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunohistoquímica , Ratones , Receptores de Complemento/químicaRESUMEN
Inhibitors of the programmed cell death-1 (PD-1/PD-L1) signaling axis are approved to treat non-small cell lung cancer (NSCLC) patients, based on their significant overall survival (OS) benefit. Using transcriptomic analysis of 891 NSCLC tumors from patients treated with either the PD-L1 inhibitor atezolizumab or chemotherapy from two large randomized clinical trials, we find a significant B cell association with extended OS with PD-L1 blockade, independent of CD8+ T cell signals. We then derive gene signatures corresponding to the dominant B cell subsets present in NSCLC from single-cell RNA sequencing (RNA-seq) data. Importantly, we find increased plasma cell signatures to be predictive of OS in patients treated with atezolizumab, but not chemotherapy. B and plasma cells are also associated with the presence of tertiary lymphoid structures and organized lymphoid aggregates. Our results suggest an important contribution of B and plasma cells to the efficacy of PD-L1 blockade in NSCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Antígeno B7-H1/genética , Antígeno B7-H1/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Humanos , Inhibidores de Puntos de Control Inmunológico , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Células Plasmáticas/patologíaRESUMEN
The Esx-1 (type VII) secretion system is critical for virulence of both Mycobacterium tuberculosis and Mycobacterium marinum, and is highly conserved between the two species. Despite its importance, there has been no direct visualization of Esx-1 secretion until now. In M. marinum, we show that secretion of Mh3864, a novel Esx-1 substrate that remains partially cell wall-associated after translocation, occurred in polar regions, indicating that Esx-1 secretion takes place in these regions. Analysis of Esx-1 secretion in infected host cells suggested that Esx-1 activity is similarly localized in vivo. A core component of the Esx-1 apparatus, Mh3870, also localized to bacterial poles, showing a preference for new poles with active cell wall peptidoglycan (PGN) synthesis. This work demonstrates that the Esx-1 secretion machine localizes to, and is active at, the bacterial poles. Thus, virulence-related protein secretion is localized in mycobacteria, suggesting new potential therapeutic targets, which are urgently needed.
Asunto(s)
Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Mycobacterium marinum/metabolismo , Factores de Virulencia/metabolismo , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , Actinas , Animales , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Línea Celular , Polaridad Celular , Pared Celular/metabolismo , Ratones , Mutagénesis , Mycobacterium marinum/genética , Peptidoglicano/metabolismo , Factores de Virulencia/genéticaRESUMEN
Hematopoietic stem cells (HSCs) have the capacity to self-renew and continuously differentiate into all blood cell lineages throughout life. At each branching point during differentiation, interactions with the environment are key in the generation of daughter cells with distinct fates. Here, we examined the role of the cell adhesion molecule JAM-C, a protein known to mediate cellular polarity during spermatogenesis, in hematopoiesis. We show that murine JAM-C is highly expressed on HSCs in the bone marrow (BM). Expression correlates with self-renewal, the highest being on long-term repopulating HSCs, and decreases with differentiation, which is maintained longest among myeloid committed progenitors. Inclusion of JAM-C as a sole marker on lineage-negative BM cells yields HSC enrichments and long-term multilineage reconstitution when transferred to lethally irradiated mice. Analysis of Jam-C-deficient mice showed that two-thirds die within 48 hours after birth. In the surviving animals, loss of Jam-C leads to an increase in myeloid progenitors and granulocytes in the BM. Stem cells and myeloid cells from fetal liver are normal in number and homing to the BM. These results provide evidence that JAM-C defines HSCs in the BM and that JAM-C plays a role in controlling myeloid progenitor generation in the BM.
Asunto(s)
Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/fisiología , Diferenciación Celular/genética , Inmunoglobulinas/genética , Inmunoglobulinas/fisiología , Células Progenitoras Mieloides/fisiología , Animales , Moléculas de Adhesión Celular/metabolismo , Linaje de la Célula/genética , Proliferación Celular , Femenino , Eliminación de Gen , Hematopoyesis/genética , Inmunoglobulinas/metabolismo , Inmunofenotipificación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Progenitoras Mieloides/metabolismoRESUMEN
Immunohistochemistry (IHC) assays provide valuable insights into protein expression patterns, the reliable interpretation of which requires well-characterized positive and negative control samples. Because appropriate tissue or cell line controls are not always available, a simple method to create synthetic IHC controls may be beneficial. Such a method is described here. It is adaptable to various antigen types, including proteins, peptides, or oligonucleotides, in a wide range of concentrations. This protocol explains the steps necessary to create synthetic antigen controls, using as an example a peptide from the human erythroblastic oncogene B2 (ERBB2/HER2) intracellular domain (ICD) recognized by a variety of diagnostically relevant antibodies. Serial dilutions of the HER2 ICD peptide in bovine serum albumin (BSA) solution are mixed with formaldehyde and heated for 10 min at 85 °C to solidify and cross-link the peptide/BSA mixture. The resulting gel can be processed, sectioned, and stained like a tissue, yielding a series of samples of known antigen concentrations spanning a wide range of staining intensities. This simple protocol is consistent with routine histology lab procedures. The method requires only that the user have a sufficient quantity of the desired antigen. Recombinant proteins, protein domains, or linear peptides that encode relevant epitopes may be synthesized locally or commercially. Laboratories generating in-house antibodies can reserve aliquots of the immunizing antigen as the synthetic control target. The opportunity to create well-defined positive controls across a wide range of concentrations allows users to assess intra- and inter-laboratory assay performance, gain insight into the dynamic range and linearity of their assays, and optimize assay conditions for their particular experimental goals.
Asunto(s)
Antígenos , Formaldehído , Epítopos , Humanos , Inmunohistoquímica , Vacunas SintéticasRESUMEN
With the advent of checkpoint inhibitors, there is increasing need to study the dynamics of CD8+ T-cells in the tumor microenviroment. In this article, we describe a semi-automated method to quantify and interrogate spatial relationships between T-cells and collagenous stroma in human and mouse tissue samples. The assay combines CD8 immunohistochemistry with modified Masson's trichrome. Slides are scanned and digital images are analyzed using an adjustable MATLAB algorithm, allowing for high-throughput quantification of cytotoxic T-cells and collagen. This method provides a flexible tool for unbiased quantification of T-cells and their interactions with tumor cells and tumor microenvironment in tissue samples.
Asunto(s)
Antígenos CD8/análisis , Ensayos Analíticos de Alto Rendimiento , Algoritmos , Animales , Humanos , Inmunohistoquímica , Ratones , Microambiente TumoralRESUMEN
Optimization and standardization of immunohistochemistry (IHC) protocols within and between laboratories requires reproducible positive and negative control samples. In many situations, suitable tissue or cell line controls are not available. We demonstrate here a method to incorporate target antigens into synthetic protein gels that can serve as IHC controls. The method can use peptides, protein domains, or whole proteins as antigens, and is compatible with a variety of fixation protocols. The resulting gels can be used to create tissue microarrays (TMAs) with a range of antigen concentrations that can be used to objectively quantify and calibrate chromogenic, fluorescent, or mass spectrometry-based IHC protocols. The method offers an opportunity to objectively quantify IHC staining results, and to optimize and standardize IHC protocols within and between laboratories. (J Histochem Cytochem 58:XXX-XXX, 2019).
Asunto(s)
Antígenos/análisis , Geles/química , Inmunohistoquímica/métodos , Animales , Formaldehído/química , Humanos , Inmunohistoquímica/normas , Ratones , Coloración y Etiquetado/métodos , Coloración y Etiquetado/normas , Análisis de Matrices Tisulares/métodos , Análisis de Matrices Tisulares/normas , Fijación del Tejido/métodos , Fijación del Tejido/normasRESUMEN
Mitochondria are complex organelles with a highly dynamic distribution and internal organization. Here, we demonstrate that mitofilin, a previously identified mitochondrial protein of unknown function, controls mitochondrial cristae morphology. Mitofilin is enriched in the narrow space between the inner boundary and the outer membranes, where it forms a homotypic interaction and assembles into a large multimeric protein complex. Down-regulation of mitofilin in HeLa cells by using specific small interfering RNA lead to decreased cellular proliferation and increased apoptosis, suggesting abnormal mitochondrial function. Although gross mitochondrial fission and fusion seemed normal, ultrastructural studies revealed disorganized mitochondrial inner membrane. Inner membranes failed to form tubular or vesicular cristae and showed as closely packed stacks of membrane sheets that fused intermittently, resulting in a complex maze of membranous network. Electron microscopic tomography estimated a substantial increase in inner:outer membrane ratio, whereas no cristae junctions were detected. In addition, mitochondria subsequently exhibited increased reactive oxygen species production and membrane potential. Although metabolic flux increased due to mitofilin deficiency, mitochondrial oxidative phosphorylation was not increased accordingly. We propose that mitofilin is a critical organizer of the mitochondrial cristae morphology and thus indispensable for normal mitochondrial function.
Asunto(s)
Membranas Intracelulares/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/química , Proteínas Musculares/química , Proteínas Musculares/fisiología , Animales , Apoptosis , Western Blotting , Proliferación Celular , Centrifugación por Gradiente de Densidad , Electroforesis en Gel de Poliacrilamida , Glicerol/farmacología , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Inmunoprecipitación , Hígado/metabolismo , Sustancias Macromoleculares/metabolismo , Ratones , Microscopía Electrónica , Proteínas Mitocondriales/fisiología , Plásmidos/metabolismo , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Retroviridae/genética , Relación Estructura-Actividad , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
Activating mutations in receptor tyrosine kinases play a critical role in oncogenesis. Despite evidence that Met kinase is deregulated in human cancer, the role of activating mutations in cancers other than renal papillary carcinoma has not been well defined. Here we report the identification of somatic intronic mutations of Met kinase that lead to an alternatively spliced transcript in lung cancer, which encodes a deletion of the juxtamembrane domain resulting in the loss of Cbl E3-ligase binding. The mutant receptor exhibits decreased ubiquitination and delayed down-regulation correlating with elevated, distinct Met expression in primary tumors harboring the deleted receptor. As a consequence, phospho-Met and downstream mitogen-activated protein kinase activation is sustained on ligand stimulation. Cells expressing the Met deletion reveal enhanced ligand-mediated proliferation and significant in vivo tumor growth. A hepatocyte growth factor competitive Met antagonist inhibits receptor activation and proliferation in tumor cells harboring the Met deletion, suggesting the important role played by ligand-dependent Met activation and the potential for anticancer therapy. These results support a critical role for Met in lung cancer and somatic mutation-driven splicing of an oncogene that leads to a different mechanism for tyrosine kinase activation through altered receptor down-regulation in human cancer.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Eliminación de Gen , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogénicas/genética , Receptores de Factores de Crecimiento/genética , Empalme Alternativo , Animales , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Neoplasias del Colon/enzimología , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Regulación hacia Abajo , Activación Enzimática , Exones , Femenino , Humanos , Intrones , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Desnudos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutación , Fosforilación , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Proteínas Proto-Oncogénicas c-met , Receptores de Factores de Crecimiento/antagonistas & inhibidores , Receptores de Factores de Crecimiento/biosíntesis , Receptores de Factores de Crecimiento/metabolismo , Transducción de Señal , Ubiquitina/metabolismoRESUMEN
In situ analysis of biomarkers is essential for clinical diagnosis and research purposes. The increasing need to understand the molecular signature of pathologies has led to the blooming of ultrasensitive and multiplexable techniques that combine in situ hybridization (ISH) and immunohistochemistry or immunocytochemistry (IHC or ICC). Most protocols are tailored to formalin-fixed paraffin embedded (FFPE) tissue sections. However, methods to perform such assays on non-adherent cell samples, such as patient blood-derived PBMCs, rare tumor samples, effusions or other body fluids, dissociated or sorted cells, are limited. Typically, a laboratory would need to invest a significant amount of time and resources to establish one such assay. Here, we describe a method that combines ultrasensitive RNAscope-ISH with ICC on cytospin cell preparations. This method allows automated, sensitive, multiplex ISH-ICC on small numbers of non-adherent cells. We provide guidelines for both chromogenic and fluorescent ISH/ICC combinations that can be performed either in fully automated or in manual settings. By using a CD8+ T cells in vitro stimulation paradigm, we demonstrate that this protocol is sensitive enough to detect subtle differences in gene expression and compares well to commonly used methods such as RT-qPCR and flow cytometry with the added benefit of visualization at the cellular level.
Asunto(s)
Linfocitos T CD8-positivos/citología , Células Dendríticas/citología , ARN/análisis , Animales , Linfocitos T CD8-positivos/química , Células Cultivadas , Células Dendríticas/química , Citometría de Flujo , Humanos , Inmunohistoquímica , Hibridación in Situ/métodos , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
RESUMEN
Geographic atrophy (GA), the advanced form of dry age-related macular degeneration (AMD), is characterized by progressive loss of retinal pigment epithelium cells and photoreceptors in the setting of characteristic extracellular deposits and remains a serious unmet medical need. While genetic predisposition to AMD is dominated by polymorphisms in complement genes, it remains unclear how complement activation contributes to retinal atrophy. Here we demonstrate that complement is activated on photoreceptor outer segments (POS) in the retina peripheral to atrophic lesions associated with GA. When exposed to human serum following outer blood-retinal barrier breakdown, POS act as potent activators of the classical and alternative complement pathway. In mouse models of retinal degeneration, classical and alternative pathway complement activation on photoreceptors contributed to the loss of photoreceptor function. This was dependent on C5a-mediated recruitment of peripheral blood monocytes but independent of resident microglia. Genetic or pharmacologic inhibition of both classical and alternative complement C3 and C5 convertases was required to reduce progressive degeneration of photoreceptor rods and cones. Our study implicates systemic classical and alternative complement proteins and peripheral blood monocytes as critical effectors of localized retinal degeneration with potential relevance for the contribution of complement activation to GA.