RESUMEN
In this study, we aimed to identify the myosin motor proteins that control trafficking at the Golgi complex. In addition to the known Golgi-associated myosins MYO6, MYO18A and MYH9 (myosin IIA), we identified MYO1C as a novel player at the Golgi in a human cell line. We demonstrate that depletion of MYO1C induces Golgi complex fragmentation and decompaction. MYO1C accumulates at dynamic structures around the Golgi complex that colocalize with Golgi-associated actin dots. MYO1C depletion leads to loss of cellular F-actin, and Golgi complex decompaction is also observed after inhibition or loss of the actin-related protein 2/3 complex, Arp2/3 (also known as ARPC). We show that the functional consequence of MYO1C depletion is a delay in the arrival of incoming transport carriers, both from the anterograde and retrograde routes. We propose that MYO1C stabilizes actin at the Golgi complex, facilitating the arrival of incoming transport carriers at the Golgi.This article has an associated First Person interview with the first author of the paper.
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Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Aparato de Golgi/metabolismo , Miosina Tipo I/metabolismo , Línea Celular , Movimiento Celular , Humanos , Miosina Tipo I/genética , Transporte de ProteínasRESUMEN
Understanding the medical effect of an ever-growing number of human variants detected is a long term challenge in genetic counseling. Functional assays, based on in vitro or in vivo evaluations of the variant effects, provide essential information, but they require robust statistical validation, as well as adapted outputs, to be implemented in the clinical decision-making process. Here, we assessed 25 pathogenic and 15 neutral missense variants of the BRCA1 breast/ovarian cancer susceptibility gene in four BRCA1 functional assays. Next, we developed a novel approach that refines the variant ranking in these functional assays. Lastly, we developed a computational system that provides a probabilistic classification of variants, adapted to clinical interpretation. Using this system, the best functional assay exhibits a variant classification accuracy estimated at 93%. Additional theoretical simulations highlight the benefit of this ready-to-use system in the classification of variants after functional assessment, which should facilitate the consideration of functional evidences in the decision-making process after genetic testing. Finally, we demonstrate the versatility of the system with the classification of siRNAs tested for human cell growth inhibition in high throughput screening.
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Neoplasias de la Mama/genética , Predisposición Genética a la Enfermedad/genética , Variación Genética/genética , Neoplasias Ováricas/genética , Proteína BRCA1/genética , Toma de Decisiones Clínicas , Femenino , Asesoramiento Genético/métodos , Pruebas Genéticas/métodos , Humanos , Mutación Missense/genéticaRESUMEN
Understanding the effect of an ever-growing number of human variants detected by genome sequencing is a medical challenge. The yeast Saccharomyces cerevisiae model has held attention for its capacity to monitor the functional impact of missense mutations found in human genes, including the BRCA1 breast and ovarian cancer susceptibility gene. When expressed in yeast, the wild-type full-length BRCA1 protein forms a single nuclear aggregate and induces a growth inhibition. Both events are modified by pathogenic mutations of BRCA1. However, the biological processes behind these events in yeast remain to be determined. Here, we show that the BRCA1 nuclear aggregation and the growth inhibition are sensitive to misfolding effects induced by missense mutations. Moreover, misfolding mutations impair the nuclear targeting of BRCA1 in yeast cells and in a human cell line. In conclusion, we establish a connection between misfolding and nuclear transport impairment, and we illustrate that yeast is a suitable model to decipher the effect of misfolding mutations.
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Proteína BRCA1/química , Proteína BRCA1/metabolismo , Pliegue de Proteína , Saccharomyces cerevisiae/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Fluorescencia , Humanos , Modelos Biológicos , Mutación/genética , Señales de Localización Nuclear , Agregado de Proteínas , Dominios Proteicos , Estabilidad Proteica , Transporte de Proteínas , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
To dissect secretory traffic, we developed the retention using selective hooks (RUSH) system. RUSH is a two-state assay based on the reversible interaction of a hook protein fused to core streptavidin and stably anchored in the donor compartment with a reporter protein of interest fused to streptavidin-binding peptide (SBP). Biotin addition causes a synchronous release of the reporter from the hook. Using the RUSH system, we analyzed different transport characteristics of various Golgi and plasma membrane reporters at physiological temperature in living cells. Using dual-color simultaneous live-cell imaging of two cargos, we observed intra- and post-Golgi segregation of cargo traffic, consistent with observation in other systems. We show preliminarily that the RUSH system is usable for automated screening. The system should help increase the understanding of the mechanisms of trafficking and enable screens for molecules that perturb pathological protein transport.
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Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Aparato de Golgi/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Microscopía Confocal/métodos , Estreptavidina/metabolismo , Transporte Biológico , Membrana Celular/ultraestructura , Aparato de Golgi/ultraestructura , Células HeLa , Humanos , Microscopía Inmunoelectrónica , Transfección/métodosRESUMEN
BACKGROUND: Aberrant activation of the Wnt/ß-catenin pathway is a major and frequent event in liver cancer, but inhibition of oncogenic ß-catenin signaling has proven challenging. The identification of genes that are synthetically lethal in ß-catenin-activated cancer cells would provide new targets for therapeutic drug design. METHODS: We transfected the parental HuH6 hepatoblastoma cell line with a doxycycline-inducible shRNA against CTNNB1 (gene coding for ß-catenin) to obtain an isogenic cell line pair with or without aberrant ß-catenin signaling. Using this hepatoblastoma isogenic cell line pair, we performed a human kinome-wide siRNA screen to identify synthetic lethal interactions with oncogenic CTNNB1. The phenotypic readouts of the screen were cell proliferation, cell cycle arrest and apoptosis, which were assessed by image-based analysis. In addition, apoptosis was assessed by flow cytometric experiments and immunoblotting. The potential synthetic lethal relationship between candidates genes identified in the screen and oncogenic CTNNB1 was also investigated in a different cellular context, a colorectal HCT116 isogenic cell line pair. RESULTS: We first determined the experimental conditions that led to the efficient expression of shRNA against CTNNB1 and maximal reduction of ß-catenin signaling activity in response to doxycycline treatment. Following high throughput screening in which 687 genes coding for kinases and proteins related to kinases (such as pseudokinases and phosphatases) were targeted, we identified 52 genes required for HuH6 survival. The silencing of five of these genes selectively impaired the viability of HuH6 cells with high ß-catenin signaling: HGS, STRADA, FES, BRAF and PKMYT1. Among these candidates, HGS depletion had the strongest inhibitory effect on cell growth and led to apoptosis specifically in HuH6 with high ß-catenin activity, while HuH6 with low ß-catenin activity were spared. In addition, HGS was identified as a potential synthetic lethal partner of oncogenic CTNNB1 in the HCT116 colorectal isogenic cell line pair. CONCLUSIONS: These results demonstrate the existence of crosstalk between ß-catenin signaling and HGS. Importantly, HGS depletion specifically affected cells with uncontrolled ß-catenin signaling activity in two different types of cancer (Hepatoblastoma HuH6 and colorectal HCT116), and thus may represent a new potential target for novel therapeutic strategies in liver and colorectal cancer.
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Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Hepatoblastoma/genética , Neoplasias Hepáticas/genética , Mutación , Fosfoproteínas/genética , ARN Interferente Pequeño/metabolismo , beta Catenina/antagonistas & inhibidores , Apoptosis , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Células HCT116 , Humanos , Fosfotransferasas/antagonistas & inhibidores , Vía de Señalización Wnt , beta Catenina/genéticaRESUMEN
Toll-like receptor (TLR) 3 is an endosomal TLR that mediates immune responses against viral infections upon activation by its ligand double-stranded RNA, a replication intermediate of most viruses. TLR3 is expressed widely in the body and activates both the innate and adaptive immune systems. However, little is known about how TLR3 intracellular trafficking and maturation are regulated. Here we show that newly synthesized endogenous TLR3 is transported through the ER and Golgi apparatus to endosomes, where it is rapidly cleaved. TLR3 protein expression is up-regulated by its own ligand, leading to the accumulation of its cleaved form. In agreement with its proposed role as a transporter, UNC93B1 expression is required for TLR3 cleavage and signaling. Furthermore, TLR3 signaling and cleavage are sensitive to cathepsin inhibition. Cleavage occurs between aa 252 and 346, and results in a functional receptor that signals upon activation. A truncated form of TLR3 lacking the N-terminal 345 aa also signals from acidic compartments in response to ligand activation. Screening of the human cathepsin family by RNA interference identified cathepsins B and H as key mediators of TLR3 processing. Taken together, our data indicate that TLR3 proteolytic processing is essential for its function, and suggest a mechanism of tight control of TLR3 signaling and thus immunity.
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Catepsina B/metabolismo , Catepsina H/metabolismo , Transducción de Señal/inmunología , Receptor Toll-Like 3/metabolismo , Análisis de Varianza , Catepsina B/inmunología , Catepsina H/inmunología , Línea Celular , Endosomas/metabolismo , Epítopos/genética , Humanos , Immunoblotting , Inmunoprecipitación , Luciferasas , Proteínas de Transporte de Membrana/metabolismo , Interferencia de ARN , ARN Bicatenario/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masas en Tándem , Receptor Toll-Like 3/inmunologíaRESUMEN
RNA interference has boosted the field of functional genomics, by making it possible to carry out 'loss-of-function' screens in cultured cells. Here, we performed a small interfering RNA screening, in three breast cancer cell lines, for 101 candidate driver genes overexpressed in amplified breast tumors and belonging to eight amplicons on chromosomes 8q and 17q, investigating their role in cell survival/proliferation. This screening identified eight driver genes that were amplified, overexpressed and critical for breast tumor cell proliferation or survival. They included the well-described oncogenic driver genes for the 17q12 amplicon, ERBB2 and GRB7. Four of six other candidate driver genes-RAD21 and EIF3H, both on chromosome 8q23, CHRAC1 on chromosome 8q24.3 and TANC2 on chromosome 17q23-were confirmed to be driver genes regulating the proliferation/survival of clonogenic breast cancer cells presenting an amplification of the corresponding region. Indeed, knockdown of the expression of these genes decreased cell viability, through both cell cycle arrest and apoptosis induction, and inhibited the formation of colonies in anchorage-independent conditions, in soft agar. Strategies for inhibiting the expression of these genes or the function of the proteins they encode are therefore of potential value for the treatment of breast cancers presenting amplifications of the corresponding genomic region.
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Neoplasias de la Mama/genética , División Celular/genética , Supervivencia Celular/genética , Transformación Celular Neoplásica/genética , Cromosomas Humanos Par 17 , Cromosomas Humanos Par 8 , ARN Interferente Pequeño/genética , Secuencia de Bases , Neoplasias de la Mama/patología , Proteínas de Ciclo Celular , Cartilla de ADN , Proteínas de Unión al ADN/genética , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Proteínas Nucleares/genética , Nucleoproteínas/genética , Fosfoproteínas/genética , Proteínas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
PURPOSE: High-throughput screening (HTS) platforms have been widely used to identify candidate anticancer drugs and drug-drug combinations; however, HTS-based identification of new drug-ionizing radiation (IR) combinations has rarely been reported. Herein, we developed an integrated approach including cell-based HTS and computational large-scale isobolographic analysis to accelerate the identification of radiosensitizing compounds acting strongly and more specifically on cancer cells. METHODS AND MATERIALS: In a 384-well plate format, 160 compounds likely to interfere with the cell response to radiation were screened on human glioblastoma (U251-MG) and cervix carcinoma (ME-180) cell lines, as well as on normal fibroblasts (CCD-19Lu). After drug exposure, cells were irradiated or not and short-term cell survival was assessed by high-throughput cell microscopy. Computational large-scale dose-response and isobolographic approach were used to identify promising synergistic drugs radiosensitizing cancer cells rather than normal cells. Synergy of a promising compound was confirmed on ME-180 cells by an independent 96-well assay protocol, and finally, by the gold-standard colony forming assay. RESULTS: We retained 4 compounds synergistic at 2 isoeffects in U251-MG and ME-180 cell lines and 11 compounds synergistically effective in only one cancer cell line. Among these 15 promising radiosensitizers, 5 compounds showed limited toxicity combined or not with IR on normal fibroblasts. CONCLUSIONS: Overall, this study demonstrated that HTS chemoradiation screening together with large-scale computational analysis is an efficient tool to identify synergistic drug-IR combinations, with concomitant assessment of unwanted toxicity on normal fibroblasts. It sparks expectations to accelerate the discovery of highly desired agents improving the therapeutic index of radiation therapy.
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Antineoplásicos , Neoplasias , Fármacos Sensibilizantes a Radiaciones , Femenino , Humanos , Ensayos Analíticos de Alto Rendimiento/métodos , Detección Precoz del Cáncer , Fármacos Sensibilizantes a Radiaciones/farmacología , Antineoplásicos/farmacología , Línea Celular , Línea Celular TumoralRESUMEN
Cell polarity is an essential and highly conserved process governing cell function. Cell polarization is generally triggered by an external signal that induces the relocation of the centrosome, thus defining the polarity axis of the cell. Here, we took advantage of B cells as a model to study cell polarity and perform a medium-throughput siRNA-based imaging screen to identify new molecular regulators of polarization. We first identified candidates based on a quantitative proteomic analysis of proteins differentially associated with the centrosome of resting non-polarized and stimulated polarized B cells. We then targeted 233 candidates in a siRNA screen and identified hits regulating the polarization of the centrosome and/or lysosomes in B cells upon stimulation. Our dataset of proteomics, images, and polarity indexes provides a valuable source of information for a broad community of scientists interested in the molecular mechanisms regulating cell polarity.
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Linfocitos B , ARN Interferente Pequeño , Centrosoma/metabolismo , Proteómica , Humanos , AnimalesRESUMEN
Eukaryotic cells, including cancer cells, secrete highly heterogeneous populations of extracellular vesicles (EVs). EVs could have different subcellular origin, composition and functional properties, but tools to distinguish between EV subtypes are scarce. Here, we tagged CD63- or CD9-positive EVs secreted by triple negative breast cancer cells with Nanoluciferase enzyme, to set-up a miniaturized method to quantify secretion of these two EV subtypes directly in the supernatant of cells. We performed a cell-based high-content screening to identify clinically-approved drugs able to affect EV secretion. One of the identified hits is Homosalate, an anti-inflammatory drug found in sunscreens which robustly increased EVs' release. Comparing EVs induced by Homosalate with those induced by Bafilomycin A1, we demonstrate that: (1) the two drugs act on EVs generated in distinct subcellular compartments, and (2) EVs released by Homosalate-, but not by Bafilomycin A1-treated cells enhance resistance to anchorage loss in another recipient epithelial tumour cell line. In conclusion, we identified a new drug modifying EV release and demonstrated that under influence of different drugs, triple negative breast cancer cells release EV subpopulations from different subcellular origins harbouring distinct functional properties.
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Vesículas Extracelulares , Neoplasias de la Mama Triple Negativas , Suplementos Dietéticos , Humanos , Salicilatos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológicoRESUMEN
Establishment of the interferon (IFN)-mediated antiviral state provides a crucial initial line of defense against viral infection. Numerous genes that contribute to this antiviral state remain to be identified. Using a loss-of-function strategy, we screened an original library of 1156 siRNAs targeting 386 individual curated human genes in stimulated microglial cells infected with Zika virus (ZIKV), an emerging RNA virus that belongs to the flavivirus genus. The screen recovered twenty-one potential host proteins that modulate ZIKV replication in an IFN-dependent manner, including the previously known IFITM3 and LY6E. Further characterization contributed to delineate the spectrum of action of these genes towards other pathogenic RNA viruses, including Hepatitis C virus and SARS-CoV-2. Our data revealed that APOL3 acts as a proviral factor for ZIKV and several other related and unrelated RNA viruses. In addition, we showed that MTA2, a chromatin remodeling factor, possesses potent flavivirus-specific antiviral functions induced by IFN. Our work identified previously unrecognized genes that modulate the replication of RNA viruses in an IFN-dependent manner, opening new perspectives to target weakness points in the life cycle of these viruses.
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Flavivirus , Interferones , Replicación Viral , Apolipoproteínas L/genética , Apolipoproteínas L/metabolismo , Flavivirus/fisiología , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Humanos , Interferones/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , SARS-CoV-2/fisiología , Virus Zika/fisiologíaRESUMEN
The steady-state localization of Golgi-resident glycosylation enzymes in the Golgi apparatus depends on a balance between anterograde and retrograde transport. Using the Retention Using Selective Hooks (RUSH) assay and high-content screening, we identified small molecules that perturb the localization of Mannosidase II (ManII) used as a model cargo for Golgi resident enzymes. In particular, we found that two compounds known as EGFR tyrosine kinase inhibitors, namely BML-265 and Tyrphostin AG1478 disrupt Golgi integrity and abolish secretory protein transport of diverse cargos, thus inducing brefeldin A-like effects. Interestingly, BML-265 and Tyrphostin AG1478 affect Golgi integrity and transport in human cells but not in rodent cells. The effects of BML-265 are reversible since Golgi integrity and protein transport are quickly restored upon washout of the compounds. BML-265 and Tyrphostin AG1478 do not lead to endosomal tubulation suggesting that, contrary to brefeldin A, they do not target the trans-Golgi ARF GEF BIG1 and BIG2. They quickly induce COPI dissociation from Golgi membranes suggesting that, in addition to EGFR kinase, the cis-Golgi ARF GEF GBF1 might also be a target of these molecules. Accordingly, overexpression of GBF1 prevents the effects of BML-265 and Tyrphostin AG1478 on Golgi integrity.
RESUMEN
Using a cell-based assay monitoring differential protein transport in the secretory pathway coupled to high-content screening, we have identified three molecules that specifically reduce the delivery of the major co-receptor for HIV-1, CCR5, to the plasma membrane. They have no effect on the closely related receptors CCR1 and CXCR4. These molecules are also potent in primary macrophages as they markedly decrease HIV entry. At the molecular level, two of these molecules inhibit the critical palmitoylation of CCR5 and thereby block CCR5 in the early secretory pathway. Our results open a clear therapeutics avenue based on trafficking control and demonstrate that preventing HIV infection can be performed at the level of its receptor delivery.
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Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/patogenicidad , Transporte de Proteínas/fisiología , Receptores CCR5/metabolismo , Secuencia de Aminoácidos , Línea Celular , Línea Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Macrófagos/metabolismo , Macrófagos/virología , Receptores CCR1/metabolismo , Receptores CXCR4/metabolismo , Vías Secretoras/fisiologíaRESUMEN
Rhabdoid tumors (RTs) are aggressive tumors of early childhood characterized by SMARCB1 inactivation. Their poor prognosis highlights an urgent need to develop new therapies. Here, we performed a high-throughput screening of approved drugs and identified broad inhibitors of tyrosine kinase receptors (RTKs), including pazopanib, and the potassium channel inhibitor clofilium tosylate (CfT), as SMARCB1-dependent candidates. Pazopanib targets were identified as PDGFRα/ß and FGFR2, which were the most highly expressed RTKs in a set of primary tumors. Combined genetic inhibition of both these RTKs only partially recapitulated the effect of pazopanib, emphasizing the requirement for broad inhibition. CfT perturbed protein metabolism and endoplasmic reticulum stress and, in combination with pazopanib, induced apoptosis of RT cells in vitro. In vivo, reduction of tumor growth by pazopanib was enhanced in combination with CfT, matching the efficiency of conventional chemotherapy. These results strongly support testing pazopanib/CfT combination therapy in future clinical trials for RTs.
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Antineoplásicos/farmacología , Descubrimiento de Drogas/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Compuestos de Amonio Cuaternario/farmacología , Tumor Rabdoide/metabolismo , Sulfonamidas/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Estrés del Retículo Endoplásmico/efectos de los fármacos , Humanos , Indazoles , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Proteína SMARCB1/metabolismoRESUMEN
A screening procedure was developed that takes advantage of the cellular normalization by micropatterning and a novel quantitative organelle mapping approach that allows unbiased and automated cell morphology comparison using black-box statistical testing. Micropatterns of extracellular matrix proteins force cells to adopt a reproducible shape and distribution of intracellular compartments avoiding strong cell-to-cell variation that is a major limitation of classical culture conditions. To detect changes in cell morphology induced by compound treatment, fluorescently labeled intracellular structures from several tens of micropatterned cells were transformed into probabilistic density maps. Then, the similarity or difference between two given density maps was quantified using statistical testing that evaluates differences directly from the data without additional analysis or any subjective decision. The versatility of this organelle mapping approach for different magnifications and its performance for different cell shapes has been assessed. Density-based analysis detected changes in cell morphology due to compound treatment in a small-scale proof-of-principle screen demonstrating its compatibility with high-throughput screening. This novel tool for high-content and high-throughput cellular phenotyping can potentially be used for a wide range of applications from drug screening to careful characterization of cellular processes.
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Forma de la Célula , Ensayos Analíticos de Alto Rendimiento , Orgánulos , Citoesqueleto/metabolismo , Humanos , Procesamiento de Imagen Asistido por ComputadorRESUMEN
High Content Screening (HCS) platforms allow screening living cells under a wide range of experimental conditions and give access to a whole panel of cellular responses to a specific treatment. The outcome is a series of cell population images. Within these images, the heterogeneity of cellular response to the same treatment leads to a whole range of observed values for the recorded cellular features. Consequently, it is difficult to compare and interpret experiments. Moreover, the definition of phenotypic classes at a cell population level remains an open question, although this would ease experiments analyses. In the present work, we tackle these two questions. The input of the method is a series of cell population images for which segmentation and cellular phenotype classification has already been performed. We propose a probabilistic model to represent and later compare cell populations. The model is able to fully exploit the HCS-specific information: "dependence structure of population descriptors" and "within-population variability". The experiments we carried out illustrate how our model accounts for this specific information, as well as the fact that the model benefits from considering them. We underline that these features allow richer HCS data analysis than simpler methods based on single cellular feature values averaged over each well. We validate an HCS data analysis method based on control experiments. It accounts for HCS specificities that were not taken into account by previous methods but have a sound biological meaning. Biological validation of previously unknown outputs of the method constitutes a future line of work.