RESUMO
Intracellular accumulation of misfolded proteins causes toxic proteinopathies, diseases without targeted therapies. Mucin 1 kidney disease (MKD) results from a frameshift mutation in the MUC1 gene (MUC1-fs). Here, we show that MKD is a toxic proteinopathy. Intracellular MUC1-fs accumulation activated the ATF6 unfolded protein response (UPR) branch. We identified BRD4780, a small molecule that clears MUC1-fs from patient cells, from kidneys of knockin mice and from patient kidney organoids. MUC1-fs is trapped in TMED9 cargo receptor-containing vesicles of the early secretory pathway. BRD4780 binds TMED9, releases MUC1-fs, and re-routes it for lysosomal degradation, an effect phenocopied by TMED9 deletion. Our findings reveal BRD4780 as a promising lead for the treatment of MKD and other toxic proteinopathies. Generally, we elucidate a novel mechanism for the entrapment of misfolded proteins by cargo receptors and a strategy for their release and anterograde trafficking to the lysosome.
Assuntos
Benzamidas/metabolismo , Compostos Bicíclicos com Pontes/farmacologia , Heptanos/farmacologia , Lisossomos/efeitos dos fármacos , Proteínas de Transporte Vesicular/metabolismo , Fator 6 Ativador da Transcrição/metabolismo , Animais , Benzamidas/química , Benzamidas/farmacologia , Compostos Bicíclicos com Pontes/uso terapêutico , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Mutação da Fase de Leitura , Heptanos/uso terapêutico , Humanos , Receptores de Imidazolinas/antagonistas & inibidores , Receptores de Imidazolinas/genética , Receptores de Imidazolinas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Rim/citologia , Rim/metabolismo , Rim/patologia , Nefropatias/metabolismo , Nefropatias/patologia , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Mucina-1/química , Mucina-1/genética , Mucina-1/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Proteínas de Transporte Vesicular/químicaRESUMO
Heart failure encompasses a heterogeneous set of clinical features that converge on impaired cardiac contractile function1,2 and presents a growing public health concern. Previous work has highlighted changes in both transcription and protein expression in failing hearts3,4, but may overlook molecular changes in less prevalent cell types. Here we identify extensive molecular alterations in failing hearts at single-cell resolution by performing single-nucleus RNA sequencing of nearly 600,000 nuclei in left ventricle samples from 11 hearts with dilated cardiomyopathy and 15 hearts with hypertrophic cardiomyopathy as well as 16 non-failing hearts. The transcriptional profiles of dilated or hypertrophic cardiomyopathy hearts broadly converged at the tissue and cell-type level. Further, a subset of hearts from patients with cardiomyopathy harbour a unique population of activated fibroblasts that is almost entirely absent from non-failing samples. We performed a CRISPR-knockout screen in primary human cardiac fibroblasts to evaluate this fibrotic cell state transition; knockout of genes associated with fibroblast transition resulted in a reduction of myofibroblast cell-state transition upon TGFß1 stimulation for a subset of genes. Our results provide insights into the transcriptional diversity of the human heart in health and disease as well as new potential therapeutic targets and biomarkers for heart failure.
Assuntos
Cardiomiopatia Dilatada , Cardiomiopatia Hipertrófica , Núcleo Celular , Perfilação da Expressão Gênica , Insuficiência Cardíaca , Análise de Célula Única , Sistemas CRISPR-Cas , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/patologia , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/patologia , Estudos de Casos e Controles , Núcleo Celular/genética , Células Cultivadas , Técnicas de Inativação de Genes , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Humanos , Miocárdio/metabolismo , Miocárdio/patologia , Miofibroblastos/metabolismo , Miofibroblastos/patologia , RNA-Seq , Transcrição Gênica , Fator de Crescimento Transformador beta1RESUMO
The identification of genetic and chemical perturbations with similar impacts on cell morphology can elucidate compounds' mechanisms of action or novel regulators of genetic pathways. Research on methods for identifying such similarities has lagged due to a lack of carefully designed and well-annotated image sets of cells treated with chemical and genetic perturbations. Here we create such a Resource dataset, CPJUMP1, in which each perturbed gene's product is a known target of at least two chemical compounds in the dataset. We systematically explore the directionality of correlations among perturbations that target the same protein encoded by a given gene, and we find that identifying matches between chemical and genetic perturbations is a challenging task. Our dataset and baseline analyses provide a benchmark for evaluating methods that measure perturbation similarities and impact, and more generally, learn effective representations of cellular state from microscopy images. Such advancements would accelerate the applications of image-based profiling of cellular states, such as uncovering drug mode of action or probing functional genomics.
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Processamento de Imagem Assistida por Computador , Humanos , Processamento de Imagem Assistida por Computador/métodos , Microscopia/métodosRESUMO
To assess telomerase as a cancer therapeutic target and determine adaptive mechanisms to telomerase inhibition, we modeled telomerase reactivation and subsequent extinction in T cell lymphomas arising in Atm(-/-) mice engineered with an inducible telomerase reverse transcriptase allele. Telomerase reactivation in the setting of telomere dysfunction enabled full malignant progression with alleviation of telomere dysfunction-induced checkpoints. These cancers possessed copy number alterations targeting key loci in human T cell lymphomagenesis. Upon telomerase extinction, tumor growth eventually slowed with reinstatement of telomere dysfunction-induced checkpoints, yet growth subsequently resumed as tumors acquired alternative lengthening of telomeres (ALT) and aberrant transcriptional networks centering on mitochondrial biology and oxidative defense. ALT+ tumors acquired amplification/overexpression of PGC-1ß, a master regulator of mitochondrial biogenesis and function, and they showed marked sensitivity to PGC-1ß or SOD2 knockdown. Genetic modeling of telomerase extinction reveals vulnerabilities that motivate coincidental inhibition of mitochondrial maintenance and oxidative defense mechanisms to enhance antitelomerase cancer therapy.
Assuntos
Mitocôndrias , Telomerase/antagonistas & inibidores , Homeostase do Telômero , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Técnicas de Silenciamento de Genes , Genes cdc , Humanos , Linfoma de Células T/genética , Linfoma de Células T/metabolismo , Linfoma de Células T/patologia , Camundongos , Mitocôndrias/metabolismo , Invasividade Neoplásica/patologia , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/genética , Espécies Reativas de Oxigênio/metabolismo , Receptores de Estrogênio/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Telomerase/genética , Telomerase/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
To determine the role of telomere dysfunction and telomerase reactivation in generating pro-oncogenic genomic events and in carcinoma progression, an inducible telomerase reverse transcriptase (mTert) allele was crossed onto a prostate cancer-prone mouse model null for Pten and p53 tumor suppressors. Constitutive telomerase deficiency and associated telomere dysfunction constrained cancer progression. In contrast, telomerase reactivation in the setting of telomere dysfunction alleviated intratumoral DNA-damage signaling and generated aggressive cancers with rearranged genomes and new tumor biological properties (bone metastases). Comparative oncogenomic analysis revealed numerous recurrent amplifications and deletions of relevance to human prostate cancer. Murine tumors show enrichment of the TGF-ß/SMAD4 network, and genetic validation studies confirmed the cooperative roles of Pten, p53, and Smad4 deficiencies in prostate cancer progression, including skeletal metastases. Thus, telomerase reactivation in tumor cells experiencing telomere dysfunction enables full malignant progression and provides a mechanism for acquisition of cancer-relevant genomic events endowing new tumor biological capabilities.
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Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Telomerase/metabolismo , Telômero/metabolismo , Animais , Neoplasias Ósseas/secundário , Linhagem Celular Tumoral , Cruzamentos Genéticos , Variações do Número de Cópias de DNA , Modelos Animais de Doenças , Feminino , Instabilidade Genômica , Humanos , Masculino , Camundongos , Proteína Supressora de Tumor p53/metabolismoRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers in western countries, with a median survival of 6 months and an extremely low percentage of long-term surviving patients. KRAS mutations are known to be a driver event of PDAC, but targeting mutant KRAS has proved challenging. Targeting oncogene-driven signalling pathways is a clinically validated approach for several devastating diseases. Still, despite marked tumour shrinkage, the frequency of relapse indicates that a fraction of tumour cells survives shut down of oncogenic signalling. Here we explore the role of mutant KRAS in PDAC maintenance using a recently developed inducible mouse model of mutated Kras (Kras(G12D), herein KRas) in a p53(LoxP/WT) background. We demonstrate that a subpopulation of dormant tumour cells surviving oncogene ablation (surviving cells) and responsible for tumour relapse has features of cancer stem cells and relies on oxidative phosphorylation for survival. Transcriptomic and metabolic analyses of surviving cells reveal prominent expression of genes governing mitochondrial function, autophagy and lysosome activity, as well as a strong reliance on mitochondrial respiration and a decreased dependence on glycolysis for cellular energetics. Accordingly, surviving cells show high sensitivity to oxidative phosphorylation inhibitors, which can inhibit tumour recurrence. Our integrated analyses illuminate a therapeutic strategy of combined targeting of the KRAS pathway and mitochondrial respiration to manage pancreatic cancer.
Assuntos
Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Mitocôndrias/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Autofagia , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Respiração Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Regulação Neoplásica da Expressão Gênica , Genes p53/genética , Glicólise , Lisossomos/metabolismo , Camundongos , Mitocôndrias/efeitos dos fármacos , Mutação/genética , Recidiva Local de Neoplasia/prevenção & controle , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fosforilação Oxidativa/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Recidiva , Transdução de Sinais , Neoplasias PancreáticasRESUMO
An ageing world population has fuelled interest in regenerative remedies that may stem declining organ function and maintain fitness. Unanswered is whether elimination of intrinsic instigators driving age-associated degeneration can reverse, as opposed to simply arrest, various afflictions of the aged. Such instigators include progressively damaged genomes. Telomerase-deficient mice have served as a model system to study the adverse cellular and organismal consequences of wide-spread endogenous DNA damage signalling activation in vivo. Telomere loss and uncapping provokes progressive tissue atrophy, stem cell depletion, organ system failure and impaired tissue injury responses. Here, we sought to determine whether entrenched multi-system degeneration in adult mice with severe telomere dysfunction can be halted or possibly reversed by reactivation of endogenous telomerase activity. To this end, we engineered a knock-in allele encoding a 4-hydroxytamoxifen (4-OHT)-inducible telomerase reverse transcriptase-oestrogen receptor (TERT-ER) under transcriptional control of the endogenous TERT promoter. Homozygous TERT-ER mice have short dysfunctional telomeres and sustain increased DNA damage signalling and classical degenerative phenotypes upon successive generational matings and advancing age. Telomerase reactivation in such late generation TERT-ER mice extends telomeres, reduces DNA damage signalling and associated cellular checkpoint responses, allows resumption of proliferation in quiescent cultures, and eliminates degenerative phenotypes across multiple organs including testes, spleens and intestines. Notably, somatic telomerase reactivation reversed neurodegeneration with restoration of proliferating Sox2(+) neural progenitors, Dcx(+) newborn neurons, and Olig2(+) oligodendrocyte populations. Consistent with the integral role of subventricular zone neural progenitors in generation and maintenance of olfactory bulb interneurons, this wave of telomerase-dependent neurogenesis resulted in alleviation of hyposmia and recovery of innate olfactory avoidance responses. Accumulating evidence implicating telomere damage as a driver of age-associated organ decline and disease risk and the marked reversal of systemic degenerative phenotypes in adult mice observed here support the development of regenerative strategies designed to restore telomere integrity.
Assuntos
Envelhecimento/metabolismo , Envelhecimento/patologia , Telomerase/deficiência , Telomerase/metabolismo , Envelhecimento/efeitos dos fármacos , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Encéfalo/anatomia & histologia , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Dano ao DNA/efeitos dos fármacos , Proteína Duplacortina , Ativação Enzimática/efeitos dos fármacos , Reativadores Enzimáticos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Bainha de Mielina/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/enzimologia , Células-Tronco Neurais/patologia , Tamanho do Órgão/efeitos dos fármacos , Fenótipo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Medicina Regenerativa , Olfato/efeitos dos fármacos , Olfato/fisiologia , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia , Telomerase/genética , Telômero/efeitos dos fármacos , Telômero/metabolismo , Telômero/patologiaRESUMO
Telomere dysfunction activates p53-mediated cellular growth arrest, senescence and apoptosis to drive progressive atrophy and functional decline in high-turnover tissues. The broader adverse impact of telomere dysfunction across many tissues including more quiescent systems prompted transcriptomic network analyses to identify common mechanisms operative in haematopoietic stem cells, heart and liver. These unbiased studies revealed profound repression of peroxisome proliferator-activated receptor gamma, coactivator 1 alpha and beta (PGC-1α and PGC-1ß, also known as Ppargc1a and Ppargc1b, respectively) and the downstream network in mice null for either telomerase reverse transcriptase (Tert) or telomerase RNA component (Terc) genes. Consistent with PGCs as master regulators of mitochondrial physiology and metabolism, telomere dysfunction is associated with impaired mitochondrial biogenesis and function, decreased gluconeogenesis, cardiomyopathy, and increased reactive oxygen species. In the setting of telomere dysfunction, enforced Tert or PGC-1α expression or germline deletion of p53 (also known as Trp53) substantially restores PGC network expression, mitochondrial respiration, cardiac function and gluconeogenesis. We demonstrate that telomere dysfunction activates p53 which in turn binds and represses PGC-1α and PGC-1ß promoters, thereby forging a direct link between telomere and mitochondrial biology. We propose that this telomere-p53-PGC axis contributes to organ and metabolic failure and to diminishing organismal fitness in the setting of telomere dysfunction.
Assuntos
Mitocôndrias/metabolismo , Mitocôndrias/patologia , Telômero/metabolismo , Telômero/patologia , Trifosfato de Adenosina/biossíntese , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Cardiomiopatias/induzido quimicamente , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Cardiomiopatias/fisiopatologia , Proliferação de Células , DNA Mitocondrial/análise , Doxorrubicina/toxicidade , Gluconeogênese , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Fígado/citologia , Fígado/metabolismo , Camundongos , Miocárdio/citologia , Miocárdio/metabolismo , RNA/genética , Espécies Reativas de Oxigênio/metabolismo , Telomerase/deficiência , Telomerase/genética , Telômero/enzimologia , Telômero/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Recurrence of multiple myeloma (MM) after therapy suggests the presence of tumor-initiating subpopulations. In our study, we performed flow cytometry-based Hoechst 33342 staining to evaluate the existence of a MM population with stem-like features known as side population (SP) cells. SP cells exhibit substantial heterogeneity in MM cell lines and primary MM cells; express CD138 antigen in MM cell lines; display higher mRNA expression and functional activity of ABCG2 transporter; and have a higher proliferation index compared with non-SP cells. We observed evidence for clonogenic potential of SP cells, as well as the ability of SP cells to regenerate original population. Moreover, SP cells revealed higher tumorigenicity compared with non-SP cells. Importantly, lenalidomide decreased the percentage and clonogenicity of SP cells, and also induced phosphorylation changes in Akt, GSK-3α/ß, MEK1, c-Jun, p53, and p70S6K in SP cells. Adherence to bone marrow stromal cells (BMSCs) increased the percentage, viability, and proliferation potential of SP cells. Lenalidomide and thalidomide abrogated this stimulatory effect of BMSCs and significantly decreased the percentage of SP cells. Our studies demonstrate a novel mechanism of action for lenalidomide, namely targeting SP fraction, providing the framework for new therapeutic strategies targeting subpopulations of MM cells including presumptive stem cells.
Assuntos
Antineoplásicos/farmacologia , Mieloma Múltiplo , Células-Tronco Neoplásicas/efeitos dos fármacos , Talidomida/análogos & derivados , Transportadores de Cassetes de Ligação de ATP/genética , Inibidores da Angiogênese/farmacologia , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/fisiologia , Divisão Celular/efeitos dos fármacos , Fracionamento Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Resistencia a Medicamentos Antineoplásicos , Humanos , Lenalidomida , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Mieloma Múltiplo/fisiopatologia , Recidiva Local de Neoplasia/prevenção & controle , Células-Tronco Neoplásicas/patologia , Transdução de Sinais/efeitos dos fármacos , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Células Estromais/fisiologia , Sindecana-1/metabolismo , Talidomida/farmacologiaRESUMO
We present the nELISA, a high-throughput, high-fidelity, and high-plex protein profiling platform. DNA oligonucleotides are used to pre-assemble antibody pairs on spectrally encoded microparticles and perform displacement-mediated detection. Spatial separation between non-cognate antibodies prevents the rise of reagent-driven cross-reactivity, while read-out is performed cost-efficiently and at high-throughput using flow cytometry. We assembled an inflammatory panel of 191 targets that were multiplexed without cross-reactivity or impact on performance vs 1-plex signals, with sensitivities as low as 0.1pg/mL and measurements spanning 7 orders of magnitude. We then performed a large-scale secretome perturbation screen of peripheral blood mononuclear cells (PBMCs), with cytokines as both perturbagens and read-outs, measuring 7,392 samples and generating ~1.5M protein datapoints in under a week, a significant advance in throughput compared to other highly multiplexed immunoassays. We uncovered 447 significant cytokine responses, including multiple putatively novel ones, that were conserved across donors and stimulation conditions. We also validated the nELISA's use in phenotypic screening, and propose its application to drug discovery.
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Cellular exposure to free fatty acids (FFAs) is implicated in the pathogenesis of obesity-associated diseases. However, there are no scalable approaches to comprehensively assess the diverse FFAs circulating in human plasma. Furthermore, assessing how FFA-mediated processes interact with genetic risk for disease remains elusive. Here, we report the design and implementation of fatty acid library for comprehensive ontologies (FALCON), an unbiased, scalable, and multimodal interrogation of 61 structurally diverse FFAs. We identified a subset of lipotoxic monounsaturated fatty acids associated with decreased membrane fluidity. Furthermore, we prioritized genes that reflect the combined effects of harmful FFA exposure and genetic risk for type 2 diabetes (T2D). We found that c-MAF-inducing protein (CMIP) protects cells from FFA exposure by modulating Akt signaling. In sum, FALCON empowers the study of fundamental FFA biology and offers an integrative approach to identify much needed targets for diverse diseases associated with disordered FFA metabolism.
Assuntos
Diabetes Mellitus Tipo 2 , Ácidos Graxos não Esterificados , Humanos , Ácidos Graxos não Esterificados/metabolismo , Ácidos Graxos , Transdução de Sinais , BiologiaRESUMO
Cellular exposure to free fatty acids (FFA) is implicated in the pathogenesis of obesity-associated diseases. However, studies to date have assumed that a few select FFAs are representative of broad structural categories, and there are no scalable approaches to comprehensively assess the biological processes induced by exposure to diverse FFAs circulating in human plasma. Furthermore, assessing how these FFA- mediated processes interact with genetic risk for disease remains elusive. Here we report the design and implementation of FALCON (Fatty Acid Library for Comprehensive ONtologies) as an unbiased, scalable and multimodal interrogation of 61 structurally diverse FFAs. We identified a subset of lipotoxic monounsaturated fatty acids (MUFAs) with a distinct lipidomic profile associated with decreased membrane fluidity. Furthermore, we developed a new approach to prioritize genes that reflect the combined effects of exposure to harmful FFAs and genetic risk for type 2 diabetes (T2D). Importantly, we found that c-MAF inducing protein (CMIP) protects cells from exposure to FFAs by modulating Akt signaling and we validated the role of CMIP in human pancreatic beta cells. In sum, FALCON empowers the study of fundamental FFA biology and offers an integrative approach to identify much needed targets for diverse diseases associated with disordered FFA metabolism. Highlights: FALCON (Fatty Acid Library for Comprehensive ONtologies) enables multimodal profiling of 61 free fatty acids (FFAs) to reveal 5 FFA clusters with distinct biological effectsFALCON is applicable to many and diverse cell typesA subset of monounsaturated FAs (MUFAs) equally or more toxic than canonical lipotoxic saturated FAs (SFAs) leads to decreased membrane fluidityNew approach prioritizes genes that represent the combined effects of environmental (FFA) exposure and genetic risk for diseaseC-Maf inducing protein (CMIP) is identified as a suppressor of FFA-induced lipotoxicity via Akt-mediated signaling.
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In image-based profiling, software extracts thousands of morphological features of cells from multi-channel fluorescence microscopy images, yielding single-cell profiles that can be used for basic research and drug discovery. Powerful applications have been proven, including clustering chemical and genetic perturbations on the basis of their similar morphological impact, identifying disease phenotypes by observing differences in profiles between healthy and diseased cells and predicting assay outcomes by using machine learning, among many others. Here, we provide an updated protocol for the most popular assay for image-based profiling, Cell Painting. Introduced in 2013, it uses six stains imaged in five channels and labels eight diverse components of the cell: DNA, cytoplasmic RNA, nucleoli, actin, Golgi apparatus, plasma membrane, endoplasmic reticulum and mitochondria. The original protocol was updated in 2016 on the basis of several years' experience running it at two sites, after optimizing it by visual stain quality. Here, we describe the work of the Joint Undertaking for Morphological Profiling Cell Painting Consortium, to improve upon the assay via quantitative optimization by measuring the assay's ability to detect morphological phenotypes and group similar perturbations together. The assay gives very robust outputs despite various changes to the protocol, and two vendors' dyes work equivalently well. We present Cell Painting version 3, in which some steps are simplified and several stain concentrations can be reduced, saving costs. Cell culture and image acquisition take 1-2 weeks for typically sized batches of ≤20 plates; feature extraction and data analysis take an additional 1-2 weeks.This protocol is an update to Nat. Protoc. 11, 1757-1774 (2016): https://doi.org/10.1038/nprot.2016.105.
Assuntos
Técnicas de Cultura de Células , Processamento de Imagem Assistida por Computador , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência , Mitocôndrias , SoftwareRESUMO
A primary obstacle in translating genetic associations with disease into therapeutic strategies is elucidating the cellular programs affected by genetic risk variants and effector genes. Here, we introduce LipocyteProfiler, a cardiometabolic-disease-oriented high-content image-based profiling tool that enables evaluation of thousands of morphological and cellular profiles that can be systematically linked to genes and genetic variants relevant to cardiometabolic disease. We show that LipocyteProfiler allows surveillance of diverse cellular programs by generating rich context- and process-specific cellular profiles across hepatocyte and adipocyte cell-state transitions. We use LipocyteProfiler to identify known and novel cellular mechanisms altered by polygenic risk of metabolic disease, including insulin resistance, fat distribution, and the polygenic contribution to lipodystrophy. LipocyteProfiler paves the way for large-scale forward and reverse deep phenotypic profiling in lipocytes and provides a framework for the unbiased identification of causal relationships between genetic variants and cellular programs relevant to human disease.
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Type 2 diabetes is marked by progressive ß-cell failure, leading to loss of ß-cell mass. Increased levels of circulating glucose and free fatty acids associated with obesity lead to ß-cell glucolipotoxicity. There are currently no therapeutic options to address this facet of ß-cell loss in obese type 2 diabetes patients. To identify small molecules capable of protecting ß-cells, we performed a high-throughput screen of 20,876 compounds in the rat insulinoma cell line INS-1E in the presence of elevated glucose and palmitate. We found 312 glucolipotoxicity-protective small molecules (1.49% hit rate) capable of restoring INS-1E viability, and we focused on 17 with known biological targets. 16 of the 17 compounds were kinase inhibitors with activity against specific families including but not limited to cyclin-dependent kinases (CDK), PI-3 kinase (PI3K), Janus kinase (JAK), and Rho-associated kinase 2 (ROCK2). 7 of the 16 kinase inhibitors were PI3K inhibitors. Validation studies in dissociated human islets identified 10 of the 17 compounds, namely, KD025, ETP-45658, BMS-536924, AT-9283, PF-03814735, torin-2, AZD5438, CP-640186, ETP-46464, and GSK2126458 that reduced glucolipotoxicity-induced ß-cell death. These 10 compounds decreased markers of glucolipotoxicity including caspase activation, mitochondrial depolarization, and increased calcium flux. Together, these results provide a path forward toward identifying novel treatments to preserve ß-cell viability in the face of glucolipotoxicity.
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Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Animais , Apoptose , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo , Palmitatos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , RatosRESUMO
Sickle cell disease (SCD) is an inherited disorder of hemoglobin (Hb); approximately 300,000 babies are born worldwide with SCD each year. In SCD, fibers of polymerized sickle Hb (HbS) form in red blood cells (RBCs), which cause RBCs to develop their characteristic "sickled" shape, resulting in hemolytic anemia and numerous vascular complications including vaso-occlusive crises. The development of novel antisickling compounds will provide new therapeutic options for patients with SCD. We developed a high-throughput "sickling assay" that is based on an automated high-content imaging system to quantify the effects of hypoxia on the shape and size of RBCs from HbSS SCD patients (SS RBCs). We used this assay to screen thousands of compounds for their ability to inhibit sickling. In the assay, voxelotor (an FDA-approved medication used to treat SCD) prevented sickling with a z'-factor > 0.4, suggesting that the assay is capable of identifying compounds that inhibit sickling. We screened the Broad Repurposing Library of 5393 compounds for their ability to prevent sickling in 4% oxygen/96% nitrogen. We identified two compounds, SNS-314 mesylate and voxelotor itself, that successfully prevented sickling. SNS-314 mesylate prevented sickling in the absence of oxygen, while voxelotor did not, suggesting that SNS-314 mesylate acts by a mechanism that is different from that of voxelotor. The sickling assay described in this study will permit the identification of additional, novel antisickling compounds, which will potentially expand the therapeutic options for SCD.
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Morphological and gene expression profiling can cost-effectively capture thousands of features in thousands of samples across perturbations by disease, mutation, or drug treatments, but it is unclear to what extent the two modalities capture overlapping versus complementary information. Here, using both the L1000 and Cell Painting assays to profile gene expression and cell morphology, respectively, we perturb human A549 lung cancer cells with 1,327 small molecules from the Drug Repurposing Hub across six doses, providing a data resource including dose-response data from both assays. The two assays capture both shared and complementary information for mapping cell state. Cell Painting profiles from compound perturbations are more reproducible and show more diversity but measure fewer distinct groups of features. Applying unsupervised and supervised methods to predict compound mechanisms of action (MOAs) and gene targets, we find that the two assays not only provide a partially shared but also a complementary view of drug mechanisms. Given the numerous applications of profiling in biology, our analyses provide guidance for planning experiments that profile cells for detecting distinct cell types, disease phenotypes, and response to chemical or genetic perturbations.
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Perfilação da Expressão Gênica , Humanos , Perfilação da Expressão Gênica/métodos , FenótipoRESUMO
Genetic and chemical perturbations impact diverse cellular phenotypes, including multiple indicators of cell health. These readouts reveal toxicity and antitumorigenic effects relevant to drug discovery and personalized medicine. We developed two customized microscopy assays, one using four targeted reagents and the other three targeted reagents, to collectively measure 70 specific cell health phenotypes including proliferation, apoptosis, reactive oxygen species, DNA damage, and cell cycle stage. We then tested an approach to predict multiple cell health phenotypes using Cell Painting, an inexpensive and scalable image-based morphology assay. In matched CRISPR perturbations of three cancer cell lines, we collected both Cell Painting and cell health data. We found that simple machine learning algorithms can predict many cell health readouts directly from Cell Painting images, at less than half the cost. We hypothesized that these models can be applied to accurately predict cell health assay outcomes for any future or existing Cell Painting dataset. For Cell Painting images from a set of 1500+ compound perturbations across multiple doses, we validated predictions by orthogonal assay readouts. We provide a web app to browse predictions: http://broad.io/cell-health-app. Our approach can be used to add cell health annotations to Cell Painting datasets.
Assuntos
Células/citologia , Previsões/métodos , Processamento de Imagem Assistida por Computador/métodos , Algoritmos , Bioensaio , Linhagem Celular , Humanos , Aprendizado de Máquina , Microscopia , FenótipoRESUMO
Mutations affecting mitochondrial coenzyme Q (CoQ) biosynthesis lead to kidney failure due to selective loss of podocytes, essential cells of the kidney filter. Curiously, neighboring tubular epithelial cells are spared early in disease despite higher mitochondrial content. We sought to illuminate noncanonical, cell-specific roles for CoQ, independently of the electron transport chain (ETC). Here, we demonstrate that CoQ depletion caused by Pdss2 enzyme deficiency in podocytes results in perturbations in polyunsaturated fatty acid (PUFA) metabolism and the Braf/Mapk pathway rather than ETC dysfunction. Single-nucleus RNA-Seq from kidneys of Pdss2kd/kd mice with nephrotic syndrome and global CoQ deficiency identified a podocyte-specific perturbation of the Braf/Mapk pathway. Treatment with GDC-0879, a Braf/Mapk-targeting compound, ameliorated kidney disease in Pdss2kd/kd mice. Mechanistic studies in Pdss2-depleted podocytes revealed a previously unknown perturbation in PUFA metabolism that was confirmed in vivo. Gpx4, an enzyme that protects against PUFA-mediated lipid peroxidation, was elevated in disease and restored after GDC-0879 treatment. We demonstrate broader human disease relevance by uncovering patterns of GPX4 and Braf/Mapk pathway gene expression in tissue from patients with kidney diseases. Our studies reveal ETC-independent roles for CoQ in podocytes and point to Braf/Mapk as a candidate pathway for the treatment of kidney diseases.
Assuntos
Ataxia/metabolismo , Indenos/farmacologia , Nefropatias/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Doenças Mitocondriais/metabolismo , Debilidade Muscular/metabolismo , Podócitos/metabolismo , Proteínas Proto-Oncogênicas B-raf/metabolismo , Pirazóis/farmacologia , Ubiquinona/deficiência , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Animais , Ataxia/tratamento farmacológico , Ataxia/genética , Ataxia/patologia , Sistemas de Liberação de Medicamentos , Células HEK293 , Humanos , Nefropatias/tratamento farmacológico , Nefropatias/genética , Nefropatias/patologia , Peroxidação de Lipídeos/genética , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Doenças Mitocondriais/tratamento farmacológico , Doenças Mitocondriais/genética , Doenças Mitocondriais/patologia , Debilidade Muscular/tratamento farmacológico , Debilidade Muscular/genética , Debilidade Muscular/patologia , Podócitos/patologia , Proteínas Proto-Oncogênicas B-raf/genética , RNA-Seq , Ubiquinona/genética , Ubiquinona/metabolismoRESUMO
Some osteoblasts embed within bone matrix, change shape, and become dendrite-bearing osteocytes. The circuitry that drives dendrite formation during "osteocytogenesis" is poorly understood. Here we show that deletion of Sp7 in osteoblasts and osteocytes causes defects in osteocyte dendrites. Profiling of Sp7 target genes and binding sites reveals unexpected repurposing of this transcription factor to drive dendrite formation. Osteocrin is a Sp7 target gene that promotes osteocyte dendrite formation and rescues defects in Sp7-deficient mice. Single-cell RNA-sequencing demonstrates defects in osteocyte maturation in the absence of Sp7. Sp7-dependent osteocyte gene networks are associated with human skeletal diseases. Moreover, humans with a SP7R316C mutation show defective osteocyte morphology. Sp7-dependent genes that mark osteocytes are enriched in neurons, highlighting shared features between osteocytic and neuronal connectivity. These findings reveal a role for Sp7 and its target gene Osteocrin in osteocytogenesis, revealing that pathways that control osteocyte development influence human bone diseases.