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Three-dimensional (3D) cell culture in vitro promises to improve representation of neuron physiology in vivo. This inspired development of a 3D culture platform for LUHMES (Lund Human Mesencephalic) dopaminergic neurons for high-throughput screening (HTS) of chemicals for neurotoxicity. Three culture platforms, adhesion (2D-monolayer), 3D-suspension, and 3D-shaken, were compared to monitor mRNA expression of seven neuronal marker genes, DCX, DRD2, ENO2, NEUROD4, SYN1, TH, and TUBB3. These seven marker genes reached similar maxima in all three formats, with the two 3D platforms showing similar kinetics, whereas several markers peaked earlier in 2D adhesion compared to both 3D culture platforms. The differentiated LUHMES (dLUHMES) neurons treated with ziram, methylmercury or thiram dynamically increased expression of metallothionein biomarker genes MT1G, MT1E and MT2A at 6 h. These gene expression increases were generally more dynamic in 2D adhesion cultures than in 3D cultures, but were generally comparable between 3D-suspension and 3D-u plate (low binding) platforms. Finally, we adapted 3D-suspension culture of dLUHMES and neural stem cells to 1536 well plates with a HTS cytotoxicity assay. This HTS assay revealed that cytotoxicity IC50 values were not significantly different between adhesion and 3D-suspension platforms for 31 of 34 (91%) neurotoxicants tested, whereas IC50 values were significantly different for at least two toxicants. In summary, the 3D-suspension culture platform for LUHMES dopaminergic neurons supported full differentiation and reproducible assay results, enabling quantitative HTS (qHTS) for cytotoxicity in 1536 well format with a Robust Z' score of 0.68.
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Neurônios Dopaminérgicos , Ensaios de Triagem em Larga Escala , Ensaios de Triagem em Larga Escala/métodos , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Humanos , Técnicas de Cultura de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Técnicas de Cultura de Células em Três Dimensões/métodos , Biomarcadores/metabolismo , Compostos de Metilmercúrio/toxicidade , Neurotoxinas/toxicidade , Linhagem Celular , Células CultivadasRESUMO
Embryoid bodies (EBs) and self-organizing organoids derived from human pluripotent stem cells (hPSCs) recapitulate tissue development in a dish and hold great promise for disease modeling and drug development. However, current protocols are hampered by cellular stress and apoptosis during cell aggregation, resulting in variability and impaired cell differentiation. Here, we demonstrate that EBs and various organoid models (e.g., brain, gut, kidney) can be optimized by using the small molecule cocktail named CEPT (chroman 1, emricasan, polyamines, trans-ISRIB), a polypharmacological approach that ensures cytoprotection and cell survival. Application of CEPT for just 24 h during cell aggregation has long-lasting consequences affecting morphogenesis, gene expression, cellular differentiation, and organoid function. Various qualification methods confirmed that CEPT treatment enhanced experimental reproducibility and consistently improved EB and organoid fitness as compared to the widely used ROCK inhibitor Y-27632. Collectively, we discovered that stress-free cell aggregation and superior cell survival in the presence of CEPT are critical quality control determinants that establish a robust foundation for bioengineering complex tissue and organ models.
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Corpos Embrioides , Células-Tronco Pluripotentes , Humanos , Corpos Embrioides/metabolismo , Reprodutibilidade dos Testes , Organoides , Diferenciação CelularRESUMO
Human gliogenesis remains poorly understood, and derivation of astrocytes from human pluripotent stem cells (hPSCs) is inefficient and cumbersome. Here, we report controlled glial differentiation from hPSCs that bypasses neurogenesis, which otherwise precedes astrogliogenesis during brain development and in vitro differentiation. hPSCs were first differentiated into radial glial cells (RGCs) resembling resident RGCs of the fetal telencephalon, and modulation of specific cell signaling pathways resulted in direct and stepwise induction of key astroglial markers (NFIA, NFIB, SOX9, CD44, S100B, glial fibrillary acidic protein [GFAP]). Transcriptomic and genome-wide epigenetic mapping and single-cell analysis confirmed RGC-to-astrocyte differentiation, obviating neurogenesis and the gliogenic switch. Detailed molecular and cellular characterization experiments uncovered new mechanisms and markers for human RGCs and astrocytes. In summary, establishment of a glia-exclusive neural lineage progression model serves as a unique serum-free platform of manufacturing large numbers of RGCs and astrocytes for neuroscience, disease modeling (e.g., Alexander disease), and regenerative medicine.
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Astrócitos , Células-Tronco Pluripotentes , Humanos , Astrócitos/metabolismo , Células Ependimogliais/metabolismo , Células-Tronco Pluripotentes/metabolismo , Neurogênese , Diferenciação Celular , Proteína Glial Fibrilar Ácida/metabolismoRESUMO
SEQUIN is a web-based application (app) that allows fast and intuitive analysis of RNA sequencing data derived for model organisms, tissues, and single cells. Integrated app functions enable uploading datasets, quality control, gene set enrichment, data visualization, and differential gene expression analysis. We also developed the iPSC Profiler, a practical gene module scoring tool that helps measure and compare pluripotent and differentiated cell types. Benchmarking to other commercial and non-commercial products underscored several advantages of SEQUIN. Freely available to the public, SEQUIN empowers scientists using interdisciplinary methods to investigate and present transcriptome data firsthand with state-of-the-art statistical methods. Hence, SEQUIN helps democratize and increase the throughput of interrogating biological questions using next-generation sequencing data with single-cell resolution.
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Software , Transcriptoma , RNA-Seq , Transcriptoma/genética , Análise de Sequência de RNA/métodos , Redes Reguladoras de GenesRESUMO
Development of new non-addictive analgesics requires advanced strategies to differentiate human pluripotent stem cells (hPSCs) into relevant cell types. Following principles of developmental biology and translational applicability, here we developed an efficient stepwise differentiation method for peptidergic and non-peptidergic nociceptors. By modulating specific cell signaling pathways, hPSCs were first converted into SOX10+ neural crest, followed by differentiation into sensory neurons. Detailed characterization, including ultrastructural analysis, confirmed that the hPSC-derived nociceptors displayed cellular and molecular features comparable to native dorsal root ganglion (DRG) neurons, and expressed high-threshold primary sensory neuron markers, transcription factors, neuropeptides, and over 150 ion channels and receptors relevant for pain research and axonal growth/regeneration studies (e.g., TRPV1, NAV1.7, NAV1.8, TAC1, CALCA, GAP43, DPYSL2, NMNAT2). Moreover, after confirming robust functional activities and differential response to noxious stimuli and specific drugs, a robotic cell culture system was employed to produce large quantities of human sensory neurons, which can be used to develop nociceptor-selective analgesics.
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Neurônios , Células-Tronco Pluripotentes , Humanos , Neurônios/metabolismo , Nociceptores , Diferenciação Celular , Transdução de Sinais , Gânglios Espinais/metabolismo , Células Receptoras SensoriaisRESUMO
Human pluripotent stem cells (hPSCs) are inherently sensitive cells. Single-cell dissociation and the establishment of clonal cell lines have been long-standing challenges. This inefficiency of cell cloning represents a major obstacle for the standardization and streamlining of gene editing in induced pluripotent stem cells for basic and translational research. Here we describe a chemically defined protocol for robust single-cell cloning using microfluidics-based cell sorting in combination with the CEPT small-molecule cocktail. This advanced strategy promotes the viability and cell fitness of self-renewing stem cells. The use of low-pressure microfluidic cell dispensing ensures gentle and rapid dispensing of single cells into 96- and 384-well plates, while the fast-acting CEPT cocktail minimizes cellular stress and maintains cell structure and function immediately after cell dissociation. The protocol also facilitates clone picking and produces genetically stable clonal cell lines from hPSCs in a safe and cost-efficient fashion. Depending on the proliferation rate of the clone derived from a single cell, this protocol can be completed in 7-14 d and requires experience with aseptic cell culture techniques. Altogether, the relative ease, scalability and robustness of this workflow should boost gene editing in hPSCs and leverage a wide range of applications, including cell line development (e.g., reporter and isogenic cell lines), disease modeling and applications in regenerative medicine.
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Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes , Humanos , Técnicas de Cultura de Células/métodos , Linhagem Celular , Diferenciação Celular , Clonagem MolecularRESUMO
This chapter was inadvertently published with an error, the ninth point under 'Materials' section must read.
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Human pluripotent stem cells (hPSCs), such as induced pluripotent stem cells (iPSCs), hold great promise for drug discovery, toxicology studies, and regenerative medicine. Here, we describe standardized protocols and experimental procedures that combine automated cell culture for scalable production of hPSCs with quantitative high-throughput screening (qHTS) in miniaturized 384-well plates. As a proof of principle, we established dose-response assessments and determined optimal concentrations of 12 small molecule compounds that are commonly used in the stem cell field. Multi-parametric analysis of readouts from diverse assays including cell viability, mitochondrial membrane potential, plasma membrane integrity, and ATP production was used to distinguish normal biological responses from cellular stress induced by small molecule treatment. Collectively, the establishment of integrated workflows for cell manufacturing, qHTS, high-content imaging, and data analysis provides an end-to-end platform for industrial-scale projects and should leverage the drug discovery process using hPSC-derived cell types.
Assuntos
Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala/métodos , HumanosRESUMO
Efficient translation of human induced pluripotent stem cells (hiPSCs) requires scalable cell manufacturing strategies for optimal self-renewal and functional differentiation. Traditional manual cell culture is variable and labor intensive, posing challenges for high-throughput applications. Here, we established a robotic platform and automated all essential steps of hiPSC culture and differentiation under chemically defined conditions. This approach allowed rapid and standardized manufacturing of billions of hiPSCs that can be produced in parallel from up to 90 different patient- and disease-specific cell lines. Moreover, we established automated multi-lineage differentiation and generated functional neurons, cardiomyocytes, and hepatocytes. To validate our approach, we compared robotic and manual cell culture operations and performed comprehensive molecular and cellular characterizations (e.g., single-cell transcriptomics, mass cytometry, metabolism, electrophysiology) to benchmark industrial-scale cell culture operations toward building an integrated platform for efficient cell manufacturing for disease modeling, drug screening, and cell therapy.
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Técnicas de Cultura de Células/métodos , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Robótica , Automação , Linhagem da Célula , Células Cultivadas , Corpos Embrioides/citologia , Hepatócitos/citologia , Hepatócitos/virologia , Células-Tronco Embrionárias Humanas/citologia , Humanos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/virologia , Neurônios/citologia , RNA-Seq , Padrões de Referência , Análise de Célula Única , Infecção por Zika virus/patologiaRESUMO
Every year cervical cancer affects more than 300,000 people, and on average one woman is diagnosed with cervical cancer every minute. Early diagnosis and classification of cervical lesions greatly boosts up the chance of successful treatments of patients, and automated diagnosis and classification of cervical lesions from Papanicolaou (Pap) smear images have become highly demanded. To the authors' best knowledge, this is the first study of fully automated cervical lesions analysis on whole slide images (WSIs) of conventional Pap smear samples. The presented deep learning-based cervical lesions diagnosis system is demonstrated to be able to detect high grade squamous intraepithelial lesions (HSILs) or higher (squamous cell carcinoma; SQCC), which usually immediately indicate patients must be referred to colposcopy, but also to rapidly process WSIs in seconds for practical clinical usage. We evaluate this framework at scale on a dataset of 143 whole slide images, and the proposed method achieves a high precision 0.93, recall 0.90, F-measure 0.88, and Jaccard index 0.84, showing that the proposed system is capable of segmenting HSILs or higher (SQCC) with high precision and reaches sensitivity comparable to the referenced standard produced by pathologists. Based on Fisher's Least Significant Difference (LSD) test (P < 0.0001), the proposed method performs significantly better than the two state-of-the-art benchmark methods (U-Net and SegNet) in precision, F-Measure, Jaccard index. For the run time analysis, the proposed method takes only 210 seconds to process a WSI and is 20 times faster than U-Net and 19 times faster than SegNet, respectively. In summary, the proposed method is demonstrated to be able to both detect HSILs or higher (SQCC), which indicate patients for further treatments, including colposcopy and surgery to remove the lesion, and rapidly processing WSIs in seconds for practical clinical usages.
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Inteligência Artificial , Tomada de Decisões Assistida por Computador , Detecção Precoce de Câncer/métodos , Lesões Intraepiteliais Escamosas/diagnóstico , Displasia do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/diagnóstico , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Teste de Papanicolaou , Esfregaço VaginalRESUMO
Human pluripotent stem cells (hPSCs) are capable of extensive self-renewal yet remain highly sensitive to environmental perturbations in vitro, posing challenges to their therapeutic use. There is an urgent need to advance strategies that ensure safe and robust long-term growth and functional differentiation of these cells. Here, we deployed high-throughput screening strategies to identify a small-molecule cocktail that improves viability of hPSCs and their differentiated progeny. The combination of chroman 1, emricasan, polyamines, and trans-ISRIB (CEPT) enhanced cell survival of genetically stable hPSCs by simultaneously blocking several stress mechanisms that otherwise compromise cell structure and function. CEPT provided strong improvements for several key applications in stem-cell research, including routine cell passaging, cryopreservation of pluripotent and differentiated cells, embryoid body (EB) and organoid formation, single-cell cloning, and genome editing. Thus, CEPT represents a unique poly-pharmacological strategy for comprehensive cytoprotection, providing a rationale for efficient and safe utilization of hPSCs.
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Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Crioprotetores/farmacologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Polifarmacologia , Técnicas de Cultura de Células , Criopreservação/métodos , Crioprotetores/química , Regulação da Expressão Gênica/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Humanos , Células-Tronco Pluripotentes/fisiologia , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismoRESUMO
Identification of toxicants that underlie neurological diseases is a neglected area awaiting a valid strategy to identify such toxicants. We sought biomarkers that respond to known neurotoxicants in LUHMES immortalized neurons and evaluated these biomarkers for use in screening libraries of environmental toxicants. LUHMES immortalized human dopaminergic neurons were surveyed by RNA sequencing following challenge with parkinsonian toxicants rotenone, 6-hydroxydopamine, MPP+, and ziram (zinc dimethyldithiocarbamate; Zn2+DDC2), as well as additional toxicants paraquat, MS275, and methylmercury. The metallothionein gene MT1G was the most dynamic gene expression response to all seven toxicants. Multiple toxicants also increased transcripts for SLC30A1 and SLC30A2 zinc secretion transporters, the SLC7A11 xCT cystine/glutamate antiporter important for glutathione synthesis, DNA damage inducible transcript 3 (DDIT3), and secreted growth factors FIBIN and CXCL12, whereas several toxicants decreased expression of the apelin growth factor (APLN). These biomarker genes revealed stress responses to many toxicants at sub-cytotoxic concentrations. Since several of these biomarker genes and prior neurological disease studies implicated disruption of metal distribution, we tested metal chelator thiram (dimethyldithiocarbamate, DDC), ziram, and several other metals and metal chelates for cytotoxicity and induction of MT1G expression. Metals and chelators that caused dynamic increases in MT1G expression also caused cytotoxicity, except Ni2+DDC2 induced MT1G at 5 µM, but lacked cytotoxicity up to 100 µM. These results bolster prior work suggesting that neurons are characteristically sensitive to depletion of glutathione or to disruption of cellular metal distribution and provide biomarkers to search for such neurotoxicants in chemical libraries.
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Substâncias Perigosas/toxicidade , Metalotioneína/biossíntese , Metalotioneína/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Biomarcadores/metabolismo , Linhagem Celular Transformada , Relação Dose-Resposta a Droga , Guanidinas/toxicidade , Humanos , Neonicotinoides/toxicidade , Nitrocompostos/toxicidadeRESUMO
Efficient translation of human induced pluripotent stem cells (hiPSCs) depends on implementing scalable cell manufacturing strategies that ensure optimal self-renewal and functional differentiation. Currently, manual culture of hiPSCs is highly variable and labor-intensive posing significant challenges for high-throughput applications. Here, we established a robotic platform and automated all essential steps of hiPSC culture and differentiation under chemically defined conditions. This streamlined approach allowed rapid and standardized manufacturing of billions of hiPSCs that can be produced in parallel from up to 90 different patient-and disease-specific cell lines. Moreover, we established automated multi-lineage differentiation to generate primary embryonic germ layers and more mature phenotypes such as neurons, cardiomyocytes, and hepatocytes. To validate our approach, we carefully compared robotic and manual cell culture and performed molecular and functional cell characterizations (e.g. bulk culture and single-cell transcriptomics, mass cytometry, metabolism, electrophysiology, Zika virus experiments) in order to benchmark industrial-scale cell culture operations towards building an integrated platform for efficient cell manufacturing for disease modeling, drug screening, and cell therapy. Combining stem cell-based models and non-stop robotic cell culture may become a powerful strategy to increase scientific rigor and productivity, which are particularly important during public health emergencies (e.g. opioid crisis, COVID-19 pandemic).
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To clarify how smoking leads to heart attack and stroke, we developed an endothelial cell model (iECs) generated from human induced Pluripotent Stem Cells (iPSC) and evaluated its responses to tobacco smoke. These iECs exhibited a uniform endothelial morphology, and expressed markers PECAM1/CD31, VWF/ von Willebrand Factor, and CDH5/VE-Cadherin. The iECs also exhibited tube formation and acetyl-LDL uptake comparable to primary endothelial cells (EC). RNA sequencing (RNA-Seq) revealed a robust correlation coefficient between iECs and EC (R = 0.76), whereas gene responses to smoke were qualitatively nearly identical between iECs and primary ECs (R = 0.86). Further analysis of transcriptional responses implicated 18 transcription factors in regulating responses to smoke treatment, and identified gene sets regulated by each transcription factor, including pathways for oxidative stress, DNA damage/repair, ER stress, apoptosis, and cell cycle arrest. Assays for 42 cytokines in HUVEC cells and iECs identified 23 cytokines that responded dynamically to cigarette smoke. These cytokines and cellular stress response pathways describe endothelial responses for lymphocyte attachment, activation of coagulation and complement, lymphocyte growth factors, and inflammation and fibrosis; EC-initiated events that collectively lead to atherosclerosis. Thus, these studies validate the iEC model and identify transcriptional response networks by which ECs respond to tobacco smoke. Our results systematically trace how ECs use these response networks to regulate genes and pathways, and finally cytokine signals to other cells, to initiate the diverse processes that lead to atherosclerosis and cardiovascular disease.
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Células Endoteliais/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Modelos Biológicos , Fumar Tabaco/efeitos adversos , Citocinas/análise , Células Endoteliais/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/patologiaRESUMO
BACKGROUND: A central challenge in toxicity testing is the large number of chemicals in commerce that lack toxicological assessment. In response, the Tox21 program is re-focusing toxicity testing from animal studies to less expensive and higher throughput in vitro methods using target/pathway-specific, mechanism-driven assays. OBJECTIVES: Our objective was to use an in-depth mechanistic study approach to prioritize and characterize the chemicals affecting mitochondrial function. METHODS: We used a tiered testing approach to prioritize for more extensive testing 622 compounds identified from a primary, quantitative high-throughput screen of 8,300 unique small molecules, including drugs and industrial chemicals, as potential mitochondrial toxicants by their ability to significantly decrease the mitochondrial membrane potential (MMP). Based on results from secondary MMP assays in HepG2 cells and rat hepatocytes, 34 compounds were selected for testing in tertiary assays that included formation of reactive oxygen species (ROS), upregulation of p53 and nuclear erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE), mitochondrial oxygen consumption, cellular Parkin translocation, and larval development and ATP status in the nematode Caenorhabditis elegans. RESULTS: A group of known mitochondrial complex inhibitors (e.g., rotenone) and uncouplers (e.g., chlorfenapyr), as well as potential novel complex inhibitors and uncouplers, were detected. From this study, we identified four not well-characterized potential mitochondrial toxicants (lasalocid, picoxystrobin, pinacyanol, and triclocarban) that merit additional in vivo characterization. CONCLUSIONS: The tier-based approach for identifying and mechanistically characterizing mitochondrial toxicants can potentially reduce animal use in toxicological testing. https://doi.org/10.1289/EHP2589.
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Poluentes Ambientais/toxicidade , Substâncias Perigosas/toxicidade , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Testes de Toxicidade/métodos , Animais , Células Hep G2 , Hepatócitos , Humanos , Ratos , Testes de Toxicidade/instrumentaçãoRESUMO
In 2009, the passing of the Family Smoking Prevention and Tobacco Control Act facilitated the establishment of the FDA Center for Tobacco Products (CTP), and gave it regulatory authority over the marketing, manufacture and distribution of tobacco products, including those termed 'modified risk'. On 4-6 April 2016, the Institute for In Vitro Sciences, Inc. (IIVS) convened a workshop conference entitled, In Vitro Exposure Systems and Dosimetry Assessment Tools for Inhaled Tobacco Products, to bring together stakeholders representing regulatory agencies, academia and industry to address the research priorities articulated by the FDA CTP. Specific topics were covered to assess the status of current in vitro smoke and aerosol/vapour exposure systems, as well as the various approaches and challenges to quantifying the complex exposures in in vitro pulmonary models developed for evaluating adverse pulmonary events resulting from tobacco product exposures. The four core topics covered were: a) Tobacco Smoke and E-Cigarette Aerosols; b) Air-Liquid Interface-In Vitro Exposure Systems; c) Dosimetry Approaches for Particles and Vapours/In Vitro Dosimetry Determinations; and d) Exposure Microenvironment/Physiology of Cells. The 2.5-day workshop included presentations from 20 expert speakers, poster sessions, networking discussions, and breakout sessions which identified key findings and provided recommendations to advance these technologies. Here, we will report on the proceedings, recommendations, and outcome of the April 2016 technical workshop, including paths forward for developing and validating non-animal test methods for tobacco product smoke and next generation tobacco product aerosol/vapour exposures. With the recent FDA publication of the final deeming rule for the governance of tobacco products, there is an unprecedented necessity to evaluate a very large number of tobacco-based products and ingredients. The questionable relevance, high cost, and ethical considerations for the use of in vivo testing methods highlight the necessity of robust in vitro approaches to elucidate tobacco-based exposures and how they may lead to pulmonary diseases that contribute to lung exposure-induced mortality worldwide.
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Fumar/efeitos adversos , Produtos do Tabaco/efeitos adversos , Testes de Toxicidade/métodos , Aerossóis , Animais , Sistemas Eletrônicos de Liberação de Nicotina/efeitos adversos , Humanos , Técnicas In Vitro , Especificidade da Espécie , Estados Unidos , United States Food and Drug AdministrationRESUMO
Optogenetic and chemogenetic control of proteins has revealed otherwise inaccessible facets of signaling dynamics. Here, we use light- or ligand-sensitive domains to modulate the structural disorder of diverse proteins, thereby generating robust allosteric switches. Sensory domains were inserted into nonconserved, surface-exposed loops that were tight and identified computationally as allosterically coupled to active sites. Allosteric switches introduced into motility signaling proteins (kinases, guanosine triphosphatases, and guanine exchange factors) controlled conversion between conformations closely resembling natural active and inactive states, as well as modulated the morphodynamics of living cells. Our results illustrate a broadly applicable approach to design physiological protein switches.
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Luz , Engenharia de Proteínas , Quinases da Família src , Regulação Alostérica/genética , Regulação Alostérica/efeitos da radiação , Sítio Alostérico , Domínio Catalítico , Ativação Enzimática/genética , Ativação Enzimática/efeitos da radiação , GTP Fosfo-Hidrolases/antagonistas & inibidores , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/efeitos da radiação , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Células HEK293 , Humanos , Ligantes , Optogenética , Domínios Proteicos/efeitos da radiação , Proteínas Proto-Oncogênicas c-vav/química , Transdução de Sinais , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/química , Quinases da Família src/genética , Quinases da Família src/efeitos da radiaçãoRESUMO
The Src kinase family comprises nine homologous members whose distinct expression patterns and cellular distributions indicate that they have unique roles. These roles have not been determined because genetic manipulation has not produced clearly distinct phenotypes, and the kinases' homology complicates generation of specific inhibitors. Through insertion of a modified FK506 binding protein (insertable FKBP12, iFKBP) into the protein kinase isoforms Fyn, Src, Lyn, and Yes, we engineered kinase analogs that can be activated within minutes in living cells (RapR analogs). Combining our RapR analogs with computational tools for quantifying and characterizing cellular dynamics, we demonstrate that Src family isoforms produce very different phenotypes, encompassing cell spreading, polarized motility, and production of long, thin cell extensions. Activation of Src and Fyn led to patterns of kinase translocation that correlated with morphological changes in temporally distinct stages. Phenotypes were dependent on N-terminal acylation, not on Src homology 3 (SH3) and Src homology 2 (SH2) domains, and correlated with movement between a perinuclear compartment, adhesions, and the plasma membrane.
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Quinases da Família src/química , Quinases da Família src/metabolismo , Acilação , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Fenômenos Biofísicos , Células COS , Chlorocebus aethiops , Ativação Enzimática , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fenótipo , Engenharia de Proteínas , Proteínas Proto-Oncogênicas c-fyn/química , Proteínas Proto-Oncogênicas c-fyn/genética , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Proteína 1A de Ligação a Tacrolimo/química , Proteína 1A de Ligação a Tacrolimo/genética , Proteína 1A de Ligação a Tacrolimo/metabolismo , Domínios de Homologia de src , Quinases da Família src/genéticaRESUMO
Understanding the heterogeneous dynamics of cellular processes requires not only tools to visualize molecular behavior but also versatile approaches to extract and analyze the information contained in live-cell movies of many cells. Automated identification and tracking of cellular features enable thorough and consistent comparative analyses in a high-throughput manner. Here, we present tools for two challenging problems in computational image analysis: (1) classification of motion for cells with complex shapes and dynamics and (2) segmentation of clustered cells and quantification of intracellular protein distributions based on a single fluorescence channel. We describe these methods and user-friendly software(1) (MATLAB applications with graphical user interfaces) so these tools can be readily applied without an extensive knowledge of computational techniques.
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Processamento de Imagem Assistida por Computador/métodos , Algoritmos , Animais , Movimento Celular , Forma Celular , Rastreamento de Células , Humanos , Microscopia de Fluorescência , Complexos Multiproteicos/metabolismo , Transporte Proteico , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Análise de Célula Única/métodos , SoftwareRESUMO
We describe an approach to selectively activate a kinase in a specific protein complex or at a specific subcellular location within living cells and within minutes. This reveals the effects of specific kinase pathways without time for genetic compensation. The new technique, dubbed rapamycin-regulated targeted activation of pathways (RapRTAP), was used to dissect the role of Src kinase interactions with FAK and p130Cas in cell motility and morphodynamics. The overall effects of Src activation on cell morphology and adhesion dynamics were first quantified, without restricting effector access. Subsets of Src-induced behaviors were then attributed to specific interactions between Src and the two downstream proteins. Activation of Src in the cytoplasm versus at the cell membrane also produced distinct phenotypes. The conserved nature of the kinase site modified for RapRTAP indicates that the technique can be applied to many kinases.