RESUMEN
Molecular mobility, localisation and spatiotemporal activity are at the core of cell biological processes and deregulation of these dynamic events can underpin disease development and progression. Recent advances in intravital imaging techniques in mice are providing new avenues to study real-time molecular behaviour in intact tissues within a live organism and to gain exciting insights into the intricate regulation of live cell biology at the microscale level. The monitoring of fluorescently labelled proteins and agents can be combined with autofluorescent properties of the microenvironment to provide a comprehensive snapshot of in vivo cell biology. In this Review, we summarise recent intravital microscopy approaches in mice, in processes ranging from normal development and homeostasis to disease progression and treatment in cancer, where we emphasise the utility of intravital imaging to observe dynamic and transient events in vivo We also highlight the recent integration of advanced subcellular imaging techniques into the intravital imaging pipeline, which can provide in-depth biological information beyond the single-cell level. We conclude with an outlook of ongoing developments in intravital microscopy towards imaging in humans, as well as provide an overview of the challenges the intravital imaging community currently faces and outline potential ways for overcoming these hurdles.
Asunto(s)
Microscopía Intravital/tendencias , Imagen Molecular/tendencias , Neoplasias/genética , Microambiente Tumoral/genética , Progresión de la Enfermedad , Humanos , Neoplasias/ultraestructuraRESUMEN
Intravital microscopy represents a more physiologically relevant method for assessing therapeutic response. However, the movement into an in vivo setting brings with it several additional considerations, the primary being the context in which drug activity is assessed. Microenvironmental factors, such as hypoxia, pH, fibrosis, immune infiltration and stromal interactions have all been shown to have pronounced effects on drug activity in a more complex setting, which is often lost in simpler two- or three-dimensional assays. Here we present a practical guide for the application of intravital microscopy, looking at the available fluorescent reporters and their respective expression systems and analysis considerations. Moving in vivo, we also discuss the microscopy set up and methods available for overlaying microenvironmental context to the experimental readouts. This enables a smooth transition into applying higher fidelity intravital imaging to improve the drug discovery process.
Asunto(s)
Técnicas Biosensibles/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , Microscopía Intravital/métodos , Animales , Humanos , Colgajos Quirúrgicos , Resultado del Tratamiento , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
This paper demonstrates multiphoton excited fluorescence imaging through a polarisation maintaining multicore fiber (PM-MCF) while the fiber is dynamically deformed using all-proximal detection. Single-shot proximal measurement of the relative optical path lengths of all the cores of the PM-MCF in double pass is achieved using a Mach-Zehnder interferometer read out by a scientific CMOS camera operating at 416 Hz. A non-linear least squares fitting procedure is then employed to determine the deformation-induced lateral shift of the excitation spot at the distal tip of the PM-MCF. An experimental validation of this approach is presented that compares the proximally measured deformation-induced lateral shift in focal spot position to an independent distally measured ground truth. The proximal measurement of deformation-induced shift in focal spot position is applied to correct for deformation-induced shifts in focal spot position during raster-scanning multiphoton excited fluorescence imaging.
RESUMEN
We describe an approach to non-invasively map spatiotemporal biochemical and physiological changes in 3D cell culture using Forster Resonance Energy Transfer (FRET) biosensors expressed in tumour spheroids. In particular, we present an improved Adenosine Monophosphate (AMP) Activated Protein Kinase (AMPK) FRET biosensor, mTurquoise2 AMPK Activity Reporter (T2AMPKAR), for fluorescence lifetime imaging (FLIM) readouts that we have evaluated in 2D and 3D cultures. Our results in 2D cell culture indicate that replacing the FRET donor, enhanced Cyan Fluorescent Protein (ECFP), in the original FRET biosensor, AMPK activity reporter (AMPKAR), with mTurquoise2 (mTq2FP), increases the dynamic range of the response to activation of AMPK, as demonstrated using the direct AMPK activator, 991. We demonstrated 3D FLIM of this T2AMPKAR FRET biosensor expressed in tumour spheroids using two-photon excitation.
Asunto(s)
Técnicas Biosensibles/métodos , Técnicas de Cultivo de Célula , Imagen Molecular/métodos , Proteínas Quinasas/aislamiento & purificación , Quinasas de la Proteína-Quinasa Activada por el AMP , Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas Fluorescentes Verdes/química , Humanos , Imagen Óptica/métodos , Esferoides Celulares/citologíaRESUMEN
Multiplexed imaging of Förster Resonance Energy Transfer (FRET)-based biosensors potentially presents a powerful approach to monitoring the spatio-temporal correlation of signalling pathways within a single live cell. Here, we discuss the potential of homo-FRET based biosensors to facilitate multiplexed imaging. We demonstrate that the homo-FRET between pleckstrin homology domains of Akt (Akt-PH) labelled with mCherry may be used to monitor 3'-phosphoinositide accumulation in live cells and show how global analysis of time resolved fluorescence anisotropy measurements can be used to quantify this accumulation. We further present multiplexed imaging readouts of calcium concentration, using fluorescence lifetime measurements of TN-L15-a CFP/YFP based hetero-FRET calcium biosensor-with 3'-phosphoinositide accumulation.
Asunto(s)
Técnicas Biosensibles/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , Transducción de Señal , Animales , Anisotropía , Calcio/metabolismo , Línea Celular , Transferencia Resonante de Energía de Fluorescencia/instrumentación , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Fosfatidilinositoles/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is characterized by increasing fibrosis, which can enhance tumor progression and spread. Here, we undertook an unbiased temporal assessment of the matrisome of the highly metastatic KPC (Pdx1-Cre, LSL-KrasG12D/+, LSL-Trp53R172H/+) and poorly metastatic KPflC (Pdx1-Cre, LSL-KrasG12D/+, Trp53fl/+) genetically engineered mouse models of pancreatic cancer using mass spectrometry proteomics. Our assessment at early-, mid-, and late-stage disease reveals an increased abundance of nidogen-2 (NID2) in the KPC model compared to KPflC, with further validation showing that NID2 is primarily expressed by cancer-associated fibroblasts (CAFs). Using biomechanical assessments, second harmonic generation imaging, and birefringence analysis, we show that NID2 reduction by CRISPR interference (CRISPRi) in CAFs reduces stiffness and matrix remodeling in three-dimensional models, leading to impaired cancer cell invasion. Intravital imaging revealed improved vascular patency in live NID2-depleted tumors, with enhanced response to gemcitabine/Abraxane. In orthotopic models, NID2 CRISPRi tumors had less liver metastasis and increased survival, highlighting NID2 as a potential PDAC cotarget.
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Proteómica , Animales , Humanos , Ratones , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/patología , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/genética , Moléculas de Adhesión Celular , Línea Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Modelos Animales de Enfermedad , Fibrosis , Gemcitabina , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/genética , Proteómica/métodosRESUMEN
Aberrant AKT activation occurs in a number of cancers, metabolic syndrome, and immune disorders, making it an important target for the treatment of many diseases. To monitor spatial and temporal AKT activity in a live setting, we generated an Akt-FRET biosensor mouse that allows longitudinal assessment of AKT activity using intravital imaging in conjunction with image stabilization and optical window technology. We demonstrate the sensitivity of the Akt-FRET biosensor mouse using various cancer models and verify its suitability to monitor response to drug targeting in spheroid and organotypic models. We also show that the dynamics of AKT activation can be monitored in real time in diverse tissues, including in individual islets of the pancreas, in the brown and white adipose tissue, and in the skeletal muscle. Thus, the Akt-FRET biosensor mouse provides an important tool to study AKT dynamics in live tissue contexts and has broad preclinical applications.
Asunto(s)
Técnicas Biosensibles , Proteínas Proto-Oncogénicas c-akt , Ratones , Animales , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Técnicas Biosensibles/métodosRESUMEN
The concept that extracellular vesicles (EVs) from the diet can be absorbed by the intestinal tract of the consuming organism, be bioavailable in various organs, and in-turn exert phenotypic changes is highly debatable. Here, we isolate EVs from both raw and commercial bovine milk and characterize them by electron microscopy, nanoparticle tracking analysis, western blotting, quantitative proteomics and small RNA sequencing analysis. Orally administered bovine milk-derived EVs survive the harsh degrading conditions of the gut, in mice, and is subsequently detected in multiple organs. Milk-derived EVs orally administered to mice implanted with colorectal and breast cancer cells reduce the primary tumor burden. Intriguingly, despite the reduction in primary tumor growth, milk-derived EVs accelerate metastasis in breast and pancreatic cancer mouse models. Proteomic and biochemical analysis reveal the induction of senescence and epithelial-to-mesenchymal transition in cancer cells upon treatment with milk-derived EVs. Timing of EV administration is critical as oral administration after resection of the primary tumor reverses the pro-metastatic effects of milk-derived EVs in breast cancer models. Taken together, our study provides context-based and opposing roles of milk-derived EVs as metastasis inducers and suppressors.
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Vesículas Extracelulares , Leche/citología , Neoplasias Experimentales/patología , Administración Oral , Animales , Disponibilidad Biológica , Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Bovinos , Línea Celular Tumoral , Proliferación Celular , Transición Epitelial-Mesenquimal , Vesículas Extracelulares/química , Vesículas Extracelulares/genética , Femenino , Humanos , Neoplasias Hepáticas Experimentales/patología , Neoplasias Hepáticas Experimentales/secundario , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Ratones Endogámicos BALB C , Neoplasias Experimentales/terapia , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/terapia , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Assessing drug response within live native tissue provides increased fidelity with regards to optimizing efficacy while minimizing off-target effects. Here, using longitudinal intravital imaging of a Rac1-Förster resonance energy transfer (FRET) biosensor mouse coupled with in vivo photoswitching to track intratumoral movement, we help guide treatment scheduling in a live breast cancer setting to impair metastatic progression. We uncover altered Rac1 activity at the center versus invasive border of tumors and demonstrate enhanced Rac1 activity of cells in close proximity to live tumor vasculature using optical window imaging. We further reveal that Rac1 inhibition can enhance tumor cell vulnerability to fluid-flow-induced shear stress and therefore improves overall anti-metastatic response to therapy during transit to secondary sites such as the lung. Collectively, this study demonstrates the utility of single-cell intravital imaging in vivo to demonstrate that Rac1 inhibition can reduce tumor progression and metastases in an autochthonous setting to improve overall survival.
Asunto(s)
Técnicas Biosensibles/métodos , Neoplasias de la Mama/patología , Proteína de Unión al GTP rac1/metabolismo , Aminoquinolinas/farmacología , Animales , Neoplasias de la Mama/diagnóstico por imagen , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Femenino , Transferencia Resonante de Energía de Fluorescencia , Humanos , Neoplasias Pulmonares/diagnóstico por imagen , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Ratones , Ratones Endogámicos BALB C , Pirimidinas/farmacología , Resistencia al Corte , Transducción de Señal , Proteína de Unión al GTP rac1/antagonistas & inhibidoresRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic, chemoresistant malignancy and is characterized by a dense, desmoplastic stroma that modulates PDAC progression. Here, we visualized transient manipulation of focal adhesion kinase (FAK), which integrates bidirectional cell-environment signaling, using intravital fluorescence lifetime imaging microscopy of the FAK-based Förster resonance energy transfer biosensor in mouse and patient-derived PDAC models. Parallel real-time quantification of the FUCCI cell cycle reporter guided us to improve PDAC response to standard-of-care chemotherapy at primary and secondary sites. Critically, micropatterned pillar plates and stiffness-tunable matrices were used to pinpoint the contribution of environmental cues to chemosensitization, while fluid flowinduced shear stress assessment, patient-derived matrices, and personalized in vivo models allowed us to deconstruct how FAK inhibition can reduce PDAC spread. Last, stratification of PDAC patient samples via Merlin status revealed a patient subset with poor prognosis that are likely to respond to FAK priming before chemotherapy.
RESUMEN
Metastasis is a dynamic succession of events involving the dissemination of tumour cells to distant sites within the body, ultimately reducing the survival of patients with cancer. To colonize distant organs and, therefore, systemically disseminate within the organism, cancer cells and associated factors exploit several bodily fluid systems, which provide a natural transportation route. Indeed, the flow mechanics of the blood and lymphatic circulatory systems can be co-opted to improve the efficiency of cancer cell transit from the primary tumour, extravasation and metastatic seeding. Flow rates, vessel size and shear stress can all influence the survival of cancer cells in the circulation and control organotropic seeding patterns. Thus, in addition to using these fluids as a means to travel throughout the body, cancer cells exploit the underlying physical forces within these fluids to successfully seed distant metastases. In this Review, we describe how circulating tumour cells and tumour-associated factors leverage bodily fluids, their underlying forces and imposed stresses during metastasis. As the contribution of bodily fluids and their mechanics raises interesting questions about the biology of the metastatic cascade, an improved understanding of this process might provide a new avenue for targeting cancer cells in transit.
Asunto(s)
Líquidos Corporales/metabolismo , Modelos Biológicos , Neoplasias/metabolismo , Neoplasias/patología , Microambiente Tumoral , Animales , Biomarcadores , Líquidos Corporales/efectos de los fármacos , Matriz Extracelular/metabolismo , Humanos , Terapia Molecular Dirigida , Metástasis de la Neoplasia , Neoplasias/etiología , Neoplasias/terapia , Células Neoplásicas Circulantes/efectos de los fármacos , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patología , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/inmunologíaRESUMEN
The small GTPase RhoA is a master regulator of signalling in cell-extracellular matrix interactions. RhoA signalling is critical to many cellular processes including migration, mechanotransduction, and is often disrupted in carcinogenesis. Investigating RhoA activity in a native tissue environment is challenging using conventional biochemical methods; we therefore developed a RhoA-FRET biosensor mouse, employing the adaptable nature of intravital imaging to a variety of settings. Mechanotransduction was explored in the context of osteocyte processes embedded in the calvaria responding in a directional manner to compression stress. Further, the migration of neutrophils was examined during in vivo "chemotaxis" in wound response. RhoA activity was tightly regulated during tissue remodelling in mammary gestation, as well as during mammary and pancreatic carcinogenesis. Finally, pharmacological inhibition of RhoA was temporally resolved by the use of optical imaging windows in fully developed pancreatic and mammary tumours in vivo. The RhoA-FRET mouse therefore constitutes a powerful tool to facilitate development of new inhibitors targeting the RhoA signalling axis.
Asunto(s)
Técnicas Biosensibles , Transferencia Resonante de Energía de Fluorescencia , Preparaciones Farmacéuticas/química , Proteína de Unión al GTP rhoA/metabolismo , Animales , Ratones , Transducción de SeñalRESUMEN
In microorganisms, evolutionarily conserved mechanisms facilitate adaptation to harsh conditions through stress-induced mutagenesis (SIM). Analogous processes may underpin progression and therapeutic failure in human cancer. We describe SIM in multiple in vitro and in vivo models of human cancers under nongenotoxic drug selection, paradoxically enhancing adaptation at a competing intrinsic fitness cost. A genome-wide approach identified the mechanistic target of rapamycin (MTOR) as a stress-sensing rheostat mediating SIM across multiple cancer types and conditions. These observations are consistent with a two-phase model for drug resistance, in which an initially rapid expansion of genetic diversity is counterbalanced by an intrinsic fitness penalty, subsequently normalizing to complete adaptation under the new conditions. This model suggests synthetic lethal strategies to minimize resistance to anticancer therapy.
Asunto(s)
Adaptación Fisiológica/genética , Antineoplásicos/farmacología , Resistencia a Antineoplásicos/genética , Mutagénesis , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Serina-Treonina Quinasas TOR/metabolismo , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Reparación del ADN/genética , Aptitud Genética , Estudio de Asociación del Genoma Completo , Humanos , Selección Genética , Transducción de Señal , Serina-Treonina Quinasas TOR/genéticaRESUMEN
Heterogeneous subtypes of cancer-associated fibroblasts (CAFs) coexist within pancreatic cancer tissues and can both promote and restrain disease progression. Here, we interrogate how cancer cells harboring distinct alterations in p53 manipulate CAFs. We reveal the existence of a p53-driven hierarchy, where cancer cells with a gain-of-function (GOF) mutant p53 educate a dominant population of CAFs that establish a pro-metastatic environment for GOF and null p53 cancer cells alike. We also demonstrate that CAFs educated by null p53 cancer cells may be reprogrammed by either GOF mutant p53 cells or their CAFs. We identify perlecan as a key component of this pro-metastatic environment. Using intravital imaging, we observe that these dominant CAFs delay cancer cell response to chemotherapy. Lastly, we reveal that depleting perlecan in the stroma combined with chemotherapy prolongs mouse survival, supporting it as a potential target for anti-stromal therapies in pancreatic cancer.
Asunto(s)
Fibroblastos Asociados al Cáncer/patología , Resistencia a Antineoplásicos/genética , Proteoglicanos de Heparán Sulfato/metabolismo , Neoplasias Pancreáticas/patología , Proteína p53 Supresora de Tumor/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Regulación Neoplásica de la Expresión Génica/genética , Ratones , Ratones Endogámicos BALB C , Invasividad Neoplásica/patología , Páncreas/patología , Neoplasias Pancreáticas/genética , Transducción de Señal/fisiología , Proteína p53 Supresora de Tumor/genéticaRESUMEN
This paper reports a handheld multiphoton fluorescence microscope designed for clinical imaging that incorporates axial motion compensation and lateral image stabilization. Spectral domain optical coherence tomography is employed to track the axial position of the skin surface, and lateral motion compensation is realised by imaging the speckle pattern arising from the optical coherence tomography beam illuminating the sample. Our system is able to correct lateral sample velocities of up to approximately 65 µm s-1 . Combined with the use of negative curvature microstructured optical fibre to deliver tunable ultrafast radiation to the handheld multiphoton scanner without the need of a dispersion compensation unit, this instrument has potential for a range of clinical applications. The system is used to compensate for both lateral and axial motion of the sample when imaging human skin in vivo.
Asunto(s)
Artefactos , Mano , Microscopía de Fluorescencia por Excitación Multifotónica/instrumentación , Movimiento , Diseño de Equipo , Antebrazo/diagnóstico por imagen , Humanos , Piel/diagnóstico por imagen , Tomografía de Coherencia ÓpticaRESUMEN
Intravital microscopy can provide unique insights into the function of biological processes in a native context. However, physiological motion caused by peristalsis, respiration and the heartbeat can present a significant challenge, particularly for functional readouts such as fluorescence lifetime imaging (FLIM), which require longer acquisition times to obtain a quantitative readout. Here, we present and benchmark Galene, a versatile multi-platform software tool for image-based correction of sample motion blurring in both time resolved and conventional laser scanning fluorescence microscopy data in two and three dimensions. We show that Galene is able to resolve intravital FLIM-FRET images of intra-abdominal organs in murine models and NADH autofluorescence of human dermal tissue imaging subject to a wide range of physiological motions. Thus, Galene can enable FLIM imaging in situations where a stable imaging platform is not always possible and rescue previously discarded quantitative imaging data.
Asunto(s)
Imagenología Tridimensional , Microscopía Intravital , Movimiento (Física) , Algoritmos , Animales , Técnicas Biosensibles , Adhesión Celular , Simulación por Computador , Transferencia Resonante de Energía de Fluorescencia , Guanosina Trifosfato/metabolismo , Humanos , Intestinos/fisiología , Ratones , Microscopía Fluorescente , Modelos Biológicos , Metástasis de la Neoplasia , Neuropéptidos/metabolismo , Neoplasias Pancreáticas/patología , Piel/anatomía & histología , Programas Informáticos , Proteína de Unión al GTP rac1/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
Application of advanced intravital imaging facilitates dynamic monitoring of pathway activity upon therapeutic inhibition. Here, we assess resistance to therapeutic inhibition of the PI3K pathway within the hypoxic microenvironment of pancreatic ductal adenocarcinoma (PDAC) and identify a phenomenon whereby pronounced hypoxia-induced resistance is observed for three clinically relevant inhibitors. To address this clinical problem, we have mapped tumor hypoxia by both immunofluorescence and phosphorescence lifetime imaging of oxygen-sensitive nanoparticles and demonstrate that these hypoxic regions move transiently around the tumor. To overlay this microenvironmental information with drug response, we applied a FRET biosensor for Akt activity, which is a key effector of the PI3K pathway. Performing dual intravital imaging of drug response in different tumor compartments, we demonstrate an improved drug response to a combination therapy using the dual mTORC1/2 inhibitor AZD2014 with the hypoxia-activated pro-drug TH-302.
Asunto(s)
Resistencia a Antineoplásicos , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal , Animales , Benzamidas , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Quimioterapia Combinada , Femenino , Transferencia Resonante de Energía de Fluorescencia , Humanos , Hipoxia , Microscopía Intravital/métodos , Ratones , Ratones Endogámicos BALB C , Morfolinas/farmacología , Morfolinas/uso terapéutico , Nanopartículas/química , Nitroimidazoles/farmacología , Nitroimidazoles/uso terapéutico , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Mostazas de Fosforamida/farmacología , Mostazas de Fosforamida/uso terapéutico , Proteínas Proto-Oncogénicas c-akt/metabolismo , Pirimidinas , Transducción de Señal/efectos de los fármacos , Trasplante Heterólogo , Microambiente TumoralRESUMEN
Organotypic co-cultures bridge the gap between standard two-dimensional culture and mouse models. Such assays increase the fidelity of pre-clinical studies, to better inform lead compound development and address the increasing attrition rates of lead compounds within the pharmaceutical industry, which are often a result of screening in less faithful two-dimensional models. Using large-scale acid-extraction techniques, we demonstrate a step-by-step process to isolate collagen I from commercially available animal byproducts. Using the well-established rat tail tendon collagen as a benchmark, we apply our novel kangaroo tail tendon collagen as an alternative collagen source for our screening-ready three-dimensional organotypic co-culture platform. Both collagen sources showed equal applicability for invasive, proliferative or survival assessment of well-established cancer models and clinically relevant patient-derived cancer cell lines. Additional readouts were also demonstrated when comparing these alternative collagen sources for stromal contributions to stiffness, organization and ultrastructure via atomic force microscopy, second harmonic generation imaging and scanning electron microscopy, among other vital biological readouts, where only minor differences were found between the preparations. Organotypic co-cultures represent an easy, affordable and scalable model to investigate drug responses within a physiologically relevant 3D platform.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Colágeno/química , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Técnicas de Cocultivo , Colágeno/aislamiento & purificación , Matriz Extracelular/metabolismo , Gefitinib/farmacología , Humanos , Macropodidae/metabolismo , Ratones , Microscopía de Fuerza Atómica , Ratas , Tendones/metabolismoRESUMEN
We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in µManager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Microscopía Fluorescente/métodos , Animales , Técnicas Biosensibles , Células COS , Chlorocebus aethiops , Humanos , Imagen Óptica , Programas Informáticos , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
The small GTPase RhoA is involved in a variety of fundamental processes in normal tissue. Spatiotemporal control of RhoA is thought to govern mechanosensing, growth, and motility of cells, while its deregulation is associated with disease development. Here, we describe the generation of a RhoA-fluorescence resonance energy transfer (FRET) biosensor mouse and its utility for monitoring real-time activity of RhoA in a variety of native tissues in vivo. We assess changes in RhoA activity during mechanosensing of osteocytes within the bone and during neutrophil migration. We also demonstrate spatiotemporal order of RhoA activity within crypt cells of the small intestine and during different stages of mammary gestation. Subsequently, we reveal co-option of RhoA activity in both invasive breast and pancreatic cancers, and we assess drug targeting in these disease settings, illustrating the potential for utilizing this mouse to study RhoA activity in vivo in real time.