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1.
Int J Mol Sci ; 22(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34205072

RESUMO

Two-photon microscopy enables monitoring cellular dynamics and communication in complex systems, within a genuine environment, such as living tissues and, even, living organisms. Particularly, its application to understand cellular interactions in the immune system has brought unique insights into pathophysiologic processes in vivo. Simultaneous multiplexed imaging is required to understand the dynamic orchestration of the multiple cellular and non-cellular tissue compartments defining immune responses. Here, we present an improvement of our previously developed method, which allowed us to achieve multiplexed dynamic intravital two-photon imaging, by using a synergistic strategy. This strategy combines a spectrally broad range of fluorophore emissions, a wave-mixing concept for simultaneous excitation of all targeted fluorophores, and an unmixing algorithm based on the calculation of spectral similarities with previously measured fluorophore fingerprints. The improvement of the similarity spectral unmixing algorithm here described is based on dimensionality reduction of the mixing matrix. We demonstrate its superior performance in the correct pixel-based assignment of probes to tissue compartments labeled by single fluorophores with similar spectral fingerprints, as compared to the full-dimensional similarity spectral unmixing approach.


Assuntos
Comunicação Celular/genética , Microambiente Celular/genética , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Algoritmos , Linhagem Celular , Corantes Fluorescentes/química , Fótons
2.
Int J Mol Sci ; 22(12)2021 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-34204595

RESUMO

Among all the proposed pathogenic mechanisms to understand the etiology of Alzheimer's disease (AD), increased oxidative stress seems to be a robust and early disease feature where many of those hypotheses converge. However, despite the significant lines of evidence accumulated, an effective diagnosis and treatment of AD are not yet available. This limitation might be partially explained by the use of cellular and animal models that recapitulate partial aspects of the disease and do not account for the particular biology of patients. As such, cultures of patient-derived cells of peripheral origin may provide a convenient solution for this problem. Peripheral cells of neuronal lineage such as olfactory neuronal precursors (ONPs) can be easily cultured through non-invasive isolation, reproducing AD-related oxidative stress. Interestingly, the autofluorescence of key metabolic cofactors such as reduced nicotinamide adenine dinucleotide (NADH) can be highly correlated with the oxidative state and antioxidant capacity of cells in a non-destructive and label-free manner. In particular, imaging NADH through fluorescence lifetime imaging microscopy (FLIM) has greatly improved the sensitivity in detecting oxidative shifts with minimal intervention to cell physiology. Here, we discuss the translational potential of analyzing patient-derived ONPs non-invasively isolated through NADH FLIM to reveal AD-related oxidative stress. We believe this approach may potentially accelerate the discovery of effective antioxidant therapies and contribute to early diagnosis and personalized monitoring of this devastating disease.


Assuntos
Doença de Alzheimer/patologia , Microscopia de Fluorescência/métodos , NAD/metabolismo , Neurônios Receptores Olfatórios/patologia , Estresse Oxidativo , Animais , Antioxidantes/metabolismo , Humanos
3.
Methods Mol Biol ; 2276: 153-163, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060039

RESUMO

The spectroscopic methods commonly used to study mitochondria bioenergetics do not show the diversity of responses within a population of mitochondria (isolated or in a cell), and/or cannot measure individual dynamics. New methodological developments are necessary in order to improve quantitative and kinetic resolutions and eventually gain further insights on individual mitochondrial responses, such as studying activities of the mitochondrial permeability transition pore (mPTP ). The work reported herein is devoted to study responses of single mitochondria within a large population after isolation from cardiomyocytes. Mitochondria were preloaded with a commonly used membrane potential sensitive dye (TMRM), they are then deposited on a plasma-treated glass coverslip and subsequently energized or inhibited by additions of usual bioenergetics effectors. Responses were analyzed by fluorescence microscopy over few thousands of mitochondria simultaneously with a single organelle resolution. We report an automatic method to analyze each image of time-lapse stacks based on the TrackMate-ImageJ plug-in and specially made Python scripts. Images are processed to eliminate defects of illumination inhomogeneity, improving by at least two orders of magnitude the signal/noise ratio. This method enables us to follow the track of each mitochondrion within the observed field and monitor its fluorescence changes, with a time resolution of 400 ms, uninterrupted over the course of the experiment. Such methodological improvement is a prerequisite to further study the role of mPTP in single mitochondria during calcium transient loading.


Assuntos
Processamento Eletrônico de Dados/métodos , Microscopia de Fluorescência/métodos , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/metabolismo , Imagem Individual de Molécula/métodos , Animais , Metabolismo Energético , Potenciais da Membrana , Miócitos Cardíacos/citologia , Miócitos Cardíacos/ultraestrutura , Ratos , Ratos Wistar
4.
Methods Mol Biol ; 2276: 173-191, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060041

RESUMO

Mitochondrial Ca2+ uptake regulates mitochondrial function and contributes to cell signaling. Accordingly, quantifying mitochondrial Ca2+ signals and elaborating the mechanisms that accomplish mitochondrial Ca2+ uptake are essential to gain our understanding of cell biology. Here, we describe the benefits and drawbacks of various established old and new techniques to assess dynamic changes of mitochondrial Ca2+ concentration ([Ca2+]mito) in a wide range of applications.


Assuntos
Cálcio/metabolismo , Microscopia de Fluorescência/métodos , Mitocôndrias/metabolismo , Técnicas de Patch-Clamp/métodos , Animais , Células Cultivadas , Corantes Fluorescentes/química , Humanos , Consumo de Oxigênio/fisiologia
5.
Methods Mol Biol ; 2276: 333-341, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060053

RESUMO

Mitochondria change their morphologies from small isolated vesicles to large continuous networks across the cell cycles. The mitochondrial network formation (MNF) plays an important role in maintaining mitochondrial DNA integrity and interchanging mitochondrial materials. The disruption of the mitochondrial network affects mitochondrial functions, such as ATP production, integration of metabolism, calcium homeostasis, and regulation of apoptosis, leading to the abnormal development and several human diseases including neurodegenerative disease. In this unit, we describe the method of studying MNF, which is driven by microtubule-dependent motor protein, by in vivo imaging and single-molecule in vitro reconstitution assays.


Assuntos
DNA Mitocondrial/metabolismo , Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Animais , Células Cultivadas , Técnicas In Vitro/métodos , Cinesina/metabolismo , Potencial da Membrana Mitocondrial , Camundongos , Microscopia de Fluorescência/métodos , Dinâmica Mitocondrial , Ratos
6.
Int J Mol Sci ; 22(11)2021 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-34067339

RESUMO

Dephosphorylation inhibitor calyculin A (cal A) has been reported to inhibit the disappearance of radiation-induced γH2AX DNA repair foci in human lymphocytes. However, other studies reported no change in the kinetics of γH2AX focus induction and loss in irradiated cells. While apoptosis might interplay with the kinetics of focus formation, it was not followed in irradiated cells along with DNA repair foci. Thus, to validate plausible explanations for significant variability in outputs of these studies, we evaluated the effect of cal A (1 and 10 nM) on γH2AX/53BP1 DNA repair foci and apoptosis in irradiated (1, 5, 10, and 100 cGy) human umbilical cord blood lymphocytes (UCBL) using automated fluorescence microscopy and annexin V-FITC/propidium iodide assay/γH2AX pan-staining, respectively. No effect of cal A on γH2AX and colocalized γH2AX/53BP1 foci induced by low doses (≤10 cGy) of γ-rays was observed. Moreover, 10 nM cal A treatment decreased the number of all types of DNA repair foci induced by 100 cGy irradiation. 10 nM cal A treatment induced apoptosis already at 2 h of treatment, independently from the delivered dose. Apoptosis was also detected in UCBL treated with lower cal A concentration, 1 nM, at longer cell incubation, 20 and 44 h. Our data suggest that apoptosis triggered by cal A in UCBL may underlie the failure of cal A to maintain radiation-induced γH2AX foci. All DSB molecular markers used in this study responded linearly to low-dose irradiation. Therefore, their combination may represent a strong biodosimetry tool for estimation of radiation response to low doses. Assessment of colocalized γH2AX/53BP1 improved the threshold of low dose detection.


Assuntos
Apoptose/efeitos dos fármacos , Sangue Fetal/efeitos dos fármacos , Histonas/metabolismo , Linfócitos/efeitos dos fármacos , Toxinas Marinhas/farmacologia , Oxazóis/farmacologia , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Relação Dose-Resposta à Radiação , Sangue Fetal/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Linfócitos/metabolismo , Microscopia de Fluorescência/métodos , Fosforilação/efeitos dos fármacos
7.
Methods Mol Biol ; 2274: 25-35, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050459

RESUMO

Visualizing progression through the cell cycle provides valuable information for the study of development, tissue maintenance, and dysregulated growth in proliferative diseases, such as cancer. Developments in fluorescent biosensors have facilitated dynamic tracking of molecular processes, including the cell cycle. The genetically encoded set of fluorescent indicators, Fucci4, enables the visualization of transitions between each cell cycle phase. Here, we describe a method to track progression through each cell cycle phase using Fucci4 in live epifluorescence imaging. In principle, this approach can be adapted to in vitro time-lapse imaging of any four spectrally resolvable fluorescent indicators.


Assuntos
Técnicas Biossensoriais/métodos , Ciclo Celular , Fluorescência , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência/métodos , Imagem Óptica/métodos , Divisão Celular , Células HeLa , Humanos
8.
Methods Mol Biol ; 2274: 155-168, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050470

RESUMO

Sirtuins (SIRTs) are a family of NAD+-dependent histone deacetylases (HDACs). In mammals, dysfunction of SIRTs is associated with age-related metabolic diseases, cancers, and even aging. Therefore, the detection of SIRT activity in living cells or tissues would be helpful for diagnosis of a wide range of SIRT-associated diseases. Here, we present methodology to measure SIRT activity in living cells by using our newly developed SIRT fluorescence probe, KST-F-DA.


Assuntos
Diagnóstico por Imagem/métodos , Corantes Fluorescentes/análise , Microscopia de Fluorescência/métodos , Sirtuína 1/metabolismo , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes/química , Células HEK293 , Humanos , Sirtuína 1/antagonistas & inibidores
9.
Methods Mol Biol ; 2274: 217-235, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050475

RESUMO

Recent extensive studies revealed that the intracellular concentration of magnesium ions (Mg2+) is one of the important factors to regulate cellular functions. To evaluate the impact of Mg2+ concentration changes on intracellular signals or events, simultaneous imaging of Mg2+ with those phenomena is a powerful technique. The present protocol describes the synthesis and evaluation of near-infrared (NIR) fluorescent Mg2+-selective probes, named KMG-500 series, and the application to simultaneous imaging of the corresponding intracellular signal transductions and molecular events. The present protocol for multicolor imaging using fluorescent probes in the NIR and visible ranges is highly useful to reveal how multiple molecular events are correlated each other in each single cell.


Assuntos
Trifosfato de Adenosina/metabolismo , Corantes Fluorescentes/química , Hipocampo/metabolismo , Magnésio/metabolismo , Imagem Molecular/métodos , Neurônios/metabolismo , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Animais , Células Cultivadas , Potencial da Membrana Mitocondrial , Microscopia de Fluorescência/métodos , Ratos , Transdução de Sinais
10.
Methods Mol Biol ; 2274: 237-243, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050476

RESUMO

Living cells dynamically change their morphology and function according to the cell cycle. Long-term observations of living cells are privileged when we spy the unique, cell cycle-driven molecular events, which cannot be obtained from short-term ones. Mg2+, a metal ion abundant in cells, has been shown to be involved in a variety of physiological phenomena by noninvasive cellular observation using fluorescence microscopy. However, long-term observation of Mg2+ in cells has been a great challenge. Herein, we present a protocol for the long-term microscopic imaging of intracellular Mg2+ levels using a small molecule-protein hybrid fluorescent probe we developed.


Assuntos
Corantes Fluorescentes/química , Magnésio/metabolismo , Imagem Molecular/métodos , Células HEK293 , Humanos , Microscopia de Fluorescência/métodos , Transdução de Sinais
11.
Methods Mol Biol ; 2274: 305-314, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050482

RESUMO

Bioluminescence resonance energy transfer (BRET) is an energy transfer phenomenon from a luciferase donor to a fluorescence acceptor and serves as an indicator of protein-protein interaction or protein proximity. BRET imaging is a powerful tool in the investigation of signaling proteins because it enables spatial analysis of such protein interactions. Here, we describe a method exerting high-resolution BRET imaging by combining bright-light output luciferases, such as NanoLuc , photon-counting EM-CCD, and unique algorithms for image correction and denoising.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Luciferases/metabolismo , Medições Luminescentes/métodos , Microscopia de Fluorescência/métodos , Imagem Óptica/métodos , Fótons , Câmaras gama , Células HEK293 , Humanos , Processamento de Imagem Assistida por Computador , Substâncias Luminescentes/química
12.
Methods Mol Biol ; 2274: 385-389, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050487

RESUMO

Confocal microscopy is a simple, super-resolution technique, which does not produce a marked increase in resolution compared to other advanced techniques, such as super-resolution nanoscopy. Here, we present a simple protocol to acquire "slightly, but easily resolved" images by pinhole closure (<1 airy unit) in a conventional confocal scanning microscope equipped with an avalanche photodiode, a detector with high sensitivity. We use murine neuroblastoma Neuro2a cells to demonstrate the image resolution obtained via this protocol without the use of any special software to enhance image quality.


Assuntos
Processamento de Imagem Assistida por Computador/métodos , Microscopia Confocal/instrumentação , Microscopia de Fluorescência/instrumentação , Imagem Molecular/métodos , Neuroblastoma/patologia , Software , Animais , Camundongos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Células Tumorais Cultivadas
13.
Methods Mol Biol ; 2274: 391-441, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050488

RESUMO

Single-molecule imaging (SMI) is a powerful method to measure the dynamics of membrane proteins on the cell membrane. The single-molecule tracking (SMT) analysis provides information about the diffusion dynamics, the oligomer size distribution, and the particle density change. The affinity and on/off-rate of a protein-protein interaction can be estimated from the dual-color SMI analysis. However, it is difficult for trainees to determine quantitative information from the SMI movies. The present protocol guides the detailed workflows to measure the drug-activated dynamics of a G protein-coupled receptor (GPCR) and metabotropic glutamate receptor 3 (mGluR3), by using the total internal reflection fluorescence microscopy (TIRFM). This tutorial guidance comprises an open-source software, named smDynamicsAnalyzer, with which one can easily analyze the SMT dataset by just following the workflows after building a designated folder structure ( https://github.com/masataka-yanagawa/IgorPro8-smDynamicsAnalyzer ).


Assuntos
Membrana Celular/metabolismo , Microscopia de Fluorescência/métodos , Preparações Farmacêuticas/administração & dosagem , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Imagem Individual de Molécula/métodos , Software , Membrana Celular/efeitos dos fármacos , Células HEK293 , Humanos , Ligação Proteica , Fluxo de Trabalho
14.
Nat Commun ; 12(1): 2668, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976151

RESUMO

Telomeres are bound by dedicated proteins, which protect them from DNA damage and regulate telomere length homeostasis. In the nematode Caenorhabditis elegans, a comprehensive understanding of the proteins interacting with the telomere sequence is lacking. Here, we harnessed a quantitative proteomics approach to identify TEBP-1 and TEBP-2, two paralogs expressed in the germline and embryogenesis that associate to telomeres in vitro and in vivo. tebp-1 and tebp-2 mutants display strikingly distinct phenotypes: tebp-1 mutants have longer telomeres than wild-type animals, while tebp-2 mutants display shorter telomeres and a Mortal Germline. Notably, tebp-1;tebp-2 double mutant animals have synthetic sterility, with germlines showing signs of severe mitotic and meiotic arrest. Furthermore, we show that POT-1 forms a telomeric complex with TEBP-1 and TEBP-2, which bridges TEBP-1/-2 with POT-2/MRT-1. These results provide insights into the composition and organization of a telomeric protein complex in C. elegans.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Animais , Animais Geneticamente Modificados , Sequência de Bases , Sítios de Ligação/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/classificação , Proteínas de Caenorhabditis elegans/genética , DNA/genética , Proteínas de Ligação a DNA/genética , Células Germinativas/metabolismo , Microscopia de Fluorescência/métodos , Complexos Multiproteicos/genética , Mutação , Filogenia , Ligação Proteica , Isoformas de Proteínas/classificação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Telômero/genética , Proteínas de Ligação a Telômeros/classificação , Proteínas de Ligação a Telômeros/genética
15.
Methods Mol Biol ; 2255: 97-117, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34033098

RESUMO

Neutrophils release web like-structures known as neutrophil extracellular traps (NETs) that ensnare and kill microorganisms. These networks are constituted of a DNA scaffold with associated antimicrobial proteins, which are released to the extracellular space as an effective mechanism to fight against invading microorganisms. In parallel with this beneficial role to avoid microbial dissemination and wall off infections, accumulating evidence supports that under certain circumstances, NETs can exert deleterious effects in inflammatory, autoimmune, and thrombotic pathologies. Research on NET properties and their role in pathophysiological processes is a rapidly evolving and expanding field. Here, we describe a combination of methods to achieve a successful in vitro NET visualization, semiquantification, and isolation.


Assuntos
Separação Celular/métodos , DNA/análise , Armadilhas Extracelulares/metabolismo , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Elastase Pancreática/análise , Peroxidase/metabolismo , Humanos , Técnicas In Vitro
16.
Nat Commun ; 12(1): 3148, 2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-34035309

RESUMO

Structured Illumination Microscopy enables live imaging with sub-diffraction resolution. Unfortunately, optical aberrations can lead to loss of resolution and artifacts in Structured Illumination Microscopy rendering the technique unusable in samples thicker than a single cell. Here we report on the combination of Adaptive Optics and Structured Illumination Microscopy enabling imaging with 150 nm lateral and 570 nm axial resolution at a depth of 80 µm through Caenorhabditis elegans. We demonstrate that Adaptive Optics improves the three-dimensional resolution, especially along the axial direction, and reduces artifacts, successfully realizing 3D-Structured Illumination Microscopy in a variety of biological samples.


Assuntos
Imageamento Tridimensional/métodos , Microscopia Intravital/métodos , Iluminação/instrumentação , Animais , Artefatos , Ascomicetos , Caenorhabditis elegans , Linhagem Celular , Imageamento Tridimensional/instrumentação , Microscopia Intravital/instrumentação , Camundongos , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Oryza/microbiologia , Reprodutibilidade dos Testes
17.
Nat Commun ; 12(1): 2860, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001878

RESUMO

Bone regenerates by activation of tissue resident stem/progenitor cells, formation of a fibrous callus followed by deposition of cartilage and bone matrices. Here, we show that mesenchymal progenitors residing in skeletal muscle adjacent to bone mediate the initial fibrotic response to bone injury and also participate in cartilage and bone formation. Combined lineage and single-cell RNA sequencing analyses reveal that skeletal muscle mesenchymal progenitors adopt a fibrogenic fate before they engage in chondrogenesis after fracture. In polytrauma, where bone and skeletal muscle are injured, skeletal muscle mesenchymal progenitors exhibit altered fibrogenesis and chondrogenesis. This leads to impaired bone healing, which is due to accumulation of fibrotic tissue originating from skeletal muscle and can be corrected by the anti-fibrotic agent Imatinib. These results elucidate the central role of skeletal muscle in bone regeneration and provide evidence that skeletal muscle can be targeted to prevent persistent callus fibrosis and improve bone healing after musculoskeletal trauma.


Assuntos
Regeneração Óssea/fisiologia , Calo Ósseo/fisiologia , Consolidação da Fratura/fisiologia , Fraturas Ósseas/fisiopatologia , Células-Tronco Mesenquimais/fisiologia , Músculo Esquelético/citologia , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência/métodos , Osteogênese/fisiologia
18.
Nat Commun ; 12(1): 3067, 2021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-34031389

RESUMO

Diffractive optical elements (DOEs) are used to shape the wavefront of incident light. This can be used to generate practically any pattern of interest, albeit with varying efficiency. A fundamental challenge associated with DOEs comes from the nanoscale-precision requirements for their fabrication. Here we demonstrate a method to controllably scale up the relevant feature dimensions of a device from tens-of-nanometers to tens-of-microns by immersing the DOEs in a near-index-matched solution. This makes it possible to utilize modern 3D-printing technologies for fabrication, thereby significantly simplifying the production of DOEs and decreasing costs by orders of magnitude, without hindering performance. We demonstrate the tunability of our design for varying experimental conditions, and the suitability of this approach to ultrasensitive applications by localizing the 3D positions of single molecules in cells using our microscale fabricated optical element to modify the point-spread-function (PSF) of a microscope.


Assuntos
Imersão , Dispositivos Ópticos , Impressão Tridimensional , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Nanotecnologia , Impressão Tridimensional/instrumentação , Sensibilidade e Especificidade
19.
Nat Commun ; 12(1): 3077, 2021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-34031402

RESUMO

Non-uniform illumination limits quantitative analyses of fluorescence imaging techniques. In particular, single molecule localization microscopy (SMLM) relies on high irradiances, but conventional Gaussian-shaped laser illumination restricts the usable field of view to around 40 µm × 40 µm. We present Adaptable Scanning for Tunable Excitation Regions (ASTER), a versatile illumination technique that generates uniform and adaptable illumination. ASTER is also highly compatible with optical sectioning techniques such as total internal reflection fluorescence (TIRF). For SMLM, ASTER delivers homogeneous blinking kinetics at reasonable laser power over fields-of-view up to 200 µm × 200 µm. We demonstrate that ASTER improves clustering analysis and nanoscopic size measurements by imaging nanorulers, microtubules and clathrin-coated pits in COS-7 cells, and ß2-spectrin in neurons. ASTER's sharp and quantitative illumination paves the way for high-throughput quantification of biological structures and processes in classical and super-resolution fluorescence microscopies.


Assuntos
Iluminação , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Imagem Óptica/instrumentação , Imagem Óptica/métodos , Imagem Individual de Molécula/instrumentação , Imagem Individual de Molécula/métodos , Algoritmos , Animais , Células COS , Chlorocebus aethiops , Lasers , Luz , Microtúbulos , Reprodutibilidade dos Testes
20.
Methods Mol Biol ; 2262: 233-250, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33977480

RESUMO

On the plasma membrane, Ras is organized into laterally segregated proteo-lipid complexes called nanoclusters. The extent of Ras nanoclustering correlates with its signaling output, positioning nanocluster as dynamic signaling gain modulators. Recent evidence suggests that stacked dimers of Ras and Raf are elemental units at least of one type of Ras nanocluster. However, it is still incompletely understood, in which physiological contexts nanoclustering is regulated and which constituents are parts of nanocluster. Nonetheless, disruption of nanoclustering faithfully diminishes Ras activity in cells, suggesting Ras nanocluster as potential drug targets.While there are several methods available to study Ras nanocluster , fluorescence or Förster resonance energy transfer (FRET ) between fluorescently labeled, nanoclustered Ras proteins is a relatively simple readout. FRET measurements using fluorescence lifetime imaging microscopy (FLIM ) have proven to be robust and sensitive to determine Ras nanoclustering changes. Loss of FRET that emerges due to nanoclustering reports on all processes upstream of Ras nanoclustering, i.e., also on proper trafficking or lipid modification of Ras. Here we report our standard FLIM-FRET protocol to measure nanoclustering-dependent FRET of Ras in mammalian cells. Importantly, nanoclustering-dependent FRET is one of the few methods that can detect differences between the Ras isoforms.


Assuntos
Membrana Celular/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Processamento de Imagem Assistida por Computador/métodos , Lipídeos de Membrana/metabolismo , Microscopia de Fluorescência/métodos , Nanopartículas/química , Proteínas ras/metabolismo , Membrana Celular/ultraestrutura , Humanos , Transdução de Sinais
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