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1.
Nat Commun ; 12(1): 4693, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34344862

RESUMO

Many cellular processes, including cell division, development, and cell migration require spatially and temporally coordinated forces transduced by cell-surface receptors. Nucleic acid-based molecular tension probes allow one to visualize the piconewton (pN) forces applied by these receptors. Building on this technology, we recently developed molecular force microscopy (MFM) which uses fluorescence polarization to map receptor force orientation with diffraction-limited resolution (~250 nm). Here, we show that structured illumination microscopy (SIM), a super-resolution technique, can be used to perform super-resolution MFM. Using SIM-MFM, we generate the highest resolution maps of both the magnitude and orientation of the pN traction forces applied by cells. We apply SIM-MFM to map platelet and fibroblast integrin forces, as well as T cell receptor forces. Using SIM-MFM, we show that platelet traction force alignment occurs on a longer timescale than adhesion. Importantly, SIM-MFM can be implemented on any standard SIM microscope without hardware modifications.


Assuntos
Microscopia de Fluorescência/métodos , Receptores de Superfície Celular/metabolismo , Animais , Fenômenos Biomecânicos , Plaquetas/metabolismo , Linfócitos T CD8-Positivos , Corantes Fluorescentes/metabolismo , Humanos , Integrinas/metabolismo , Camundongos , Sondas Moleculares/metabolismo , Células NIH 3T3 , Paxilina/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Imagem com Lapso de Tempo
2.
Molecules ; 26(16)2021 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-34443565

RESUMO

Formaldehyde (FA) is a colorless, flammable, foul-smelling chemical used in building materials and in the production of numerous household chemical goods. Herein, a fluorescent chemosensor for FA is designed and prepared using a selective organ-targeting probe containing naphthalimide as a fluorophore and hydrazine as a FA-binding site. The amine group of the hydrazine reacts with FA to form a double bond and this condensation reaction is accompanied by a shift in the absorption band of the probe from 438 nm to 443 nm upon the addition of FA. Further, the addition of FA is shown to enhance the emission band at 532 nm relative to the very weak fluorescent emission of the probe itself. Moreover, a high specificity is demonstrated towards FA over other competing analytes such as the calcium ion (Ca2+), magnesium ion (Mg2+), acetaldehyde, benzaldehyde, salicylaldehyde, glucose, glutathione, sodium sulfide (Na2S), sodium hydrosulfide (NaHS), hydrogen peroxide (H2O2), and the tert-butylhydroperoxide radical. A typical two-photon dye incorporated into the probe provides intense fluorescence upon excitation at 800 nm, thus demonstrating potential application as a two-photon fluorescent probe for FA sensing. Furthermore, the probe is shown to exhibit a fast response time for the sensing of FA at room temperature and to facilitate intense fluorescence imaging of breast cancer cells upon exposure to FA, thus demonstrating its potential application for the monitoring of FA in living cells. Moreover, the presence of the phenylsulfonamide group allows the probe to visualize dynamic changes in the targeted Golgi apparatus. Hence, the as-designed probe is expected to open up new possibilities for unique interactions with organ-specific biological molecules with potential application in early cancer cell diagnosis.


Assuntos
Corantes Fluorescentes/metabolismo , Formaldeído/metabolismo , Complexo de Golgi/metabolismo , Naftalimidas/metabolismo , Células HeLa , Humanos , Fótons
3.
Nat Commun ; 12(1): 5083, 2021 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-34426577

RESUMO

AMPA receptor (AMPAR) abundance and positioning at excitatory synapses regulates the strength of transmission. Changes in AMPAR localisation can enact synaptic plasticity, allowing long-term information storage, and is therefore tightly controlled. Multiple mechanisms regulating AMPAR synaptic anchoring have been described, but with limited coherence or comparison between reports, our understanding of this process is unclear. Here, combining synaptic recordings from mouse hippocampal slices and super-resolution imaging in dissociated cultures, we compare the contributions of three AMPAR interaction domains controlling transmission at hippocampal CA1 synapses. We show that the AMPAR C-termini play only a modulatory role, whereas the extracellular N-terminal domain (NTD) and PDZ interactions of the auxiliary subunit TARP γ8 are both crucial, and each is sufficient to maintain transmission. Our data support a model in which γ8 accumulates AMPARs at the postsynaptic density, where the NTD further tunes their positioning. This interplay between cytosolic (TARP γ8) and synaptic cleft (NTD) interactions provides versatility to regulate synaptic transmission and plasticity.


Assuntos
Região CA1 Hipocampal/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Receptores de AMPA/metabolismo , Sinapses/metabolismo , Sequência de Aminoácidos , Animais , Corantes Fluorescentes/metabolismo , Imageamento Tridimensional , Camundongos Endogâmicos C57BL , Modelos Neurológicos , Mutação/genética , Plasticidade Neuronal , Neurônios/metabolismo , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Receptores de AMPA/química , Transmissão Sináptica
4.
ACS Appl Mater Interfaces ; 13(33): 39076-39087, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34378375

RESUMO

Fluorophores with multifunctional properties known as rare-earth-doped nanoparticles (RENPs) are promising candidates for bioimaging, therapy, and drug delivery. When applied in vivo, these nanoparticles (NPs) have to retain long blood-circulation time, bypass elimination by phagocytic cells, and successfully arrive at the target area. Usually, NPs in a biological medium are exposed to proteins, which form the so-called "protein corona" (PC) around the NPs and influence their targeted delivery and accumulation in cells and tissues. Different surface coatings change the PC size and composition, subsequently deciding the fate of the NPs. Thus, detailed studies on the PC are of utmost importance to determine the most suitable NP surface modification for biomedical use. When it comes to RENPs, these studies are particularly scarce. Here, we investigate the PC composition and its impact on the cellular uptake of citrate-, SiO2-, and phospholipid micelle-coated RENPs (LiYF4:Yb3+,Tm3+). We observed that the PC of citrate- and phospholipid-coated RENPs is relatively stable and similar in the adsorbed protein composition, while the PC of SiO2-coated RENPs is larger and highly dynamic. Moreover, biocompatibility, accumulation, and cytotoxicity of various RENPs in cancer cells have been evaluated. On the basis of the cellular imaging, supported by the inhibition studies, it was revealed that RENPs are internalized by endocytosis and that specific endocytic routes are PC composition dependent. Overall, these results are essential to fill the gaps in the fundamental understanding of the nano-biointeractions of RENPs, pertinent for their envisioned application in biomedicine.


Assuntos
Materiais Revestidos Biocompatíveis/química , Corantes Fluorescentes/química , Nanopartículas Metálicas/química , Metais Terras Raras/química , Coroa de Proteína/metabolismo , Dióxido de Silício/química , Adsorção , Neoplasias da Mama , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Ácido Cítrico/química , Materiais Revestidos Biocompatíveis/metabolismo , Endocitose , Corantes Fluorescentes/metabolismo , Humanos , Tamanho da Partícula , Fosfolipídeos/química , Propriedades de Superfície
5.
FASEB J ; 35(9): e21863, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34411334

RESUMO

Organic anion-transporting polypeptides, OATP1B1, OATP1B3, and OATP2B1 are multispecific membrane proteins mediating the hepatocellular uptake of structurally diverse endo- and exogenous compounds, including various kinds of drugs. Co-administration of OATP1B/2B1 substrates may lead to altered pharmacokinetics or even toxicity. Therefore, the study of the interaction with these OATPs is essential in drug development and is recommended by international regulatory agencies, the FDA, EMA, and PMDA. In general, radiolabeled indicators are used to measure drug interactions of OATPs, and, lately, fluorescent probes are also gaining wider application in OATP tests. However, all of the currently available methods (either radioactive or fluorescence-based) comprise multiple steps, including the removal of the indicator in the end of the experiment. Hence, they are not ideally suited for high-throughput screening. In the current study, in order to find an indicator allowing real-time assessment of hepatic OATP function, we searched for an activatable fluorogenic OATP substrate. Here, we show that 8-acetoxypyrene-1,3,6-trisulfonate (Ace), a fluorogenic derivative of the hepatic OATP substrate pyranine (8-hydroxypyrene-1,3,6-trisulfonate) enters the cells via OATP1B1/3 or OATP2B1 function. In living cells, Ace is then converted into highly fluorescent pyranine, allowing "no-wash" measurement of OATP function and drug interactions. Furthermore, we demonstrate that Ace can be used in an indirect assay termed as competitive counterflow suitable to distinguish between transported substrates and inhibitors of OATP1B1. The fluorescence-based methods described here are unique and open the way toward high-throughput screening of interactions between new molecular entities and OATPs.


Assuntos
Corantes Fluorescentes/análise , Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/metabolismo , Animais , Sulfonatos de Arila/análise , Sulfonatos de Arila/química , Sulfonatos de Arila/metabolismo , Linhagem Celular , Sobrevivência Celular , Corantes Fluorescentes/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Fígado/metabolismo
6.
Theranostics ; 11(16): 7767-7778, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34335963

RESUMO

Background: Lipid droplets (LDs) establish a considerable number of contact sites with mitochondria to enable energy transfer and communication. In this study, we developed a fluorescent biosensor to image LD-mitochondria interactions at the nanoscale and further explored the function of LD-mediated matrix transmission in processes involving multi-organelle interactions. Methods: A fluorescent probe called C-Py (C21H19N3O2, 7-(diethylamino) coumarin-3-vinyl-4-pyridine acetonitrile) was designed and synthesized. Colocalization of C-Py and the commercial LD stain Nile Red was analyzed in HeLa cells. The fluorescence stability and signal to background ratio of C-Py under structured illumination microscopy (SIM) were compared to those of the commercial probe BODIPY493/503. The cytotoxicity of C-Py was assessed using CCK-8 assays. The uptake pattern of C-Py in HeLa cells was then observed under various temperatures, metabolic levels, and endocytosis levels. Contact sites between LDs and various organelles, such as mitochondria, nuclei, and cell membrane, were imaged and quantitated using SIM. Physical changes to the contact sites between LDs and mitochondria were monitored after lipopolysaccharide induction. Results: A LD-targeted fluorescent biosensor, C-Py, with good specificity, low background signal, excellent photostability, low cytotoxicity, and high cellular permeability was developed for tracking LD contact sites with multiple organelles using SIM. Using C-Py, the subcellular distribution and dynamic processes of LDs in living cells were observed under SIM. The formation of contact sites between LDs and multiple organelles was visualized at a resolution below ~200 nm. The number of LD-mitochondria contact sites formed was decreased by lipopolysaccharide treatment inducing an inflammatory environment. Conclusions: C-Py provides strategies for the design of ultra-highly selective biosensors and a new tool for investigating the role and regulation of LDs in living cells at the nanoscale.


Assuntos
Gotículas Lipídicas/metabolismo , Mitocôndrias/metabolismo , Imagem Individual de Molécula/métodos , Técnicas Biossensoriais/métodos , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Células HeLa , Humanos , Metabolismo dos Lipídeos/fisiologia
7.
Chem Commun (Camb) ; 57(63): 7786-7789, 2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34264259

RESUMO

Herein, we propose a dual-responsive fluorescent nanoprobe to visualize the cross-talk between O2 and adenosine triphosphate (ATP) in living cells. We hope it will be a helpful tool for the further understanding of cellular metabolism and further facilitating risk warning in the process of adaptation to consistent environmental pressures in premalignant lesions.


Assuntos
Trifosfato de Adenosina/metabolismo , Corantes Fluorescentes/metabolismo , Nanopartículas/metabolismo , Oxigênio/metabolismo , Trifosfato de Adenosina/química , Corantes Fluorescentes/química , Humanos , Células MCF-7 , Nanopartículas/química , Oxigênio/química
9.
Chem Commun (Camb) ; 57(64): 7902-7905, 2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34286752

RESUMO

A cocktail [1 + 2] dual-fluorescent probe system was developed to realize the real-time visualization of dynamic iron state changes between Fe2+ and Fe3+ at the cellular level and in multicellular organisms, providing insights into the effect of DMT1 and ferroportin on iron regulation.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Corantes Fluorescentes/química , Ferro/análise , Proteínas de Transporte de Cátions/química , Corantes Fluorescentes/metabolismo , Células Hep G2 , Humanos , Ferro/metabolismo , Estrutura Molecular
10.
Int J Mol Sci ; 22(14)2021 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-34298874

RESUMO

Polyphosphate (polyP), a biopolymer of inorganic phosphate, is widely distributed in living organisms. In platelets, polyP is released upon activation and plays important roles in coagulation and tissue regeneration. However, the lack of a specific quantification method has delayed the in-depth study of polyP. The fluorescent dye 4',6-diamidine-2-phenylindole dihydrochloride (DAPI) has recently received attention as a promising probe for the visualization and quantification of cellular polyP levels. In this study, we further optimized quantification conditions and applied this protocol in quantification of platelet polyP levels in a Japanese population. Blood samples were collected from non-smoking, healthy Japanese subjects (23 males, 23 females). Washed platelets were fixed and probed with DAPI for fluorometric determination. PolyP levels per platelet count were significantly higher in women than that in men. A moderate negative correlation between age and polyP levels was found in women. Responsiveness to CaCl2 stimulation was also significantly higher in women than that in men. Overall, our optimized protocol requires neither purification nor degradation steps, reducing both the time and bias for reproducible quantification. Thus, we suggest that despite its low specificity, this DAPI-based protocol would be useful in routine laboratory testing to quantify platelet polyP levels efficiently and economically.


Assuntos
Plaquetas/metabolismo , Corantes Fluorescentes/metabolismo , Indóis/metabolismo , Polifosfatos/metabolismo , Adulto , Grupo com Ancestrais do Continente Asiático , Feminino , Fluorescência , Fluorometria/métodos , Humanos , Masculino
11.
Methods Mol Biol ; 2314: 231-245, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34235655

RESUMO

Mycobacteria are intrinsically resistant to most antimicrobials, which is generally attributed to the impermeability of their cell wall that considerably limits drug uptake. Moreover, like in other pathogenic bacteria, active efflux systems have been widely characterized from diverse mycobacterial species in laboratory conditions, showing that they can promote resistance by extruding noxious compounds prior to their reaching their intended targets. Therefore, the intracellular concentration of a given compound is determined by the balance between permeability, influx, and efflux.Given the urgent need to discover and develop novel antimycobacterial compounds in order to design effective therapeutic strategies, the contributions to drug resistance made by the controlled permeability of the cell wall and the increased activity of efflux pumps must be determined. In this chapter, we will describe a method that allows (1) the measuring of permeability and the quantification of general efflux activity of mycobacteria, by the study of the transport (influx and efflux) of fluorescent compounds, such as ethidium bromide; and (2) the screening of compounds in search of agents that increase the permeability of the cell wall and efflux inhibitors that could restore the effectiveness of antimicrobials that are subject to efflux.


Assuntos
Proteínas de Bactérias/metabolismo , Permeabilidade da Membrana Celular , Etídio/metabolismo , Fluorometria/métodos , Mycobacterium/metabolismo , Antibacterianos/farmacologia , Transporte Biológico , Farmacorresistência Bacteriana Múltipla , Corantes Fluorescentes/metabolismo , Testes de Sensibilidade Microbiana , Mycobacterium/efeitos dos fármacos , Mycobacterium/crescimento & desenvolvimento
12.
Molecules ; 26(12)2021 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-34205289

RESUMO

The inadvertent severing of a ureter during surgery occurs in as many as 4.5% of colorectal surgeries. To help prevent this issue, several near-infrared (NIR) dyes have been developed to assist surgeons with identifying ureter location. However, the majority of these dyes exhibit at least some issue that precludes their widespread usage such as high levels of uptake in other tissues, overlapping emission wavelengths with other NIR dyes used for other fluorescence-guided surgeries, and/or rapid excretion times through the ureters. To overcome these limitations, we have synthesized and characterized the spectral properties and biodistribution of a new series of PEGylated UreterGlow derivatives. The most promising dye, UreterGlow-11 was shown to almost exclusively excrete through the kidneys/ureters with detectable fluorescence observed for at least 12 h. Additionally, while the excitation wavelength is similar to that of other NIR dyes used for cancer resections, the emission is shifted by ~30 nm allowing for discrimination between the different fluorescence-guided surgery probes. In conclusion, these new UreterGlow dyes show promising optical and biodistribution characteristics and are good candidates for translation into the clinic.


Assuntos
Abdome/cirurgia , Imagem Óptica/métodos , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Ureter/cirurgia , Animais , Fluorescência , Corantes Fluorescentes/metabolismo , Humanos , Rim/cirurgia , Camundongos , Distribuição Tecidual/fisiologia , Ureter/metabolismo
13.
Int J Mol Sci ; 22(13)2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201658

RESUMO

The death of photoreceptor cells is induced by continuous light exposure. However, it is unclear whether light damage was induced in retinal ganglion cells with photosensitivity by transduction of optogenetic genes. In this study, we evaluated the phototoxicities of continuous light exposure on retinal ganglion cells after transduction of the optogenetic gene mVChR1 using an adeno-associated virus vector. Rats were exposed to continuous light for a week, and visually evoked potentials (VEPs) were recorded. The intensities of continuous light (500, 1000, 3000, and 5000 lx) increased substantially after VEP recordings. After the final recording of VEPs, retinal ganglion cells (RGCs) were retrogradely labeled with a fluorescein tracer, FluoroGold, and the number of retinal ganglion cells was counted under a fluorescent microscope. There was no significant reduction in the amplitudes of VEPs and the number of RGCs after exposure to any light intensity. These results indicated that RGCs were photosensitive after the transduction of optogenetic genes and did not induce any phototoxicity by continuous light exposure.


Assuntos
Optogenética/métodos , Células Ganglionares da Retina/fisiologia , Rodopsina/genética , Animais , Dependovirus/genética , Potenciais Evocados Visuais , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Células HEK293 , Humanos , Luz/efeitos adversos , Técnicas de Patch-Clamp , Estimulação Luminosa , Ratos , Células Ganglionares da Retina/patologia , Rodopsina/metabolismo , Estilbamidinas/química , Estilbamidinas/metabolismo , Transdução Genética , Volvox/genética
14.
ACS Appl Mater Interfaces ; 13(26): 30337-30349, 2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34159778

RESUMO

The efficient, versatile, and straightforward synthesis of the first N-alkyl analogues of induline 3B (8a and 8b) is reported. Thanks to the introduction of lipophilic substituents and their attractive photophysical properties (far-red emission and production of singlet oxygen), phenazinium 8b can be used as a theranostic agent and shows, at very low concentrations (100 nM), a remarkable ability to (i) image cells and zebrafish embryos with high quality under both mono- (514 nm) and biphotonic (790 and 810 nm) excitations, (ii) efficiently and quickly penetrate cancer cells rather than healthy fibroblasts, and (iii) induce a total or almost total cancer cell death in vitro and in vivo after illumination (λexc = 540-560 nm). The molecular structure of 8b is based on a triamino-phenazinium core only, with no need for additional components, highlighting the emergence of a minimalistic and versatile class of fluorescent probes for targeted photodynamic cancer therapy.


Assuntos
Antineoplásicos/uso terapêutico , Corantes Fluorescentes/uso terapêutico , Fenazinas/uso terapêutico , Fármacos Fotossensibilizantes/uso terapêutico , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Antineoplásicos/efeitos da radiação , Linhagem Celular Tumoral , Fibroblastos/metabolismo , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Corantes Fluorescentes/efeitos da radiação , Humanos , Luz , Neoplasias/diagnóstico por imagem , Neoplasias/tratamento farmacológico , Fenazinas/síntese química , Fenazinas/metabolismo , Fenazinas/efeitos da radiação , Fotoquimioterapia , Fármacos Fotossensibilizantes/síntese química , Fármacos Fotossensibilizantes/metabolismo , Fármacos Fotossensibilizantes/efeitos da radiação , Medicina de Precisão/métodos , Oxigênio Singlete/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-Zebra
15.
Methods Mol Biol ; 2310: 113-159, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34096002

RESUMO

Mitochondria are dynamic organelles that participate in a broad array of molecular functions within the cell. They are responsible for maintaining the appropriate energetic levels and control the cellular homeostasis throughout the generation of intermediary metabolites. Preserving a healthy and functional mitochondrial population is of fundamental importance throughout the life of the cells under pathophysiological conditions. Hence, cells have evolved fine-tuned mechanisms of quality control that help to preserve the right amount of functional mitochondria to meet the demand of the cell. The specific recycling of mitochondria by autophagy, termed mitophagy, represents the primary contributor to mitochondrial quality control. During this process, damaged or unnecessary mitochondria are recognized and selectively degraded. In the past few years, the knowledge in mitophagy has seen rapid progress, and a growing body of evidence confirms that mitophagy holds a central role in controlling cellular functions and the progression of various human diseases.In this chapter, we will discuss the pathophysiological roles of mitophagy and provide a general overview of the current methods used to monitor and quantify mitophagy. We will also outline the main established approaches to investigate the mitochondrial function, metabolism, morphology, and protein damage.


Assuntos
Doenças Cardiovasculares/patologia , Microscopia Confocal , Microscopia de Fluorescência , Mitocôndrias/patologia , Dinâmica Mitocondrial , Mitofagia , Neoplasias/patologia , Doenças Neurodegenerativas/patologia , Animais , Biomarcadores/metabolismo , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/metabolismo , Linhagem Celular , Metabolismo Energético , Corantes Fluorescentes/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Transfecção
16.
Methods Mol Biol ; 2310: 287-299, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34096009

RESUMO

The dynamism of mitochondria, considered as complex and motile organelles, is brought about by mitochondria ability to undergo cycles of fission and fusion events, whose fine balance determines their morphology in a specific physiological context. A huge body of evidence makes it possible to associate mitochondrial organization to regulation of an increasing number of key cellular processes, such as biosynthetic pathways, oxidative phosphorylation and ATP production, calcium buffering, mtDNA homeostasis, autophagy, and cell death. Here, we review the recently developed imaging methods for studying mitochondrial dynamics, including live-cell imaging, by using mitochondrial-targeted fluorescent proteins. In more details, we focus our attention on two different protocols in the T cell model, an example of nonadherent cells, which present some particularities and difficulties in the analysis of mitochondrial shape. Also, we discuss some examples of mouse models carrying mitochondria-targeted fluorescent proteins, which allow to investigate the mitochondrial morphology in vivo.


Assuntos
Microscopia Confocal , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Linfócitos T/metabolismo , Animais , Fracionamento Celular , Corantes Fluorescentes/metabolismo , Humanos , Células Jurkat , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos Transgênicos , Microscopia de Vídeo , Mitocôndrias/genética , Mitocôndrias/imunologia , Linfócitos T/imunologia , Fatores de Tempo , Imagem com Lapso de Tempo
17.
Chem Commun (Camb) ; 57(56): 6911-6914, 2021 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-34152336

RESUMO

A coumarin-based two-photon (TP) fluorescent off-on probe has been developed for detecting tyrosinase activity. High selectivity, sensitivity and biocompatibility enable the probes to successfully image tyrosinase activity in live cells and tissues using TP microscopy.


Assuntos
Cumarínicos/química , Corantes Fluorescentes/química , Monofenol Mono-Oxigenase/análise , Animais , Linhagem Celular Tumoral , Cumarínicos/metabolismo , Desenho de Fármacos , Corantes Fluorescentes/metabolismo , Humanos , Limite de Detecção , Camundongos , Microscopia de Fluorescência por Excitação Multifotônica , Monofenol Mono-Oxigenase/metabolismo , Fótons , Ratos
18.
Am J Physiol Cell Physiol ; 321(1): C187-C198, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34106786

RESUMO

Ca2+ signaling plays a critical role in the regulation of hepatic metabolism by hormones including insulin. Changes in cytoplasmic Ca2+ regulate synthesis and posttranslational modification of key signaling proteins in the insulin pathways. Emerging evidence suggests that hepatocyte intracellular Ca2+ signaling is altered in lipid-loaded liver cells isolated from obese rodent models. The mechanisms of altered Ca2+-insulin and insulin-Ca2+ signaling pathways in obesity remain poorly understood. Here, we show that the kinetics of insulin-initiated intracellular (initial) Ca2+ release from endoplasmic reticulum is significantly impaired in steatotic hepatocytes from obese Alström syndrome mice. Furthermore, exenatide, a glucagon-like peptide-1 (GLP-1) analog, reversed lipid-induced inhibition of intracellular Ca2+ release kinetics in steatotic hepatocytes, without affecting the total content of intracellular Ca2+ released. Exenatide reversed the lipid-induced inhibition of intracellular Ca2+ release, at least partially, via lipid reduction in hepatocytes, which then restored hormone-regulated cytoplasmic Ca2+ signaling and insulin sensitivity. This data provides additional evidence for the important role of Ca2+ signaling pathways in obesity-associated impaired hepatic lipid homeostasis and insulin signaling. It also highlights a potential advantage of GLP-1 analogs when used to treat type 2 diabetes associated with hepatic steatosis.


Assuntos
Síndrome de Alstrom/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Exenatida/farmacologia , Hipoglicemiantes/farmacologia , Fígado/efeitos dos fármacos , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Obesidade/tratamento farmacológico , Síndrome de Alstrom/metabolismo , Síndrome de Alstrom/patologia , Animais , Glicemia/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Modelos Animais de Doenças , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Corantes Fluorescentes/metabolismo , Fura-2/metabolismo , Peptídeo 1 Semelhante ao Glucagon/análogos & derivados , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Insulina/metabolismo , Resistência à Insulina , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Transgênicos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Obesidade/metabolismo , Obesidade/patologia , Ácido Palmítico/farmacologia
19.
Nat Cell Biol ; 23(6): 642-651, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34059812

RESUMO

In the last decade, DNA-based tension sensors have made significant contributions to the study of the importance of mechanical forces in many biological systems. Albeit successful, one shortcoming of these techniques is their inability to reversibly measure receptor forces in a higher regime (that is, >20 pN), which limits our understanding of the molecular details of mechanochemical transduction in living cells. Here, we developed a reversible shearing DNA-based tension probe (RSDTP) for probing molecular piconewton-scale forces between 4 and 60 pN transmitted by cells. Using these probes, we can easily distinguish the differences in force-bearing integrins without perturbing adhesion biology and reveal that a strong force-bearing integrin cluster can serve as a 'mechanical pivot' to maintain focal adhesion architecture and facilitate its maturation. The benefits of the RSDTP include a high dynamic range, reversibility and single-molecule sensitivity, all of which will facilitate a better understanding of the molecular mechanisms of mechanobiology.


Assuntos
Sondas de DNA/metabolismo , Adesões Focais/metabolismo , Integrinas/metabolismo , Mecanotransdução Celular , Microscopia de Fluorescência , Microscopia de Vídeo , Animais , Técnicas Biossensoriais , Adesão Celular , Movimento Celular , Sondas de DNA/genética , Corantes Fluorescentes/metabolismo , Adesões Focais/genética , Integrinas/genética , Camundongos , Células NIH 3T3 , Nanotecnologia , Conformação de Ácido Nucleico , Estresse Mecânico , Fatores de Tempo
20.
Chem Commun (Camb) ; 57(54): 6628-6631, 2021 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-34124718

RESUMO

A ratiometric fluorescent probe with a large emission shift was developed for the accurate measurement of hydrogen peroxide (H2O2) in sophisticated pyroptosis signaling pathways. The results reported here demonstrate that H2O2, as a principal member of ROS, is a critical upstream signaling molecule in regulating pyroptosis.


Assuntos
Corantes Fluorescentes/metabolismo , Peróxido de Hidrogênio/metabolismo , Piroptose , Transdução de Sinais , Linhagem Celular , Sobrevivência Celular , Humanos
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