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1.
Methods Cell Biol ; 187: 57-72, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38705630

RESUMO

Correlative light and electron microscopy (CLEM) can provide valuable information about a biological sample by giving information on the specific localization of a molecule of interest within an ultrastructural context. In this work, we describe a simple CLEM method to obtain high-resolution images of neurotransmitter receptor distribution in synapses by electron microscopy (EM). We use hippocampal organotypic slices from a previously reported mouse model expressing a modified AMPA receptor (AMPAR) subunit that binds biotin at the surface (Getz et al., 2022). This tag can be recognized by StreptAvidin-Fluoronanogold™ conjugates (SA-FNG), which reach receptors at synapses (synaptic cleft is 50-100nm thick). By using pre-embedding labeling, we found that SA-FNG reliably bind synaptic receptors and penetrate around 10-15µm in depth in live tissue. However, the silver enhancement was only reaching the surface of the slices. We show that permeabilization with triton is highly effective at increasing the in depth-gold amplification and that the membrane integrity is well preserved. Finally, we also apply high-resolution electron tomography, thus providing important information about the 3D organization of surface AMPA receptors in synapses at the nanoscale.


Assuntos
Hipocampo , Receptores de AMPA , Sinapses , Animais , Camundongos , Hipocampo/metabolismo , Hipocampo/citologia , Receptores de AMPA/metabolismo , Sinapses/metabolismo , Sinapses/ultraestrutura , Proteínas de Membrana/metabolismo , Ouro/química , Microscopia Eletrônica/métodos , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo
2.
J Physiol ; 602(8): 1637-1654, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38625711

RESUMO

The eukaryotic cell is highly compartmentalized with organelles. Owing to their function in transporting metabolites, metabolic intermediates and byproducts of metabolic activity, organelles are important players in the orchestration of cellular function. Recent advances in optical methods for interrogating the different aspects of organellar activity promise to revolutionize our ability to dissect cellular processes with unprecedented detail. The transport activity of organelles is usually coupled to the transport of charged species; therefore, it is not only associated with the metabolic landscape but also entangled with membrane potentials. In this context, the targeted expression of fluorescent probes for interrogating organellar membrane potential (Ψorg) emerges as a powerful approach, offering less-invasive conditions and technical simplicity to interrogate cellular signalling and metabolism. Different research groups have made remarkable progress in adapting a variety of optical methods for measuring and monitoring Ψorg. These approaches include using potentiometric dyes, genetically encoded voltage indicators, hybrid fluorescence resonance energy transfer sensors and photoinduced electron transfer systems. These studies have provided consistent values for the resting potential of single-membrane organelles, such as lysosomes, the Golgi and the endoplasmic reticulum. We can foresee the use of dynamic measurements of Ψorg to study fundamental problems in organellar physiology that are linked to serious cellular disorders. Here, we present an overview of the available techniques, a survey of the resting membrane potential of internal membranes and, finally, an open-source mathematical model useful to interpret and interrogate membrane-bound structures of small volume by using the lysosome as an example.


Assuntos
Lisossomos , Organelas , Potenciais da Membrana , Organelas/metabolismo , Lisossomos/metabolismo , Retículo Endoplasmático/metabolismo , Corantes Fluorescentes/análise , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo
3.
Anal Chim Acta ; 1304: 342572, 2024 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-38637042

RESUMO

BACKGROUND: Adenosine 5'-triphosphate (ATP) plays an important role in cell metabolism and has been regarded as an indicator of cell survival and damage. Golgi apparatus participates in the signal transduction processes of substance transport, ion homeostasis and stress when extracellular substances enter cells. Till now, there is no fluorescent probe for monitoring Golgi ATP level fluctuation and visualizing the configuration change of the Golgi apparatus during the inhibition of glycolysis. RESULTS: Herein, we report the synthesis of a novel water-soluble cationic polythiophene derivative (PEMTEA) that can be employed as a fluorescent sensor for measuring ATP in the Golgi apparatus. PEMTEA self-assembles into PT-NP nanoparticles in aqueous solution with a diameter of approximately 2 nm. PT-NP displays high sensitivity and superb selectivity towards ATP with a detection limit of 90 nM and a linear detection range from 0 to 3.0 µM. The nanoparticles show low toxicity to HepG2 cells and good photostability in the Golgi apparatus. With the stimulation of Ca2+, PT-NP was practically applied to real-time monitor of endogenous ATP levels in the Golgi apparatus through fluorescence microscopy. Finally, we studied the relationship between the concentration of ATP and configuration of the Golgi apparatus during the inhibition of glycolysis using PT-NP. SIGNIFICANCE: We have demonstrated that PT-NP can not only indicate the fluctuation and distribution of ATP in the Golgi apparatus, but also give the information of the configuration change of the Golgi apparatus at the single-cell level during the inhibition of glycolysis.


Assuntos
Corantes Fluorescentes , Nanopartículas , Corantes Fluorescentes/metabolismo , Água/metabolismo , Complexo de Golgi/metabolismo , Trifosfato de Adenosina/metabolismo , Polímeros , Glicólise
4.
J Am Chem Soc ; 146(14): 9564-9574, 2024 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-38557024

RESUMO

The serotonergic transmitter system plays fundamental roles in the nervous system in neurotransmission, synaptic plasticity, pathological processes, and therapeutic effects of antidepressants and psychedelics, as well as in the gastrointestinal and circulatory systems. We introduce a novel small molecule fluorescent agent, termed SERTlight, that specifically labels serotonergic neuronal cell bodies, dendrites, and axonal projections as a serotonin transporter (SERT) fluorescent substrate. SERTlight was developed by an iterative molecular design process, based on an aminoethyl-quinolone system, to integrate structural elements that impart SERT substrate activity, sufficient fluorescent brightness, and a broad absence of pharmacological activity, including at serotonin (5-hydroxytryptamine, 5HT) receptors, other G protein-coupled receptors (GPCRs), ion channels, and monoamine transporters. The high labeling selectivity is not achieved by high affinity binding to SERT itself but rather by a sufficient rate of SERT-mediated transport of SERTlight, resulting in accumulation of these molecules in 5HT neurons and yielding a robust and selective optical signal in the mammalian brain. SERTlight provides a stable signal, as it is not released via exocytosis nor by reverse SERT transport induced by 5HT releasers such as MDMA. SERTlight is optically, pharmacologically, and operationally orthogonal to a wide range of genetically encoded sensors, enabling multiplexed imaging. SERTlight enables labeling of distal 5HT axonal projections and simultaneous imaging of the release of endogenous 5HT using the GRAB5HT sensor, providing a new versatile molecular tool for the study of the serotonergic system.


Assuntos
Corantes Fluorescentes , Serotonina , Animais , Serotonina/metabolismo , Corantes Fluorescentes/metabolismo , Neurônios/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Encéfalo/metabolismo , Mamíferos/metabolismo
5.
ACS Nano ; 18(15): 10596-10608, 2024 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-38557034

RESUMO

Continuously monitoring neurotransmitter dynamics can offer profound insights into neural mechanisms and the etiology of neurological diseases. Here, we present a miniaturized implantable fluorescence probe integrated with metal-organic frameworks (MOFs) for deep brain dopamine sensing. The probe is assembled from physically thinned light-emitting diodes (LEDs) and phototransistors, along with functional surface coatings, resulting in a total thickness of 120 µm. A fluorescent MOF that specifically binds dopamine is introduced, enabling a highly sensitive dopamine measurement with a detection limit of 79.9 nM. A compact wireless circuit weighing only 0.85 g is also developed and interfaced with the probe, which was later applied to continuously monitor real-time dopamine levels during deep brain stimulation in rats, providing critical information on neurotransmitter dynamics. Cytotoxicity tests and immunofluorescence analysis further suggest a favorable biocompatibility of the probe for implantable applications. This work presents fundamental principles and techniques for integrating fluorescent MOFs and flexible electronics for brain-computer interfaces and may provide more customized platforms for applications in neuroscience, disease tracing, and smart diagnostics.


Assuntos
Dopamina , Estruturas Metalorgânicas , Ratos , Animais , Dopamina/análise , Estruturas Metalorgânicas/metabolismo , Corantes Fluorescentes/metabolismo , Fluorescência , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Neurotransmissores/metabolismo
6.
Photobiomodul Photomed Laser Surg ; 42(4): 314-320, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38536111

RESUMO

Background: Acinetobacter baumannii, a nosocomial pathogen, poses a major public health problem due to generating resistance to several antimicrobial agents. Antimicrobial photodynamic inactivation (APDI) employs a nontoxic dye as a photosensitizer (PS) and light to produce reactive oxygen species that destroy bacterial cells. The intracellular concentration of PS could be affected by factors such as the function of efflux pumps to emit PS from the cytosol. Objective: To evaluate the augmentation effect of an efflux pump inhibitor, verapamil, three multidrug-resistant A. baumannii were subjected to APDI by erythrosine B (EB). Methods and results: The combination of EB and verapamil along with irradiation at 530 nm induced a lethal effect and more than 3 log colony-forming unit reduction to all A. baumannii strains in planktonic state. In contrast, EB and irradiation alone could produce only a sublethal effect on two of the strains. Conclusions: These data suggest that verapamil increases the intracellular concentration of EB, which potentiates the lethal efficacy of APDI. Verapamil could be applied with EB and green light to improve their antimicrobial efficacy against A. baumannii-localized infections.


Assuntos
Acinetobacter baumannii , Farmacorresistência Bacteriana Múltipla , Eritrosina , Corantes Fluorescentes , Fármacos Fotossensibilizantes , Verapamil , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/efeitos da radiação , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/efeitos da radiação , Fármacos Fotossensibilizantes/farmacologia , Verapamil/farmacologia , Eritrosina/metabolismo , Corantes Fluorescentes/metabolismo , Luz
7.
Spectrochim Acta A Mol Biomol Spectrosc ; 313: 124095, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38490121

RESUMO

Cell apoptosis is a crucial physiological process playing central roles in key biological and pathological activities. However, the current fluorescent probes for the detection of late apoptosis were "off-on" probes, which were facilely interfered by false positive signals caused by inhomogeneous staining and other factors. Herein, a unique fluorescent probe (NPn) discriminating late apoptosis from early apoptosis and heathy status with two different sets of fluorescent signals have been prepared, to overcome the possible false positive signals. NPn was designed impermeable to biomembranes and simultaneously with high affinity to DNA/RNA, which localized on the plasma membranes of living and early apoptotic cells, while relocated to the nucleus in late apoptotic cells. The hydrophilic amine unit and small ion radius were responsive for its membrane impermeability, which was confirmed with two control molecules without amine group. Using the probe, we have successfully evaluated the cell apoptosis induced by ultraviolet irradiation, rotenone, colchicine, and paclitaxel, demonstrating its potential application in biological researches.


Assuntos
Apoptose , Corantes Fluorescentes , Corantes Fluorescentes/metabolismo , Membrana Celular/metabolismo , Paclitaxel/metabolismo , Aminas
8.
Talanta ; 273: 125894, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38461644

RESUMO

Hydrogen peroxide (H2O2) is a reactive oxygen species (ROS) that can be used as a marker for the occurrence of oxidative stress in the organism. Lysosomes serve as intracellular digestive sites, and when the concentration of H2O2 in them is abnormal, lysosomal function is often impaired, leading to the development of diseases. Hydrogen sulfide (H2S) acts as a gaseous signaling molecule that scavenges H2O2 from cells and tissues, thereby maintaining the redox environment of the body. However, most of the reported hydrogen peroxide fluorescent probes so far can only detect H2O2, but cannot maintain the intracellular redox environment. In this paper, an H2O2 fluorescent probe LN-HOD with lysosomal targeting properties was designed and synthesized by combining the H2O2 recognition site with a naphthylamine fluorophore via a thiocarbamate moiety. The probe has the advantages of large Stokes shift (110 nm), high sensitivity and good H2S release capability. The probe LN-HOD can be used to detect H2O2 in cells, zebrafish and plant roots. In addition, LN-HOD detects changes in the concentration of H2O2 in plant roots when Arabidopsis is stressed by cadmium ion (Cd2+). And through its ability to release H2S, it can help to remove excess H2O2 and maintain the redox environment in cells, zebrafish and plant roots. The present work provides new ideas for the detection and assisted removal of H2O2, which contributes to the in-depth study of the cellular microenvironment in organisms.


Assuntos
Corantes Fluorescentes , Sulfeto de Hidrogênio , Animais , Humanos , Corantes Fluorescentes/metabolismo , Peróxido de Hidrogênio/metabolismo , Peixe-Zebra , Sulfeto de Hidrogênio/metabolismo , Oxirredução , Lisossomos/metabolismo , Células HeLa
9.
J Am Chem Soc ; 146(14): 10093-10102, 2024 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-38545938

RESUMO

Real-time monitoring of the development of atherosclerosis (AS) is key to the management of cardiovascular disease (CVD). However, existing laboratory approaches lack sensitivity and specificity, mostly due to the dearth of reliable AS biomarkers. Herein, we developed an in vivo fluorescent labeling strategy that allows specific staining of the foam cell-derived extracellular vesicles (EVs) in atherosclerotic plaques, which are released into the blood as circulating biomarkers for in vitro detection of AS. This strategy relies on a self-assembled nanoprobe that could recognize foam cells specifically, where the probe is degraded by the intracellular HClO to produce a trifluoromethyl-bearing boron-dipyrromethene fluorophore (termed B-CF3), a lipophilic dye that can be transferred to the exosomal membranes. These circulating B-CF3-stained EVs can be detected directly on a fluorescence spectrometer or microplate reader without resorting to any sophisticated analytical method. This liquid-biopsy format enables early detection and real-time differentiation of lesion vulnerability during AS progression, facilitating effective CVD management.


Assuntos
Aterosclerose , Vesículas Extracelulares , Humanos , Células Espumosas/metabolismo , Células Espumosas/patologia , Vesículas Extracelulares/metabolismo , Biomarcadores/metabolismo , Corantes Fluorescentes/metabolismo , Aterosclerose/diagnóstico por imagem , Aterosclerose/metabolismo
10.
Anal Chem ; 96(11): 4709-4715, 2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38457637

RESUMO

The varied functions of lipid droplets, which encompass the regulation of lipid and energy homeostasis, as well as their association with the occurrence of various metabolic diseases, are intricately linked to their dynamic properties. Super-resolution imaging techniques have emerged to decipher physiological processes and molecular mechanisms on the nanoscale. However, achieving long-term dynamic super-resolution imaging faces challenges due to the need for fluorescent probes with high photostability. This paper introduces LD-CF, a "buffering probe" for imaging lipid droplet dynamics using structured illumination microscopy (SIM). The polarity-sensitive LD-CF eliminates background fluorescence with a "cyan filter" strategy, enabling wash-free imaging of lipid droplets. In the fluorescent "off" state outside droplets, the probes act as a "buffering pool", replacing photobleached probes inside droplets and enabling photostable long-term SIM imaging. With this probe, three modes of lipid droplet fusion were observed, including the discovery of fusion from large to small lipid droplets. Fluorescence intensity tracking also revealed the direction of lipid transport during the lipid droplet fusion.


Assuntos
Corantes Fluorescentes , Gotículas Lipídicas , Corantes Fluorescentes/metabolismo , Gotículas Lipídicas/metabolismo , Microscopia de Fluorescência/métodos , Transporte Biológico , Lipídeos
11.
J Mater Chem B ; 12(12): 3022-3030, 2024 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-38426244

RESUMO

Lipid droplets (LDs) are cytoplasmic lipid-rich organelles with important roles in lipid storage and metabolism, cell signaling and membrane biosynthesis. Additionally, multiple diseases, such as obesity, fatty liver, cardiovascular diseases and cancer, are related to the metabolic disorders of LDs. In various cancer cells, LD accumulation is associated with resistance to cell death, reduced effectiveness of chemotherapeutic drugs, and increased proliferation and aggressiveness. In this work, we present a new viscosity-sensitive, green-emitting BODIPY probe capable of distinguishing between ordered and disordered lipid phases and selectively internalising into LDs of live cells. Through the use of fluorescence lifetime imaging microscopy (FLIM), we demonstrate that LDs in live cancer (A549) and non-cancer (HEK 293T) cells have vastly different microviscosities. Additionally, we quantify the microviscosity changes in LDs under the influence of DNA-damaging chemotherapy drugs doxorubicin and etoposide. Finally, we show that doxorubicin and etoposide have different effects on the microviscosities of LDs in chemotherapy-resistant A549 cancer cells.


Assuntos
Compostos de Boro , Gotículas Lipídicas , Neoplasias , Gotículas Lipídicas/metabolismo , Corantes Fluorescentes/farmacologia , Corantes Fluorescentes/metabolismo , Etoposídeo/metabolismo , Lipídeos , Doxorrubicina/farmacologia , Doxorrubicina/metabolismo , Neoplasias/diagnóstico por imagem , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo
12.
Sci Rep ; 14(1): 6419, 2024 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-38494537

RESUMO

Extracellular vesicles (EVs) have crucial roles in hemostasis and coagulation. They sustain coagulation by exposing phosphatidylserine and initiate clotting by surface expression of tissue factor (TF) under inflammatory conditions. As their relevance as biomarkers of coagulopathy is increasingly recognized, there is a need for the sensitive and reliable detection of TF+ EVs, but their flow cytometric analysis is challenging and has yielded controversial findings for TF expression on EVs in the vascular system. We investigated the effect of different fluorochrome-to-protein (F/P) ratios of anti-TF-fluorochrome conjugates on the flow cytometric detection of TF+ EVs from activated monocytes, mesenchymal stem cells (MSCs), and in COVID-19 plasma. Using a FITC-labeled anti-TF antibody (clone VD8), we show that the percentage of TF+ EVs declined with decreasing F/P ratios. TF was detected on 7.6%, 5.4%, and 1.1% of all EVs derived from activated monocytes at F/P ratios of 7.7:1, 6.6:1, and 5.2:1. A similar decline was observed for EVs from MSCs and for EVs in plasma, whereas the detection of TF on cells remained unaffected by different F/P ratios. We provide clear evidence that next to the antibody clone, the F/P ratio affects the flow cytometric detection of TF+ EVs and should be carefully controlled.


Assuntos
Vesículas Extracelulares , Tromboplastina , Tromboplastina/metabolismo , Corantes Fluorescentes/metabolismo , Coagulação Sanguínea , Vesículas Extracelulares/metabolismo
13.
Cell Calcium ; 119: 102873, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38537433

RESUMO

Calcium signaling is a critical process required for cellular mechanisms such as cardiomyocyte contraction. The inability of the cell to properly activate or regulate calcium signaling can lead to contractile dysfunction. In isolated cardiomyocytes, calcium signaling has been primarily studied using calcium fluorescent dyes, however these dyes have limited applicability to whole organs. Here, we crossed the Salsa6f mouse which expresses a genetically encoded ratiometric cytosolic calcium indicator with a cardiomyocyte specific inducible cre to temporally-induce expression and studied cytosolic calcium transients in isolated cardiomyocytes and modified Langendorff heart preparations. Isolated cardiomyocytes expressing Salsa6f or Fluo-4AM loaded were compared. We also crossed the Salsa6f mouse with a floxed Polycystin 2 (PC2) mouse to test the feasibility of using the Salsa6f mouse to measure calcium transients in PC2 heterozygous or homozygous knock out mice. Although there are caveats in the applicability of the Salsa6f mouse, there are clear advantages to using the Salsa6f mouse to measure whole heart calcium signals.


Assuntos
Cálcio , Miócitos Cardíacos , Camundongos , Animais , Cálcio/metabolismo , Miócitos Cardíacos/metabolismo , Sinalização do Cálcio/fisiologia , Corantes Fluorescentes/metabolismo , Contração Miocárdica/fisiologia
14.
Curr Opin Chem Biol ; 79: 102439, 2024 04.
Artigo em Inglês | MEDLINE | ID: mdl-38432145

RESUMO

Fluorescent dyes synergize with advanced microscopy for researchers to investigate the location and dynamic processes of biomacromolecules with high spatial and temporal resolution. However, the instability of fluorescent dyes, including photobleaching and photoconversion, represent fundamental limits for super-resolution and time-lapse imaging. In this review, we discuss the latest advances in improving the photostability of fluorescent dyes. We summarize the primary photobleaching processes of cyanine and rhodamine dyes and highlight a range of strategies developed in recent years to strengthen these fluorophores. Additionally, we discuss the influence of protein microenvironments and labeling methods on the photostability of fluorophores. We aim to inspire next-generation robust and bright fluorophores that ultimately enable the routine practice of time-lapse super-resolution imaging of live cells.


Assuntos
Corantes Fluorescentes , Proteínas , Corantes Fluorescentes/metabolismo , Fotodegradação , Microscopia de Fluorescência/métodos , Rodaminas
15.
Sci Rep ; 14(1): 4517, 2024 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-38402255

RESUMO

PURPOSE Cathepsin B (Cat B) is a cysteine lysosomal protease that is upregulated in many inflammatory diseases and widely expressed in the brain. Here, we used a Cat B activatable near-infrared (NIR) imaging probe to measure glial activation in vivo in the formalin test, a standard orofacial inflammatory pain model. The probe's efficacy was quantified with immunohistochemical analysis of the somatosensory cortex. PROCEDURES Three different concentrations of Cat B imaging probe (30, 50, 100 pmol/200 g bodyweight) were injected intracisternally into the foramen magnum of rats under anesthesia. Four hours later formalin (1.5%, 50 µl) was injected into the upper lip and the animal's behaviors recorded for 45 min. Subsequently, animals were repeatedly scanned using the IVIS Spectrum (8, 10, and 28 h post imaging probe injection) to measure extracellular Cat B activity. Aldehyde fixed brain sections were immunostained with antibodies against microglial marker Iba1 or astrocytic GFAP and detected with fluorescently labeled secondary antibodies to quantify co-localization with the fluorescent probe. RESULTS The Cat B imaging probe only slightly altered the formalin test results. Nocifensive behavior was only reduced in phase 1 in the 100 pmol group. In vivo measured fluorescence efficiency was highest in the 100 pmol group 28 h post imaging probe injection. Post-mortem immunohistochemical analysis of the somatosensory cortex detected the greatest amount of NIR fluorescence localized on microglia and astrocytes in the 100 pmol imaging probe group. Sensory neuron neuropeptide and cell injury marker expression in ipsilateral trigeminal ganglia was not altered by the presence of fluorescent probe. CONCLUSIONS These data demonstrate a concentration- and time-dependent visualization of extracellular Cat B in activated glia in the formalin test using a NIR imaging probe. Intracisternal injections are well suited for extracellular CNS proteinase detection in conditions when the blood-brain barrier is intact.


Assuntos
Catepsina B , Corantes Fluorescentes , Ratos , Animais , Catepsina B/metabolismo , Medição da Dor , Corantes Fluorescentes/metabolismo , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Microglia/metabolismo , Dor Facial/metabolismo , Formaldeído/metabolismo
16.
Talanta ; 271: 125716, 2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38301373

RESUMO

Mitochondrial copper signaling pathway plays a role in Alzheimer's disease (AD), especially in relevant Amyloid-ß oligomers (AßOs) neurotoxicity and mitochondrial dysfunction. Clarifying the relationship between mitochondrial copper homeostasis and both of mitochondrial dysfunction and AßOs neurotoxicity is important for understanding AD pathogenesis. Herein, we designed and synthesized a ratiometric fluorescent probe CHC-NS4 for Cu(I). CHC-NS4 possesses excellent ratiometric response, high selectivity to Cu(I) and specific ability to target mitochondria. Under mitochondrial dysfunction induced by oligomycin, mitochondrial Cu(I) levels gradually increased, which may be related to inhibition of ATP7A-mediated Cu(I) exportation and/or high expression of COX. On this basis, CHC-NS4 was further utilized to visualize the fluctuations of mitochondrial Cu(I) levels during progression of AD model cells induced by AßOs. It was found that mitochondrial Cu(I) levels were gradually elevated during the AD progression, which depended on not only AßOs concentration but also incubation time. Moreover, endocytosis maybe served as a prime pathway mode for mitochondrial Cu(I) dyshomeostasis induced by AßOs during AD progression. These results have provided a novel inspiration into mitochondrial copper biology in AD pathogenesis.


Assuntos
Doença de Alzheimer , Doenças Mitocondriais , Humanos , Doença de Alzheimer/metabolismo , Cobre/metabolismo , Peptídeos beta-Amiloides/metabolismo , Corantes Fluorescentes/metabolismo , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo
17.
Angew Chem Int Ed Engl ; 63(13): e202318340, 2024 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-38303099

RESUMO

Copper dysmetabolism is associated with various neurodegenerative disorders, making high-spatiotemporal-resolution imaging of Cu2+ in the brain essential for understanding the underlying pathophysiological processes. Nevertheless, the current probes encounter obstacles in crossing the blood-brain barrier (BBB) and providing high-spatial-resolution in deep tissues. Herein, we present a photoacoustic probe capable of imaging Cu2+ dynamics in the mouse brain with high-spatiotemporal-resolution. The probe demonstrates selective ratiometric and reversible responses to Cu2+ , while also efficiently crossing the BBB. Using the probe as the imaging agent, we successfully visualized Cu2+ in the brain of Parkinson's disease (PD) model mouse with a remarkable micron-level resolution. The imaging results revealed a significant increase in Cu2+ levels in the cerebral cortex as PD progresses, highlighting the close association between Cu2+ alternations in the region and the disease. We also demonstrated that the probe can be used to monitor changes in Cu2+ distribution in the PD model mouse brain during L-dopa intervention. Mechanism studies suggest that the copper dyshomeostasis in the PD mouse brain was dominated by the expression levels of divalent metal transporter 1. The application of our probe in imaging Cu2+ dynamics in the mouse brain offers valuable insights into the copper-related molecular mechanisms underlying neurodegenerative diseases.


Assuntos
Cobre , Doenças Neurodegenerativas , Animais , Camundongos , Cobre/metabolismo , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Barreira Hematoencefálica/metabolismo , Espectrometria de Fluorescência , Diagnóstico por Imagem , Doenças Neurodegenerativas/metabolismo , Corantes Fluorescentes/metabolismo
18.
J Mater Chem B ; 12(11): 2761-2770, 2024 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-38380679

RESUMO

Real-time tracking of dynamic changes in the three-dimensional morphology of the cell plasma membrane is of great importance for a deeper understanding of physiological processes related to the cell plasma membrane. However, there is a lack of imaging dyes that can specifically be used for a long term labelling of plasma membranes, especially for plant cells. Here, we have used molecular engineering strategies to develop a series of target-activated multicolour fluorescent dyes that can be used for long-term and three-dimensional imaging of plant cell plasma membranes. By combining different electron acceptors and donors, four molecular backbones with different emission colours from green to NIR have been obtained. In the designed styrene-based dyes, referred to as the SD dyes, several functional groups were introduced into the backbones to achieve the properties of target-activated fluorescence, rapid and wash-free staining, high plasma membrane targeting ability and long-term imaging function. Using onion epidermal cells as a platform, these dye molecules can provide high-quality imaging of the plasma membrane for up to 6 hours, providing a powerful tool for long-term monitoring of plasma membrane-related biological events. Calcium-mediated apoptosis of plant cells has been tracked for the first time by monitoring the morphological changes of the plasma membrane in real time using SD dyes. These dyes also exhibit excellent 3D imaging performance of the plasma membrane and were further used to track in real time the 3D morphological changes of the plasma membrane during plasmolysis of plant cells, providing a powerful imaging tool for three-dimensional (3D) biology. This work provides a set of multi-colour dye tools for long-term and three-dimensional imaging of plant cell plasma membranes, and also provides molecular design principles for guiding the transmembrane transport of small molecules.


Assuntos
Corantes Fluorescentes , Imageamento Tridimensional , Corantes Fluorescentes/metabolismo , Membrana Celular/metabolismo , Apoptose , Coloração e Rotulagem
19.
Tissue Eng Part C Methods ; 30(4): 183-192, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38411508

RESUMO

The utmost aim of regenerative medicine is to promote the regeneration of injured tissues using stem cells. Amniotic mesenchymal stem cells (AmMSCs) have been used in several studies mainly because of their easy isolation from amniotic tissue postpartum and immunomodulatory and angiogenic properties and the low level of rejection. These cells share characteristics of both embryonic/fetal and adult stem cells and are particularly advantageous because they do not trigger tumorigenic activity when injected into immunocompromised animals. The large-scale use of AmMSCs for cellular therapies would greatly benefit from fluorescence labeling studies to validate their tracking in future therapies. This study evaluated the fluorophore positivity, fluorescence intensity, and longevity of canine AmMSCs. For this purpose, canine AmMSCs from the GDTI/USP biobank were submitted to three labeling conditions, two commercial fluorophores [CellTrace CFSE Cell Proliferation kit - CTrace, and CellTracker Green CMFDA - CTracker (CellTracker Green CMFDA, CT, #C2925, Molecular Probes®; Life Technologies)] and green fluorescent protein (GFP) expression after lentiviral transduction, to select the most suitable tracer in terms of adequate persistence and easy handling and analysis that could be used in studies of domestic animals. Fluorescence was detected in all groups; however, the patterns were different. Specifically, CTrace and CTracker fluorescence was detected 6 h after labeling, while GFP was visualized no earlier than 48 h after transduction. Flow cytometry analysis revealed more than 70% of positive cells on day 7 in the CTrace and CTracker groups, while fluorescence decreased significantly to 10% or less on day 20. Variations between repetitions were observed in the GFP group under the present conditions. Our results showed earlier fluorescence detection and more uniform results across repetitions for the commercial fluorophores. In contrast, fluorescence persisted for more extended periods in the GFP group. These results indicate a promising direction for assessing the roles of canine AmMSCs in regenerative medicine without genomic integration.


Assuntos
Fluoresceínas , Células-Tronco Mesenquimais , Células-Tronco , Feminino , Animais , Cães , Células-Tronco/metabolismo , Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Células-Tronco Mesenquimais/metabolismo , Corantes Fluorescentes/metabolismo , Diferenciação Celular
20.
Biol Chem ; 405(5): 297-309, 2024 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-38353111

RESUMO

G proteins are interacting partners of G protein-coupled receptors (GPCRs) in eukaryotic cells. Upon G protein activation, the ability of the Gα subunit to exchange GDP for GTP determines the intracellular signal transduction. Although various studies have successfully shown that both Gαs and Gαi have an opposite effect on the intracellular cAMP production, with the latter being commonly described as "more active", the functional analysis of Gαs is a comparably more complicated matter. Additionally, the thorough investigation of the ubiquitously expressed variants of Gαs, Gαs(short) and Gαs(long), is still pending. Since the previous experimental evaluation of the activity and function of the Gαs isoforms is not consistent, the focus was laid on structural investigations to understand the GTPase activity. Herein, we examined recombinant human Gαs by applying an established methodological setup developed for Gαi characterization. The ability for GTP binding was evaluated with fluorescence and fluorescence anisotropy assays, whereas the intrinsic hydrolytic activity of the isoforms was determined by a GTPase assay. Among different nucleotide probes, BODIPY FL GTPγS exhibited the highest binding affinity towards the Gαs subunit. This work provides a deeper understanding of the Gαs subunit and provides novel information concerning the differences between the two protein variants.


Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP , Humanos , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Nucleotídeos de Guanina/metabolismo , Nucleotídeos de Guanina/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Guanosina Trifosfato/metabolismo
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