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1.
Nat Methods ; 20(4): 617-622, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-36823329

RESUMEN

In deep-tissue multiphoton microscopy, diffusion and scattering of fluorescent photons, rather than ballistic emanation from the focal point, have been a confounding factor. Here we report on a 2.17-g miniature three-photon microscope (m3PM) with a configuration that maximizes fluorescence collection when imaging in highly scattering regimes. We demonstrate its capability by imaging calcium activity throughout the entire cortex and dorsal hippocampal CA1, up to 1.2 mm depth, at a safe laser power. It also enables the detection of sensorimotor behavior-correlated activities of layer 6 neurons in the posterior parietal cortex in freely moving mice during single-pellet reaching tasks. Thus, m3PM-empowered imaging allows the study of neural mechanisms in deep cortex and subcortical structures, like the dorsal hippocampus and dorsal striatum, in freely behaving animals.


Asunto(s)
Hipocampo , Microscopía de Fluorescencia por Excitación Multifotónica , Ratones , Animales , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Corteza Cerebral , Colorantes , Fotones
2.
Proc Natl Acad Sci U S A ; 119(7)2022 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-35135885

RESUMEN

The medial entorhinal cortex (MEC) creates a map of local space, based on the firing patterns of grid, head-direction (HD), border, and object-vector (OV) cells. How these cell types are organized anatomically is debated. In-depth analysis of this question requires collection of precise anatomical and activity data across large populations of neurons during unrestrained behavior, which neither electrophysiological nor previous imaging methods fully afford. Here, we examined the topographic arrangement of spatially modulated neurons in the superficial layers of MEC and adjacent parasubiculum using miniaturized, portable two-photon microscopes, which allow mice to roam freely in open fields. Grid cells exhibited low levels of co-occurrence with OV cells and clustered anatomically, while border, HD, and OV cells tended to intermingle. These data suggest that grid cell networks might be largely distinct from those of border, HD, and OV cells and that grid cells exhibit strong coupling among themselves but weaker links to other cell types.


Asunto(s)
Mapeo Encefálico/métodos , Corteza Entorrinal/anatomía & histología , Corteza Entorrinal/fisiología , Microscopía/instrumentación , Animales , Masculino , Ratones , Miniaturización , Actividad Motora , Neuronas/fisiología
3.
Nat Methods ; 18(1): 46-49, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33408404

RESUMEN

We have developed a miniature two-photon microscope equipped with an axial scanning mechanism and a long-working-distance miniature objective to enable multi-plane imaging over a volume of 420 × 420 × 180 µm3 at a lateral resolution of ~1 µm. Together with the detachable design that permits long-term recurring imaging, our miniature two-photon microscope can help decipher neuronal mechanisms in freely behaving animals.


Asunto(s)
Encéfalo/diagnóstico por imagen , Encéfalo/fisiología , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Miniaturización/métodos , Neuroimagen/métodos , Animales , Conducta Animal , Encéfalo/citología , Técnicas Citológicas , Locomoción , Masculino , Ratones , Ratones Endogámicos C57BL
4.
J Neurochem ; 164(3): 270-283, 2023 02.
Artículo en Inglés | MEDLINE | ID: mdl-36281555

RESUMEN

Over the past decade, novel optical imaging tools have been developed for imaging neuronal activities along with the evolution of fluorescence indicators with brighter expression and higher sensitivity. Miniature microscopes, as revolutionary approaches, enable the imaging of large populations of neuron ensembles in freely behaving rodents and mammals, which allows exploring the neural basis of behaviors. Recent progress in two-photon miniature microscopes and mesoscale single-photon miniature microscopes further expand those affordable methods to navigate neural activities during naturalistic behaviors. In this review article, two-photon miniature microscopy techniques are summarized historically from the first documented attempt to the latest ones, and comparisons are made. The driving force behind and their potential for neuroscientific inquiries are also discussed. Current progress in terms of the mesoscale, i.e., the large field-of-view miniature microscopy technique, is addressed as well. Then, pipelines for registering single cells from the data of two-photon and large field-of-view miniature microscopes are discussed. Finally, we present the potential evolution of the techniques.


Asunto(s)
Microscopía , Imagen Óptica , Animales , Imagen Óptica/métodos , Mamíferos , Neuronas/metabolismo , Conducta Animal/fisiología
5.
Nat Methods ; 17(11): 1139-1146, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-32989318

RESUMEN

The ability to directly measure acetylcholine (ACh) release is an essential step toward understanding its physiological function. Here we optimized the GRABACh (GPCR-activation-based ACh) sensor to achieve substantially improved sensitivity in ACh detection, as well as reduced downstream coupling to intracellular pathways. The improved version of the ACh sensor retains the subsecond response kinetics, physiologically relevant affinity and precise molecular specificity for ACh of its predecessor. Using this sensor, we revealed compartmental ACh signals in the olfactory center of transgenic flies in response to external stimuli including odor and body shock. Using fiber photometry recording and two-photon imaging, our ACh sensor also enabled sensitive detection of single-trial ACh dynamics in multiple brain regions in mice performing a variety of behaviors.


Asunto(s)
Acetilcolina/metabolismo , Técnicas Biosensibles/métodos , Encéfalo/metabolismo , Animales , Animales Modificados Genéticamente , Conducta Animal/fisiología , Colinérgicos/farmacología , Drosophila/genética , Drosophila/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Ratones , Cuerpos Pedunculados/metabolismo , Neuronas/metabolismo , Corteza Olfatoria/metabolismo , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismo , Corteza Somatosensorial/metabolismo
6.
Basic Res Cardiol ; 118(1): 45, 2023 10 11.
Artículo en Inglés | MEDLINE | ID: mdl-37819607

RESUMEN

A hallmark of heart failure is a metabolic switch away from fatty acids ß-oxidation (FAO) to glycolysis. Here, we show that succinate dehydrogenase (SDH) is required for maintenance of myocardial homeostasis of FAO/glycolysis. Mice with cardiomyocyte-restricted deletion of subunit b or c of SDH developed a dilated cardiomyopathy and heart failure. Hypertrophied hearts displayed a decrease in FAO, while glucose uptake and glycolysis were augmented, which was reversed by enforcing FAO fuels via a high-fat diet, which also improved heart failure of mutant mice. SDH-deficient hearts exhibited an increase in genome-wide DNA methylation associated with accumulation of succinate, a metabolite known to inhibit DNA demethylases, resulting in changes of myocardial transcriptomic landscape. Succinate induced DNA hypermethylation and depressed the expression of FAO genes in myocardium, leading to imbalanced FAO/glycolysis. Inhibition of succinate by α-ketoglutarate restored transcriptional profiles and metabolic disorders in SDH-deficient cardiomyocytes. Thus, our findings reveal the essential role for SDH in metabolic remodeling of failing hearts, and highlight the potential of therapeutic strategies to prevent cardiac dysfunction in the setting of SDH deficiency.


Asunto(s)
Insuficiencia Cardíaca , Succinato Deshidrogenasa , Ratones , Animales , Succinato Deshidrogenasa/genética , Succinato Deshidrogenasa/metabolismo , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/metabolismo , Homeostasis , Succinatos/metabolismo , ADN/metabolismo , Epigénesis Genética
7.
Opt Express ; 31(20): 32925-32934, 2023 Sep 25.
Artículo en Inglés | MEDLINE | ID: mdl-37859084

RESUMEN

Development of miniature two-photon microscopy (m2PM) has made it possible to observe fine structure and activity of neurons in the brain of freely moving animals. However, the imaging field-of-view of existing m2PM is still significantly smaller than that of miniature single-photon microscopy. Here we report that, through the design of low-magnification objective, large field-of-view scan lens and small tilt angle microscanner, a 2.5-g m2PM achieved a field-of-view of 1000 × 788 µm2, comparable to that of a typical single-photon miniscope. We demonstrated its capability by imaging neurons, dendrites and spines in the millimeter field-of-view, and simultaneous recording calcium activities, through a gradient-index lens, of approximately 400 neurons in the dorsal hippocampal CA1 in a freely moving mouse. Integrated with a detachable 1.2-g fast z-scanning module, it enables a 1000 × 788 × 500 µm3 volumetric neuronal imaging in the cerebral cortex. Thus, millimeter FOV m2PM provides a powerful tool for deciphering neuronal population dynamics in experimental paradigms allowing for animal's free movement.


Asunto(s)
Encéfalo , Microscopía , Ratones , Animales , Microscopía/métodos , Encéfalo/diagnóstico por imagen , Encéfalo/fisiología , Cabeza , Neuronas/fisiología , Neuroimagen
8.
Cell ; 134(2): 279-90, 2008 Jul 25.
Artículo en Inglés | MEDLINE | ID: mdl-18662543

RESUMEN

In quiescent cells, mitochondria are the primary source of reactive oxygen species (ROS), which are generated by leakiness of the electron transport chain (ETC). High levels of ROS can trigger cell death, whereas lower levels drive diverse and important cellular functions. We show here by employing a newly developed mitochondrial matrix-targeted superoxide indicator, that individual mitochondria undergo spontaneous bursts of superoxide generation, termed "superoxide flashes." Superoxide flashes occur randomly in space and time, exhibit all-or-none properties, and provide a vital source of superoxide production across many different cell types. Individual flashes are triggered by transient openings of the mitochondrial permeability transition pore stimulating superoxide production by the ETC. Furthermore, we observe a flurry of superoxide flash activity during reoxygenation of cardiomyocytes after hypoxia, which is inhibited by the cardioprotective compound adenosine. We propose that superoxide flashes could serve as a valuable biomarker for a wide variety of oxidative stress-related diseases.


Asunto(s)
Mitocondrias/metabolismo , Superóxidos/metabolismo , Adenoviridae/genética , Animales , Hipoxia de la Célula , Línea Celular Tumoral , Células Cultivadas , Humanos , Proteínas Luminiscentes/metabolismo , Células Musculares/metabolismo , Miocitos Cardíacos/metabolismo , Neuronas/metabolismo , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo
9.
Small ; 18(12): e2105989, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-35088522

RESUMEN

Biomedical imaging technology (like digital subtraction angiography (DSA)) based on contrast agents has been widely employed in the diagnosis of vascular-related diseases. While the DSA achieves the high-resolution observation of specified vessels and their downstream perfusion at the cost of invasive, radioactive operation and hepatorenal toxicity. To address those problems, this study develops arterial labeling ultrasound (US) subtraction angiography (ALUSA) based on a new perfluorobutane (PFB) nanodroplets with a lower vaporization threshold through spontaneous nucleation. The nanodroplets can be selectively vaporized to microbubbles, indicating a highly echogenic signal at B-mode images only using a diagnostic transducer. By labeling a single blood vessel for nanodroplets vaporization and tracking its downstream blood perfusion in segmental renal arteries at a frame rate of 500 Hz. The results demonstrate the color-coded super-resolution ALUSA image, exhibiting the downstream arcuate and interlobular arteries of each segmental renal artery with a resolution of 36 µm in a rabbit kidney. Furthermore, ALUSA could offer the vascular structures, blood flow velocity, and direction of their primary supply vessels in the mouse breast tumor. ALUSA fills the gap of noninvasive labeling angiography in US and opens a broad vista in the diagnosis and treatment of tumor and vascular-related diseases.


Asunto(s)
Acústica , Microburbujas , Angiografía de Substracción Digital , Animales , Arterias , Ratones , Conejos , Ultrasonografía/métodos
10.
J Cell Sci ; 132(6)2019 03 25.
Artículo en Inglés | MEDLINE | ID: mdl-30814332

RESUMEN

STIM1- and Orai1-mediated store-operated Ca2+ entry (SOCE) constitutes the major Ca2+ influx in almost all electrically non-excitable cells. However, little is known about the spatiotemporal organization at the elementary level. Here, we developed Orai1-tethered or palmitoylated biosensor GCaMP6f to report subplasmalemmal Ca2+ signals. We visualized spontaneous discrete and long-lasting transients ('Ca2+ glows') arising from STIM1-Orai1 in invading melanoma cells. Ca2+ glows occurred preferentially in single invadopodia and at sites near the cell periphery under resting conditions. Re-addition of external Ca2+ after store depletion elicited spatially synchronous Ca2+ glows, followed by high-rate discharge of asynchronous local events. Knockout of STIM1 or expression of the dominant-negative Orai1-E106A mutant markedly decreased Ca2+ glow frequency, diminished global SOCE and attenuated invadopodial formation. Functionally, invadopodial Ca2+ glows provided high Ca2+ microdomains to locally activate Ca2+/calmodulin-dependent Pyk2 (also known as PTK2B), which initiates the SOCE-Pyk2-Src signaling cascade required for invasion. Overall, the discovery of elemental Ca2+ signals of SOCE not only unveils a previously unappreciated gating mode of STIM1-Orai1 channels in situ, but also underscores a critical role of the spatiotemporal dynamics of SOCE in orchestrating complex cell behaviors such as invasion. This article has an associated First Person interview with the first author of the paper.


Asunto(s)
Señalización del Calcio/fisiología , Quinasa 2 de Adhesión Focal/metabolismo , Proteínas de Neoplasias/metabolismo , Células Neoplásicas Circulantes/metabolismo , Proteína ORAI1/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Técnicas Biosensibles , Calcio/metabolismo , Canales de Calcio/metabolismo , Línea Celular , Membrana Celular/metabolismo , Células HEK293 , Humanos , Microscopía Confocal , Imagen Molecular/métodos
11.
Biochem Soc Trans ; 49(6): 2581-2589, 2021 12 17.
Artículo en Inglés | MEDLINE | ID: mdl-34854917

RESUMEN

The store-operated calcium (Ca2+) entry (SOCE) is the Ca2+ entry mechanism used by cells to replenish depleted Ca2+ store. The dysregulation of SOCE has been reported in metastatic cancer. It is believed that SOCE promotes migration and invasion by remodeling the actin cytoskeleton and cell adhesion dynamics. There is recent evidence supporting that SOCE is critical for the spatial and the temporal coding of Ca2+ signals in the cell. In this review, we critically examined the spatiotemporal control of SOCE signaling and its implication in the specificity and robustness of signaling events downstream of SOCE, with a focus on the spatiotemporal SOCE signaling during cancer cell migration, invasion and metastasis. We further discuss the limitation of our current understanding of SOCE in cancer metastasis and potential approaches to overcome such limitation.


Asunto(s)
Calcio/metabolismo , Metástasis de la Neoplasia , Neoplasias/metabolismo , Señalización del Calcio , Humanos , Transporte Iónico , Neoplasias/patología
12.
Nat Methods ; 14(7): 713-719, 2017 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-28553965

RESUMEN

Developments in miniaturized microscopes have enabled visualization of brain activities and structural dynamics in animals engaging in self-determined behaviors. However, it remains a challenge to resolve activity at single dendritic spines in freely behaving animals. Here, we report the design and application of a fast high-resolution, miniaturized two-photon microscope (FHIRM-TPM) that accomplishes this goal. With a headpiece weighing 2.15 g and a hollow-core photonic crystal fiber delivering 920-nm femtosecond laser pulses, the FHIRM-TPM is capable of imaging commonly used biosensors (GFP and GCaMP6) at high spatiotemporal resolution (0.64 µm laterally and 3.35 µm axially, 40 Hz at 256 × 256 pixels for raster scanning and 10,000 Hz for free-line scanning). We demonstrate the microscope's robustness with hour-long recordings of neuronal activities at the level of spines in mice experiencing vigorous body movements.


Asunto(s)
Conducta Animal , Encéfalo/diagnóstico por imagen , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Miniaturización/métodos , Animales , Encéfalo/citología , Encéfalo/fisiología , Técnicas Citológicas/métodos , Ratones
13.
FASEB J ; 33(12): 13310-13322, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31530015

RESUMEN

Mitochondria are fundamental organelles for cellular and systemic metabolism, and their dysfunction has been implicated in the development of diverse metabolic diseases. Boosted mitochondrial metabolism might be able to protect against metabolic stress and prevent metabolic disorders. Here we show that NADH:ubiquinone oxidoreductase (NDU)-FAB1, also known as mitochondrial acyl carrier protein, acts as a novel enhancer of mitochondrial metabolism and protects against obesity and insulin resistance. Mechanistically, NDUFAB1 coordinately enhances lipoylation and activation of pyruvate dehydrogenase mediated by the mitochondrial fatty acid synthesis pathway and increases the assembly of respiratory complexes and supercomplexes. Skeletal muscle-specific ablation of NDUFAB1 causes systemic disruption of glucose homeostasis and defective insulin signaling, leading to growth arrest and early death within 5 postnatal days. In contrast, NDUFAB1 overexpression effectively protects mice against obesity and insulin resistance when the animals are challenged with a high-fat diet. Our findings indicate that NDUFAB1 could be a novel mitochondrial target to prevent obesity and insulin resistance by enhancing mitochondrial metabolism.-Zhang, R., Hou, T., Cheng, H., Wang, X. NDUFAB1 protects against obesity and insulin resistance by enhancing mitochondrial metabolism.


Asunto(s)
Complejo I de Transporte de Electrón/fisiología , Resistencia a la Insulina , Insulina/metabolismo , Mitocondrias/metabolismo , Músculo Esquelético/patología , Obesidad/prevención & control , Sustancias Protectoras/farmacología , Animales , Dieta Alta en Grasa/efectos adversos , Metabolismo Energético , Glucosa/metabolismo , Homeostasis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mitocondrias/patología , Músculo Esquelético/metabolismo , Obesidad/etiología , Obesidad/metabolismo , Obesidad/patología , Transducción de Señal
14.
Nature ; 508(7494): 128-32, 2014 Apr 03.
Artículo en Inglés | MEDLINE | ID: mdl-24522532

RESUMEN

It has been theorized for decades that mitochondria act as the biological clock of ageing, but the evidence is incomplete. Here we show a strong coupling between mitochondrial function and ageing by in vivo visualization of the mitochondrial flash (mitoflash), a frequency-coded optical readout reflecting free-radical production and energy metabolism at the single-mitochondrion level. Mitoflash activity in Caenorhabditis elegans pharyngeal muscles peaked on adult day 3 during active reproduction and on day 9 when animals started to die off. A plethora of genetic mutations and environmental factors inversely modified the lifespan and the day-3 mitoflash frequency. Even within an isogenic population, the day-3 mitoflash frequency was negatively correlated with the lifespan of individual animals. Furthermore, enhanced activity of the glyoxylate cycle contributed to the decreased day-3 mitoflash frequency and the longevity of daf-2 mutant animals. These results demonstrate that the day-3 mitoflash frequency is a powerful predictor of C. elegans lifespan across genetic, environmental and stochastic factors. They also support the notion that the rate of ageing, although adjustable in later life, has been set to a considerable degree before reproduction ceases.


Asunto(s)
Caenorhabditis elegans/metabolismo , Longevidad , Mitocondrias/metabolismo , Superóxidos/metabolismo , Envejecimiento/metabolismo , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Proteínas de Caenorhabditis elegans/genética , Muerte , Metabolismo Energético , Ambiente , Glioxilatos/metabolismo , Organismos Hermafroditas , Longevidad/genética , Longevidad/fisiología , Masculino , Modelos Biológicos , Músculos/citología , Mutación , Estrés Oxidativo , Receptor de Insulina/genética , Reproducción , Procesos Estocásticos , Superóxidos/análisis , Factores de Tiempo
15.
J Cell Sci ; 130(15): 2620-2630, 2017 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-28630166

RESUMEN

Prohibitins (PHBs; prohibitin 1, PHB1 or PHB, and prohibitin 2, PHB2) are evolutionarily conserved and ubiquitously expressed mitochondrial proteins. PHBs form multimeric ring complexes acting as scaffolds in the inner mitochondrial membrane. Mitochondrial flashes (mitoflashes) are newly discovered mitochondrial signaling events that reflect electrical and chemical excitations of the organelle. Here, we investigate the possible roles of PHBs in the regulation of mitoflash signaling. Downregulation of PHBs increases mitoflash frequency by up to 5.4-fold due to elevated basal reactive oxygen species (ROS) production in the mitochondria. Mechanistically, PHB deficiency impairs the formation of mitochondrial respiratory supercomplexes (RSCs) without altering the abundance of individual respiratory complex subunits. These impairments induced by PHB deficiency are effectively rescued by co-expression of PHB1 and PHB2, indicating that the multimeric PHB complex acts as the functional unit. Furthermore, downregulating other RSC assembly factors, including SCAFI (also known as COX7A2L), RCF1a (HIGD1A), RCF1b (HIGD2A), UQCC3 and SLP2 (STOML2), all activate mitoflashes through elevating mitochondrial ROS production. Our findings identify the PHB complex as a new regulator of RSC formation and mitoflash signaling, and delineate a general relationship among RSC formation, basal ROS production and mitoflash biogenesis.


Asunto(s)
Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Represoras/deficiencia , Transducción de Señal , Proteínas del Complejo de Cadena de Transporte de Electrón/genética , Células HeLa , Humanos , Mitocondrias/genética , Proteínas Mitocondriales/genética , Prohibitinas
17.
Arch Biochem Biophys ; 666: 8-15, 2019 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-30898545

RESUMEN

Mitochondrial flashes (mitoflashes) represent fundamental biochemical and biophysical dynamics of the organelle, involving sudden depolarization of mitochondrial membrane potential (ΔΨm), bursting production of reactive oxygen species (ROS), and accelerated extrusion of matrix protons. Here we investigated temperature dependence of mitoflash biogenesis as well as ΔΨm oscillations, a subset of which overlapping with mitoflashes, in both cardiac myocytes and isolated respiring cardiac mitochondria. Unexpectedly, we found that mitoflash biogenesis was essentially temperature-independent in intact cardiac myocytes, evidenced by the constancy of frequency as well as amplitude and rise speed over 5 °C-40 °C. Moderate temperature dependence was found in single mitochondria charged by respiratory substrates, where mitoflash frequency was decreased over 5 °C-20 °C with Q10 of 0.74 for Complex I substrates and 0.83 for Complex II substrate. In contrast, ΔΨm oscillation frequency displayed a negative temperature dependence at 5 °C-20 °C with Q10 of 0.82 in intact cells, but a positive temperature dependence at 25 °C - 40 °C with Q10 of 1.62 in isolated mitochondria charged with either Complex I or Complex II substrates. Moreover, the recovery speed of individual mitoflashes exhibited mild temperature dependence (Q10 = 1.14-1.22). These results suggest a temperature compensation of mitoflash frequency at both the mitochondrial and extra-organelle levels, and underscore that mitoflashes and ΔΨm oscillations are related but distinctly different mitochondrial functional dynamics.


Asunto(s)
Mitocondrias Cardíacas/metabolismo , Biogénesis de Organelos , Temperatura , Animales , Homeostasis , Potencial de la Membrana Mitocondrial , Ratones , Ratones Transgénicos , Dinámicas Mitocondriales , Miocitos Cardíacos/metabolismo , Especies Reactivas de Oxígeno/metabolismo
18.
Methods ; 109: 12-20, 2016 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-27288722

RESUMEN

Mitochondrion is an organelle critically responsible for energy production and intracellular signaling in eukaryotic cells and its dysfunction often accompanies and contributes to human disease. Superoxide is the primary reactive oxygen species (ROS) produced in mitochondria. In vivo detection of superoxide has been a challenge in biomedical research. Here we describe the methods used to characterize a circularly permuted yellow fluorescent protein (cpYFP) as a biosensor for mitochondrial superoxide and pH dynamics. In vitro characterization reveals the high selectivity of cpYFP to superoxide over other ROS species and its dual sensitivity to pH. Confocal and two-photon imaging in conjunction with transgenic expression of the biosensor cpYFP targeted to the mitochondrial matrix detects mitochondrial flash events in living cells, perfused intact hearts, and live animals. The mitochondrial flashes are discrete and stochastic single mitochondrial events triggered by transient mitochondrial permeability transition (tMPT) and composed of a bursting superoxide signal and a transient alkalization signal. The real-time monitoring of single mitochondrial flashes provides a unique tool to study the integrated dynamism of mitochondrial respiration, ROS production, pH regulation and tMPT kinetics under diverse physiological and pathophysiological conditions.


Asunto(s)
Técnicas Biosensibles/métodos , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/aislamiento & purificación , Superóxidos/aislamiento & purificación , Animales , Proteínas Bacterianas/química , Transporte Biológico , Humanos , Proteínas Luminiscentes/química , Microscopía Confocal/métodos , Especies Reactivas de Oxígeno/química , Transducción de Señal , Superóxidos/química
19.
Handb Exp Pharmacol ; 240: 403-422, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28233181

RESUMEN

Mitochondrial flashes (mitoflashes) are recently discovered mitochondrial activity which reflects chemical and electrical excitation of the organelle. Emerging evidence indicates that mitoflashes represent highly regulated, elementary signaling events that play important roles in physiological and pathophysiological processes in eukaryotes. Furthermore, they are regulated by mitochondrial ROS, Ca2+, and protons, and are intertwined with mitochondrial metabolic processes. As such, targeting mitoflash activity may provide a novel means for the control of mitochondrial metabolism and signaling in health and disease. In this brief review, we summarize salient features and mechanisms of biogenesis of mitoflashes, and synthesize data on mitoflash biology in the context of metabolism, cell differentiation, stress response, disease, and ageing.


Asunto(s)
Células Eucariotas/fisiología , Mitocondrias/fisiología , Orgánulos/fisiología , Transducción de Señal/fisiología , Animales , Calcio/metabolismo , Humanos , Proteínas de Transporte de Membrana Mitocondrial , Poro de Transición de la Permeabilidad Mitocondrial , Especies Reactivas de Oxígeno/metabolismo
20.
Biophys J ; 111(2): 386-394, 2016 Jul 26.
Artículo en Inglés | MEDLINE | ID: mdl-27463140

RESUMEN

Emerging evidence indicates that mitochondrial flashes (mitoflashes) are highly conserved elemental mitochondrial signaling events. However, which signal controls their ignition and how they are integrated with other mitochondrial signals and functions remain elusive. In this study, we aimed to further delineate the signal components of the mitoflash and determine the mitoflash trigger mechanism. Using multiple biosensors and chemical probes as well as label-free autofluorescence, we found that the mitoflash reflects chemical and electrical excitation at the single-organelle level, comprising bursting superoxide production, oxidative redox shift, and matrix alkalinization as well as transient membrane depolarization. Both electroneutral H(+)/K(+) or H(+)/Na(+) antiport and matrix proton uncaging elicited immediate and robust mitoflash responses over a broad dynamic range in cardiomyocytes and HeLa cells. However, charge-uncompensated proton transport, which depolarizes mitochondria, caused the opposite effect, and steady matrix acidification mildly inhibited mitoflashes. Based on a numerical simulation, we estimated a mean proton lifetime of 1.42 ns and diffusion distance of 2.06 nm in the matrix. We conclude that nanodomain protons act as a novel, to our knowledge, trigger of mitoflashes in energized mitochondria. This finding suggests that mitoflash genesis is functionally and mechanistically integrated with mitochondrial energy metabolism.


Asunto(s)
Mitocondrias/metabolismo , Protones , Animales , Metabolismo Energético/efectos de los fármacos , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Ionóforos/farmacología , Masculino , Mitocondrias/efectos de los fármacos , Ratas , Ratas Sprague-Dawley
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