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Stress granules (SGs) are cytoplasmic messenger ribonucleoprotein granules transiently formed in stressed mammalian cells. Although SG components have been well characterized, detailed insights into the molecular behavior inside SGs remain unresolved. We investigated nanoscale dynamics and localization of endogenous mRNAs in SGs combining single mRNA tracking and super-resolution localization microscopy. First, we developed a methodology for tracking single mRNAs within SGs, revealing that although mRNAs in SGs are mainly stationary (â¼40%), they also move in a confined (â¼25%) or freely diffusing (â¼35%) manner. Second, the super-resolution localization microscopy showed that the mRNAs in SGs are heterogeneously distributed and partially form high-density clusters. Third, we simultaneously performed single mRNA tracking and super-resolution microscopy in SGs, demonstrating that single mRNA trajectories are mainly found around high-density clusters. Finally, a quantitative analysis of mRNA localization and dynamics during stress removal was conducted using live super-resolution imaging and single-molecule tracking. These results suggest that SGs have a highly organized structure that enables dynamic regulation of the mRNAs at the nanoscale, which is responsible for the ordered formation and the wide variety of functions of SGs.
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Neuronal differentiation-the development of neurons from neural stem cells-involves neurite outgrowth and is a key process during the development and regeneration of neural functions. In addition to various chemical signaling mechanisms, it has been suggested that thermal stimuli induce neuronal differentiation. However, the function of physiological subcellular thermogenesis during neuronal differentiation remains unknown. Here we create methods to manipulate and observe local intracellular temperature, and investigate the effects of noninvasive temperature changes on neuronal differentiation using neuron-like PC12 cells. Using quantitative heating with an infrared laser, we find an increase in local temperature (especially in the nucleus) facilitates neurite outgrowth. Intracellular thermometry reveals that neuronal differentiation is accompanied by intracellular thermogenesis associated with transcription and translation. Suppression of intracellular temperature increase during neuronal differentiation inhibits neurite outgrowth. Furthermore, spontaneous intracellular temperature elevation is involved in neurite outgrowth of primary mouse cortical neurons. These results offer a model for understanding neuronal differentiation induced by intracellular thermal signaling.
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Diferenciación Celular , Neuronas , Transducción de Señal , Temperatura , Animales , Células PC12 , Neuronas/fisiología , Neuronas/citología , Ratones , Ratas , Proyección Neuronal , Neurogénesis/fisiología , Neuritas/metabolismo , Neuritas/fisiología , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Células-Madre Neurales/fisiología , Termometría/métodos , Termogénesis/fisiologíaRESUMEN
MicroRNA (miRNA) plays physiologically and pathologically important roles in post-transcriptional regulation. Although miRNA has been suggested to dynamically interact with cellular organelles, the dynamicity of intracellular miRNA behavior has remained unclear. Here, by introducing fluorescently labeled pre-miRNA into living cells, we improved the miRNA visualization method using exogenous miRNA precursors. Through the combination of our miRNA visualization method and single-molecule sensitive fluorescence microscopy, we quantitatively analyzed the process of miRNA maturation. Furthermore, single-particle tracking of fluorescent miRNA in cells revealed the directed movements of miRNA on cytoskeletal components (i.e., microtubules and actin filaments). Our results also suggest that cytoskeleton-dependent miRNA trafficking is associated with the interaction of miRNAs with the nucleus and the endoplasmic reticulum/Golgi apparatus. Our method should facilitate the elucidation of the mechanism and physiological significance of the subcellular localization and organelle interaction of miRNA.
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To understand the mechanism of intracellular thermal transport, thermal properties must be elucidated, particularly thermal conductivity and specific heat capacity. However, these properties have not been extensively studied. In this study, we developed a cellular temperature measurement device with a high temperature resolution of 1.17 m °C under wet conditions and with the ability to introduce intracellular local heating using a focused infrared laser to cultured cells on the device surface. Using this device, we evaluated the thermal properties of single cells based on their temperature signals and responses. Measurements were taken using on-chip-integrated microthermistors with high temperature resolution at varying surrounding temperatures and frequencies of local infrared irradiation on cells prepared on the sensors. Frequency spectra were used to determine the intensities of the temperature signals with respect to heating times. Signal intensities at 37 °C and a frequency lower than 2 Hz were larger than those at 25 °C, which were similar to those of water. The apparent thermal conductivity and specific heat capacity, which were determined at different surrounding temperatures and local heating frequencies, were lower than and similar to those of water at 37 °C and 25 °C, respectively. Our results indicate that the thermal properties of cells depend on both temperatures and physiological activities in addition to local heating frequencies.
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Intracellular temperature affects a wide range of cellular functions in living organisms. However, it remains unclear whether temperature in individual animal cells is controlled autonomously as a response to fluctuations in environmental temperature. Using two distinct intracellular thermometers, we find that the intracellular temperature of steady-state Drosophila S2 cells is maintained in a manner dependent on Δ9-fatty acid desaturase DESAT1, which introduces a double bond at the Δ9 position of the acyl moiety of acyl-CoA. The DESAT1-mediated increase of intracellular temperature is caused by the enhancement of F1Fo-ATPase-dependent mitochondrial respiration, which is coupled with thermogenesis. We also reveal that F1Fo-ATPase-dependent mitochondrial respiration is potentiated by cold exposure through the remodeling of mitochondrial cristae structures via DESAT1-dependent unsaturation of mitochondrial phospholipid acyl chains. Based on these findings, we propose a cell-autonomous mechanism for intracellular temperature control during environmental temperature changes.
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Ácido Graso Desaturasas , Fosfolípidos , Adenosina Trifosfatasas , Animales , Drosophila , Estearoil-CoA Desaturasa , TemperaturaRESUMEN
Conventional thermal biology has elucidated the physiological function of temperature homeostasis through spontaneous thermogenesis and responses to variations in environmental temperature in organisms. In addition to research on individual physiological phenomena, the molecular mechanisms of fever and physiological events such as temperature-dependent sex determination have been intensively addressed. Thermosensitive biomacromolecules such as heat shock proteins (HSPs) and transient receptor potential (TRP) channels were systematically identified, and their sophisticated functions were clarified. Complementarily, recent progress in intracellular thermometry has opened new research fields in thermal biology. High-resolution intracellular temperature mapping has uncovered thermogenic organelles, and the thermogenic functions of brown adipocytes were ascertained by the combination of intracellular thermometry and classic molecular biology. In addition, intracellular thermometry has introduced a new concept, "thermal signaling", in which temperature variation within biological cells acts as a signal in a cascade of intriguing biological events.
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Adipocitos Marrones/metabolismo , Tejido Adiposo Pardo/metabolismo , Transducción de Señal , Termogénesis , Termometría , Orgánulos/fisiologíaRESUMEN
Eukaryotic transcription is epigenetically regulated by chromatin structure and post-translational modifications (PTMs). For example, lysine acetylation in histone H4 is correlated with activation of RNA polymerase I-, II- and III-driven transcription from chromatin templates, which requires prior chromatin remodeling. However, quantitative understanding of the contribution of particular PTM states to the sequential steps of eukaryotic transcription has been hampered partially because reconstitution of a chromatin template with designed PTMs is difficult. In this study, we reconstituted a di-nucleosome with site-specifically acetylated or unmodified histone H4, which contained two copies of the Xenopus somatic 5S rRNA gene with addition of a unique sequence detectable by hybridization-assisted fluorescence correlation spectroscopy. Using a Xenopus oocyte nuclear extract, we analyzed the time course of accumulation of nascent 5S rRNA-derived transcripts generated on chromatin templates in vitro. Our mathematically described kinetic model and fitting analysis revealed that tetra-acetylation of histone H4 at K5/K8/K12/K16 increases the rate of transcriptionally competent chromatin formation â¼3-fold in comparison with the absence of acetylation. We provide a kinetic model for quantitative evaluation of the contribution of epigenetic modifications to chromatin transcription.
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Cromatina/genética , Epigénesis Genética , Procesamiento Proteico-Postraduccional/genética , Transcripción Genética , Acetilación , Animales , Histonas/genética , Lisina/genética , Nucleosomas/genética , ARN Ribosómico 5S/genética , Xenopus laevis/genéticaRESUMEN
Mitochondria play a central role in the function of brown adipocytes (BAs). Although mitochondrial biogenesis, which is indispensable for thermogenesis, is regulated by coordination between nuclear DNA transcription and mitochondrial DNA transcription, the molecular mechanisms of mitochondrial development during BA differentiation are largely unknown. Here, we show the importance of the ER-resident sensor PKR-like ER kinase (PERK) in the mitochondrial thermogenesis of brown adipose tissue. During BA differentiation, PERK is physiologically phosphorylated independently of the ER stress. This PERK phosphorylation induces transcriptional activation by GA-binding protein transcription factor α subunit (GABPα), which is required for mitochondrial inner membrane protein biogenesis, and this novel role of PERK is involved in maintaining the body temperatures of mice during cold exposure. Our findings demonstrate that mitochondrial development regulated by the PERK-GABPα axis is indispensable for thermogenesis in brown adipose tissue.
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Tejido Adiposo Pardo/metabolismo , Retículo Endoplásmico/metabolismo , eIF-2 Quinasa/metabolismo , Adipocitos Marrones/metabolismo , Animales , Diferenciación Celular/genética , ADN Mitocondrial/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos ICR , Mitocondrias/metabolismo , Biogénesis de Organelos , Fosforilación , Transducción de Señal/genética , Termogénesis/fisiología , Transcripción Genética/genéticaRESUMEN
Brain edema is characterized by an increase in net brain water content, which results in an increase in brain volume. Although brain edema is associated with a high fatality rate, the cellular and molecular processes of edema remain largely unclear. Here, we developed an in vitro model of ischemic stroke-induced edema in which male mouse brain slices were treated with oxygen-glucose deprivation (OGD) to mimic ischemia. We continuously measured the cross-sectional area of the brain slice for 150 min under macroscopic microscopy, finding that OGD induces swelling of brain slices. OGD-induced swelling was prevented by pharmacologically blocking or genetically knocking out the transient receptor potential vanilloid 4 (TRPV4), a member of the thermosensitive TRP channel family. Because TRPV4 is activated at around body temperature and its activation is enhanced by heating, we next elevated the temperature of the perfusate in the recording chamber, finding that hyperthermia induces swelling via TRPV4 activation. Furthermore, using the temperature-dependent fluorescence lifetime of a fluorescent-thermosensitive probe, we confirmed that OGD treatment increases the temperature of brain slices through the activation of glutamate receptors. Finally, we found that brain edema following traumatic brain injury was suppressed in TRPV4-deficient male mice in vivo Thus, our study proposes a novel mechanism: hyperthermia activates TRPV4 and induces brain edema after ischemia.SIGNIFICANCE STATEMENT Brain edema is characterized by an increase in net brain water content, which results in an increase in brain volume. Although brain edema is associated with a high fatality rate, the cellular and molecular processes of edema remain unclear. Here, we developed an in vitro model of ischemic stroke-induced edema in which mouse brain slices were treated with oxygen-glucose deprivation. Using this system, we showed that the increase in brain temperature and the following activation of the thermosensitive cation channel TRPV4 (transient receptor potential vanilloid 4) are involved in the pathology of edema. Finally, we confirmed that TRPV4 is involved in brain edema in vivo using TRPV4-deficient mice, concluding that hyperthermia activates TRPV4 and induces brain edema after ischemia.
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Edema Encefálico/etiología , Isquemia Encefálica/complicaciones , Fiebre/etiología , Canales Catiónicos TRPV/metabolismo , Animales , Edema Encefálico/metabolismo , Isquemia Encefálica/metabolismo , Fiebre/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Temperature influences the activities of living organisms at various levels. Cells not only detect environmental temperature changes through their unique temperature-sensitive molecular machineries but also muster an appropriate response to the temperature change to maintain their inherent functions. Despite the fundamental involvement of temperature in physiological phenomena, the mechanism by which cells produce and use heat is largely unknown. Recently, fluorescent thermosensors that function as thermometers in live cells have attracted much attention in biology. These new tools, made of various temperature-sensitive molecules, have allowed for intracellular thermometry at the single-cell level. Intriguing spatiotemporal temperature variations, including organelle-specific thermogenesis, have been revealed with these fluorescent thermosensors, which suggest an intrinsic connection between temperature and cell functions. Moreover, fluorescent thermosensors have shown that intracellular temperature changes at the microscopic level are largely different from those assumed for a water environment at the macroscopic level. Thus, the employment of fluorescent thermosensors will uncover novel mechanisms of intracellular temperature-assisted physiological functions.
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Técnicas Biosensibles/métodos , Orgánulos/metabolismo , Termometría/métodos , Xantenos/química , Animales , Proteínas Fluorescentes Verdes/química , Humanos , Puntos Cuánticos/químicaRESUMEN
Temperature is a fundamental physical parameter that plays an important role in biological reactions and events. Although thermometers developed previously have been used to investigate several important phenomena, such as heterogeneous temperature distribution in a single living cell and heat generation in mitochondria, the development of a thermometer with a sensitivity over a wide temperature range and rapid response is still desired to quantify temperature change in not only homeotherms but also poikilotherms from the cellular level to in vivo. To overcome the weaknesses of the conventional thermometers, such as a limitation of applicable species and a low temporal resolution, owing to the narrow temperature range of sensitivity and the thermometry method, respectively, we developed a genetically encoded ratiometric fluorescent temperature indicator, gTEMP, by using two fluorescent proteins with different temperature sensitivities. Our thermometric method enabled a fast tracking of the temperature change with a time resolution of 50 ms. We used this method to observe the spatiotemporal temperature change between the cytoplasm and nucleus in cells, and quantified thermogenesis from the mitochondria matrix in a single living cell after stimulation with carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, which was an uncoupler of oxidative phosphorylation. Moreover, exploiting the wide temperature range of sensitivity from 5°C to 50°C of gTEMP, we monitored the temperature in a living medaka embryo for 15 hours and showed the feasibility of in vivo thermometry in various living species.
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Proteínas Luminiscentes/genética , Termómetros , Células HeLa , Humanos , Mitocondrias/metabolismo , Espectrometría de Fluorescencia , Factores de TiempoRESUMEN
Recent studies suggest that adult humans have active brown or beige adipocytes, the activation of which might be a therapeutic strategy for the treatment of diverse metabolic diseases. Here we show that the protein kinase ASK1 regulates brown and beige adipocytes function. In brown or white adipocytes, the PKA-ASK1-p38 axis is activated in response to cAMP signalling and contributes to the cell-autonomous induction of genes, including Ucp1. Global and fat-specific ASK1 deficiency leads to impaired metabolic responses, including thermogenesis and oxygen consumption, at the cell and whole-body levels, respectively. Our data thus indicate that the ASK1 signalling axis is a regulator of brown and beige adipocyte gene expression and function.
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Adipocitos Marrones/metabolismo , Tejido Adiposo Pardo/metabolismo , MAP Quinasa Quinasa Quinasa 5/metabolismo , Transducción de Señal , Termogénesis/genética , Células 3T3-L1 , Adipocitos Marrones/citología , Adipocitos Blancos/citología , Adipocitos Blancos/metabolismo , Tejido Adiposo Pardo/citología , Tejido Adiposo Blanco/citología , Tejido Adiposo Blanco/metabolismo , Animales , Diferenciación Celular , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Femenino , Regulación de la Expresión Génica , Células HEK293 , Humanos , Canales Iónicos/genética , Canales Iónicos/metabolismo , MAP Quinasa Quinasa Quinasa 5/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Consumo de Oxígeno/genética , Proteína Desacopladora 1 , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Single-molecule localization microscopy is used to construct super-resolution images, but generally requires prior intense laser irradiation and in some cases additives, such as thiols, to induce on-off switching of fluorophores. These requirements limit the potential applications of this methodology. Here, we report a first-in-class spontaneously blinking fluorophore based on an intramolecular spirocyclization reaction. Optimization of the intramolecular nucleophile and rhodamine-based fluorophore (electrophile) provide a suitable lifetime for the fluorescent open form, and equilibrium between the open form and the non-fluorescent closed form. We show that this spontaneously blinking fluorophore is suitable for single-molecule localization microscopy imaging deep inside cells and for tracking the motion of structures in living cells. We further demonstrate the advantages of this fluorophore over existing methodologies by applying it to nuclear pore structures located far above the coverslip with a spinning-disk confocal microscope and for repetitive time-lapse super-resolution imaging of microtubules in live cells for up to 1 h.
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Colorantes Fluorescentes/química , Compuestos de Espiro/química , Ciclización , Células HeLa , Humanos , Microscopía Confocal , Fotólisis , Rodaminas/química , Espectrometría de Fluorescencia , Imagen de Lapso de TiempoRESUMEN
Dysregulation of oxidative phosphorylation is associated with increased mitochondrial reactive oxygen species production and some of the most prevalent human diseases including obesity, cancer, diabetes, neurodegeneration, and heart disease. Chemical 'mitochondrial uncouplers' are lipophilic weak acids that transport protons into the mitochondrial matrix via a pathway that is independent of ATP synthase, thereby uncoupling nutrient oxidation from ATP production. Mitochondrial uncouplers also lessen the proton motive force across the mitochondrial inner membrane and thereby increase the rate of mitochondrial respiration while decreasing production of reactive oxygen species. Thus, mitochondrial uncouplers are valuable chemical tools that enable the measurement of maximal mitochondrial respiration and they have been used therapeutically to decrease mitochondrial reactive oxygen species production. However, the most widely used protonophore uncouplers such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and 2,4-dinitrophenol have off-target activity at other membranes that lead to a range of undesired effects including plasma membrane depolarization, mitochondrial inhibition, and cytotoxicity. These unwanted properties interfere with the measurement of mitochondrial function and result in a narrow therapeutic index that limits their usefulness in the clinic. To identify new mitochondrial uncouplers that lack off-target activity at the plasma membrane we screened a small molecule chemical library. Herein we report the identification and validation of a novel mitochondrial protonophore uncoupler (2-fluorophenyl){6-[(2-fluorophenyl)amino](1,2,5-oxadiazolo[3,4-e]pyrazin-5-yl)}amine, named BAM15, that does not depolarize the plasma membrane. Compared to FCCP, an uncoupler of equal potency, BAM15 treatment of cultured cells stimulates a higher maximum rate of mitochondrial respiration and is less cytotoxic. Furthermore, BAM15 is bioactive in vivo and dose-dependently protects mice from acute renal ischemic-reperfusion injury. From a technical standpoint, BAM15 represents an effective new tool that allows the study of mitochondrial function in the absence of off-target effects that can confound data interpretation. From a therapeutic perspective, BAM15-mediated protection from ischemia-reperfusion injury and its reduced toxicity will hopefully reignite interest in pharmacological uncoupling for the treatment of the myriad of diseases that are associated with altered mitochondrial function.
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An environment-sensitive fluorophore can change its maximum emission wavelength (λ(em)), fluorescence quantum yield (Φ(f)), and fluorescence lifetime in response to the surrounding environment. We have developed two new intramolecular charge-transfer-type environment-sensitive fluorophores, DBThD-IA and DBSeD-IA, in which the oxygen atom of a well-established 2,1,3-benzoxadiazole environment-sensitive fluorophore, DBD-IA, has been replaced by a sulfur and selenium atom, respectively. DBThD-IA is highly fluorescent in n-hexane (Φ(f) =0.81, λ(em) =537â nm) with excitation at 449â nm, but is almost nonfluorescent in water (Φ(f) =0.037, λ(em) =616â nm), similarly to DBD-IA (Φ(f) =0.91, λ(em) =520â nm in n-hexane; Φ(f) =0.027, λ(em) =616â nm in water). A similar variation in fluorescence properties was also observed for DBSeD-IA (Φ(f) =0.24, λ(em) =591â nm in n-hexane; Φ(f) =0.0046, λ(em) =672â nm in water). An intensive study of the solvent effects on the fluorescence properties of these fluorophores revealed that both the polarity of the environment and hydrogen bonding with solvent molecules accelerate the nonradiative relaxation of the excited fluorophores. Time-resolved optoacoustic and phosphorescence measurements clarified that both intersystem crossing and internal conversion are involved in the nonradiative relaxation processes of DBThD-IA and DBSeD-IA. In addition, DBThD-IA exhibits a 10-fold higher photostability in aqueous solution than the original fluorophore DBD-IA, which allowed us to create a new robust molecular nanogel thermometer for intracellular thermometry.
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Azoles/química , Colorantes Fluorescentes/química , Compuestos de Organoselenio/química , Tiadiazoles/química , Estructura Molecular , Procesos FotoquímicosRESUMEN
We developed a simple preparation procedure for the protein encapsulated nanoparticle and used the nanoparticle for spatiotemporal activity control of various proteins. We succeeded in the local protein activation within cells by light using the nanoparticle.
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Cápsulas/química , Células/efectos de la radiación , Luz , Nanopartículas/química , Proteínas/química , Cápsulas/síntesis química , Cápsulas/efectos de la radiación , Células/metabolismo , Microscopía Electrónica de TransmisiónRESUMEN
Cellular functions are fundamentally regulated by intracellular temperature, which influences biochemical reactions inside a cell. Despite the important contributions to biological and medical applications that it would offer, intracellular temperature mapping has not been achieved. Here we demonstrate the first intracellular temperature mapping based on a fluorescent polymeric thermometer and fluorescence lifetime imaging microscopy. The spatial and temperature resolutions of our thermometry were at the diffraction limited level (200 nm) and 0.18-0.58 °C. The intracellular temperature distribution we observed indicated that the nucleus and centrosome of a COS7 cell, both showed a significantly higher temperature than the cytoplasm and that the temperature gap between the nucleus and the cytoplasm differed depending on the cell cycle. The heat production from mitochondria was also observed as a proximal local temperature increase. These results showed that our new intracellular thermometry could determine an intrinsic relationship between the temperature and organelle function.
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Núcleo Celular/fisiología , Citoplasma/fisiología , Microscopía Fluorescente/métodos , Temperatura , Animales , Células COS , Línea Celular , Centrosoma/fisiología , Chlorocebus aethiops , Células HeLa , Humanos , TermómetrosRESUMEN
In response to environmental stress, cytoplasmic mRNAs aggregate to form stress granules (SGs). SGs have mainly been studied indirectly using protein markers, but the real-time behavior of endogenous mRNAs in SGs remains uncertain. Here, we visualized endogenous cytoplasmic poly(A)(+) mRNAs in living mammalian cells using a linear antisense 2'-O-methyl RNA probe. In arsenite-stressed cells, endogenous mRNAs aggregated in granules that colocalized with SGs marked by TIA-1-GFP. Moreover, analysis of mRNA dynamics using fluorescence recovery after photobleaching showed that approximately one-third of the endogenous mRNAs in SGs was immobile, another one-third was diffusive, and the remaining one-third was in equilibrium between binding to and dissociating from SGs, with a time constant of approximately 300 seconds. These dynamic characteristics of mRNAs were independent of the duration of stress and microtubule integrity. Similar characteristics were also observed from fos mRNA labeled with an antisense 2'-O-methyl RNA probe. Our results revealed the behavior of endogenous mRNAs, and indicated that SGs act as dynamic harbors of untranslated poly(A)(+) mRNAs.
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Gránulos Citoplasmáticos/metabolismo , ARN Mensajero/metabolismo , Estrés Fisiológico , Animales , Arsenitos/farmacología , Células COS , Chlorocebus aethiops , Citoplasma/efectos de los fármacos , Difusión , Recuperación de Fluorescencia tras Fotoblanqueo , Microinyecciones , Microtúbulos/metabolismo , Plásmidos/genética , Plásmidos/metabolismo , Poli A/metabolismo , Sondas ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Compuestos de Sodio/farmacología , Factores de Tiempo , TransfecciónRESUMEN
Visualization and monitoring of endogenous mRNA in the cytoplasm of living cells promises a significant comprehension of refined post-transcriptional regulation. Fluorescently labeled linear antisense oligonucleotides can bind to natural mRNA in a sequence-specific way and, therefore, provide a powerful tool in probing endogenous mRNA. Here, we investigated the feasibility of using linear antisense probes to monitor the variable and dynamic expression of endogenous cytoplasmic mRNAs. Two linear antisense 2'-O-methyl RNA probes, which have different interactive fluorophores at the 5'-end of one probe and at the 3'-end of the other, were used to allow fluorescence resonance energy transfer (FRET) upon hybridization to the target mRNA. By characterizing the formation of the probe-mRNA hybrids in living cells, we found that the probe composition and concentration are crucial parameters in the visualization of endogenous mRNA with high specificity. Furthermore, rapid hybridization (within 1 min) of the linear antisense probe enabled us to visualize dynamic processes of endogenous c-fos mRNA, such as fast elevation of levels after gene induction and the localization of c-fos mRNA in stress granules in response to cellular stress. Thus, our approach provides a basis for real time monitoring of endogenous cytoplasmic mRNA in living cells.
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Citoplasma/química , Transferencia Resonante de Energía de Fluorescencia , Sondas ARN/química , ARN sin Sentido/química , ARN Mensajero/análisis , Animales , Células COS , Chlorocebus aethiops , Gránulos Citoplasmáticos/química , Colorantes Fluorescentes , Cinética , Microscopía Fluorescente , Proteínas Proto-Oncogénicas c-fos/análisis , Proteínas Proto-Oncogénicas c-fos/genética , Estabilidad del ARN , ARN Mensajero/biosíntesisRESUMEN
The first methodology to measure intracellular temperature is described. A highly hydrophilic fluorescent nanogel thermometer developed for this purpose stays in the cytoplasm and emits stronger fluorescence at a higher temperature. Thus, intracellular temperature variations associated with biological processes can be monitored by this novel thermometer with a temperature resolution of better than 0.5 degrees C.