RESUMEN

Fluorescence imaging provides a powerful technique to observe biomolecular dynamics in living systems, if fluorescent biosensors for the relevant biomolecules become available. Here, we describe a highly sensitive, cell-based biosensor to visualize nitric oxide (NO) released from living cells. Nitric oxide (NO) is a gaseous molecule that is involved in a broad range of physiological and toxicological processes in cardiovascular and central nervous systems, etc. This chapter describes how to make optical measurements of NO release from living cells using the cell-based fluorescent biosensor.

Asunto(s)

Técnicas Biosensibles/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , Óxido Nítrico/metabolismo , Pruebas de Toxicidad/métodos , Animales , Línea Celular , Células Cultivadas , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/metabolismo

RESUMEN

We introduce a method to evaluate the activities of cytokines based on the nuclear transport of NF-κB. A pair of bioluminescent indicators was made for conferring cytokine sensitivity to cervical carcinoma-derived HeLa cells. The principle is based on reconstitution of split fragments of Renilla reniformis luciferase (RLuc) by protein splicing with a DnaE intein from Synechocystis sp. PCC6803. The bioluminescence intensity of thus reconstituted RLuc in the HeLa cells was used as a measure of the activities for cytokines. With the present method, we evaluated the activities of various cytokines based on the nuclear transport of NF-κB in human cervical carcinoma-derived HeLa cells carrying the indicators. The present approach to evaluating the activities of cytokines may provide a potential clinical value in monitoring drug activity and directing treatment for various diseases related with NF-κB. The method highlights the experimental procedure from our original publications, Anal. Biochem. 2006, 359, 147-149 and Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 11542. The summary of the method is: •Cytokine activities are determined within 2 h after stimulation.•Temporarily inactivated split-luciferase fragments are reconstituted by protein splicing.•Nucleartrafficking of NF-κB was illuminated for gauging the ligand-driven activity.

RESUMEN

The present protocol introduces multicolor imaging of bifacial activities of an estrogen. For the multicolor imaging, the authors fabricated two single-chain probes emitting green or red bioluminescence (named Simer-G and -R, respectively) from click beetle luciferase (CBLuc) green and red: Simer-R consists of the ligand binding domain of estrogen receptor (ER LBD) and the Src homology-2 (SH2) domain of Src, which are sandwiched between split-CBLuc red (CBLuc-R). On the other hand, Simer-G emitting red light consists of the ER LBD and a common consensus sequence of coactivators (LXXLL motif), which are inserted between split-CBLuc green (CBLuc-G). This probe set creates fingerprinting spectra from the characteristic green and red bioluminescence in response to agonistic and antagonistic activities of a ligand of interest. The present protocol further provides a unique methodology to calculate characteristic estrogenicity scores of various ligands from the spectra.

Asunto(s)

Estrógenos/metabolismo , Imagen Molecular/métodos , Animales , Línea Celular , Estrógenos/genética , Expresión Génica , Orden Génico , Vectores Genéticos/genética , Humanos , Ligandos , Mediciones Luminiscentes/métodos , Sondas Moleculares/química , Sondas Moleculares/genética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Receptores de Estrógenos/química , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo

RESUMEN

Protein phosphorylation by intracellular kinases plays one of the most pivotal roles in signaling pathways within cells. To reveal the biological processes related to the kinase proteins, electrophoresis, immunocytochemistry, and in vitro kinase assay have been used. However, these conventional methods do not provide enough information about spatial and temporal dynamics of the signal transduction based on protein phosphorylation and dephosphorylation in living cells. To overcome the limitation for investigating the kinase signaling, we developed genetically encoded fluorescent indicators for visualizing the protein phosphorylation in living cells. Using these indicators, we visualized under a fluorescence microscope when, where, and how the protein kinases are activated in single living cells.

Asunto(s)

Proteínas Bacterianas/genética , Técnicas Biosensibles , Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas Fluorescentes Verdes/genética , Microscopía Intravital/métodos , Proteínas Luminiscentes/genética , Microscopía Fluorescente/métodos , Proteínas Quinasas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/genética , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/análisis , Células CHO , Cricetinae , Cricetulus , Genes Sintéticos , Ingeniería Genética , Proteínas Fluorescentes Verdes/análisis , Humanos , Proteínas Luminiscentes/análisis , Modelos Moleculares , Datos de Secuencia Molecular , Fosforilación , Conformación Proteica , Receptor de Insulina/antagonistas & inhibidores , Receptor de Insulina/metabolismo , Proteínas Recombinantes de Fusión/análisis , Transducción de Señal

RESUMEN

The elucidation of lipid dynamics on the cytosolic and lumenal sides of a single endomembrane has been challenging in living cells because of the lack of appropriate methods. Diacylglycerol (DAG) is a lipid second messenger that is produced by enzymes that reside on both the cytosolic and lumenal sides of the endomembrane. In the present study, we attempted to observe both the cytosolic and lumenal DAG dynamics at endomembranes including the Golgi apparatus and the endoplasmic reticulum in Madin-Darby canine kidney (MDCK) cells. We developed a Förster resonance energy transfer (FRET)-based probe to detect DAG at the luminal side (lumenal DAG) of endomembranes. In combination with the FRET-based cytosolic DAG probe that has already been established, it was found that lumenal DAG is generated in a calcium-dependent manner by thapsigargin, which increases cytosolic calcium concentrations. In contrast, DAG production at the cytosolic side of endomembranes did not occur under the same experimental conditions. The thapsigargin-induced DAG generation was abolished by treatment with an inhibitor of sphingomyelin synthase (SMS) and phosphatidylcholine-specific phospholipase C (PC-PLC), which produce lumenal DAG. Thus, we have established a successful method for monitoring both cytosolic and lumenal DAG dynamics at the endomembrane in living cells.

Asunto(s)

Citosol/metabolismo , Diglicéridos/metabolismo , Animales , Membrana Celular/metabolismo , Perros , Transferencia Resonante de Energía de Fluorescencia , Células de Riñón Canino Madin Darby

Asunto(s)

Química/historia , Universidades/historia , Historia del Siglo XX , Historia del Siglo XXI , Tokio

RESUMEN

Electron-donating molecular tips were used for the observation of single-walled carbon nanotubes (SWNTs). Defects in SWNTs were selectively visualized at the atomic scale on the basis of charge-transfer interaction with the molecular tip.

Asunto(s)

Electrones , Microscopía de Túnel de Rastreo/métodos , Nanotubos de Carbono/química , Compuestos de Anilina/química , Oro/química , Compuestos de Sulfhidrilo

RESUMEN

Localization of mRNAs plays pivotal roles in different cell types, including neurons and the cells in the developing stages. To visualize the dynamic movements of mRNAs in living cells, many methods have been emerged in the past decade. However, it has not been realized to visualize endogenous mRNAs with genetically encoded fluorescent probes. We recently developed fluorescent protein-based RNA probes for characterizing the localization and dynamics of mRNAs in single living cells. The probes consist of two RNA-binding domains of human PUMILIO1, each connected with split fragments of a fluorescent protein capable of reconstitution upon binding to a target mRNA. The probes are modified to specifically recognize a 16-base sequence of an mRNA of interest and to target into organelles by means of a short signal peptide. We have shown that ND6 mRNA is concentrated particularly on mitochondrial DNA (mtDNA) and movement of the mRNA is restricted in mitochondria. The probes provide a general means to study spatial and temporal mRNA localization and dynamics in intracellular compartments in living cells.

Asunto(s)

Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Imagen Molecular/métodos , ARN Mensajero/análisis , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Animales , Secuencia de Bases , Células COS , Supervivencia Celular , Chlorocebus aethiops , Ensayo de Cambio de Movilidad Electroforética , Células HeLa , Humanos , Inmunoprecipitación , Proteínas Luminiscentes/química , Microscopía Fluorescente , Modelos Moleculares , Mutación , Conformación Proteica , Ingeniería de Proteínas , Estructura Terciaria de Proteína , Transporte de ARN , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Fluorescencia

RESUMEN

Protein-protein interaction is one of the most pivotal roles of proteins in living organisms. Association/dissociation of proteins reflects responses to intrinsic or extrinsic perturbations of signaling pathways, involved in gene expression, cell division, cell differentiation, and apoptosis. For further understanding of the biological processes, it is important to monitor protein-protein interactions in model organisms. In particular, Escherichia coli-based methods are suitable to assess large libraries of proteins. Many of these proteins cannot be used in yeast due to toxicity or poor expression. Herein we describe a general method based on an intein-mediated protein reconstitution system (PRS) to detect protein-protein interactions in bacterial cells. The PRS-based approach requires no other agents including enzymes, substrates, and ATP. Another advantage is that matured green fluorescent protein (GFP) accumulates in a targeted cell till degraded. These allow highly sensitive screening of protein-protein interactions.

Asunto(s)

Escherichia coli/metabolismo , Proteínas Fluorescentes Verdes/biosíntesis , Inteínas , Escherichia coli/genética , Proteínas Fluorescentes Verdes/genética

RESUMEN

Imaging of RNAs in single cells revealed their localized transcription and specific function. Such information cannot be obtained from bulk measurements. This unit contains a protocol of an imaging method capable of visualizing endogenous RNAs bound to genetically encoded fluorescent probes in single living cells. The protocol includes methods of design and construction of the probes, their characterization, and imaging a target RNA in living cells. The methods for RNA imaging are generally applicable to many kinds of RNAs and may allow for elucidating novel functions of localized RNAs and understanding their dynamics in living cells. Curr. Protoc. Chem. Biol. 3:27-37 © 2011 by John Wiley & Sons, Inc.

RESUMEN

This paper reviews chemically selective imaging at the single-atom/molecule level by molecular STM tips. The molecular tips enable the recognition of a particular chemical species on the basis of chemical interactions with a sample molecule, including hydrogen-bond, metal-coordination, and charge-transfer interactions. The chemical selectivity can be tailored by designing functional groups of the tip molecules. Moreover, the rational design of the molecular tip allows sophisticated chemical recognition. The discrimination of DNA bases and chiral recognition on a single molecule basis are thereby achieved. The molecular tips also revealed rectified electron transmission within an electron donor-acceptor molecular pair. Self-assembled monolayers, carboxylated carbon nanotube, and conducting polymers can be utilized for the preparation of molecular tips. This technique may be coined "intermolecular tunneling microscopy" as its principle goes, and is of general significance for novel molecular imaging of chemical identities at the membrane and solid surfaces.

Asunto(s)

Técnicas de Química Analítica , Microscopía de Túnel de Rastreo , Adsorción , Carbono/química , ADN/análisis , Electrones , Enlace de Hidrógeno , Metales/análisis , Nanotecnología , Nanotubos de Carbono , Compuestos Orgánicos/análisis , Porfirinas/análisis

RESUMEN

The present study demonstrates a single-molecular bioluminescent probe carrying functional peptides in the N-terminal domain of the androgen receptor (AR NTD) with an improved sensorial property to androgens. The N-terminal peptides in AR were genetically fused to the ligand binding domain of AR (AR LBD) with a flexible linker, and then sandwiched between the N- and C-terminal fragments of split-firefly luciferase (FLuc) dissected at D415. We found that the proline-rich region in AR NTD efficiently interacts with AR LBD and exerts (i) an enhanced signal-to-background ratio and (ii) discrimination between agonists and antagonists with (iii) a 100-times improved sensitivity to androgens, upon comparison with previous references. A deletion mutation to the proline-rich region in AR revealed that this region is critical for the transcriptional activities. The quantum yields of these single-chain probes were estimated to be 37.8 +/- 0.6%. This monomeric AR LBD-peptide binding is necessary, and sufficient for discriminating an agonist and an antagonist, where the dimerization of AR LBD is not involved. The present study guides a fundamental methodology on how to discriminate weak protein-peptide binding, and provides a new insight into the contribution of functional peptides in AR NTD to the initial activation of monomeric AR.

Asunto(s)

Andrógenos/metabolismo , Sustancias Luminiscentes/química , Sustancias Luminiscentes/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Receptores Androgénicos/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Antagonistas de Andrógenos/farmacología , Animales , Línea Celular , Dihidrotestosterona/farmacología , Genes Reporteros/genética , Humanos , Cinética , Ligandos , Límite de Detección , Estructura Terciaria de Proteína , Receptores Androgénicos/metabolismo , Reproducibilidad de los Resultados

RESUMEN

Programmed cell death, apoptosis, is a crucial process involved in pathogenesis and progression of diseases, which is executed by cysteine aspartyl proteases (caspases). The caspase activities in living subjects and their regulation with small chemical compounds are of great interest for screening drug candidates or pathological agents. We describe a novel genetically encoded bioluminescent indicator for real-time imaging of caspase-3 activities in living cells and animals. The indicator is composed of an engineered firefly luciferase, of which the N- and C-terminal ends are linked with a substrate sequence of caspase-3 (Asp-Glu-Val-Asp). When activated caspase-3 digests the substrate sequence, the cyclized luciferase recovers its activity. The indicator provides a general means of evaluating effects of cytotoxic compounds or novel pharmacological chemicals and apoptosis.

Asunto(s)

Apoptosis , Luciferasas/genética , Animales , Secuencia de Bases , Caspasa 3/metabolismo , Cartilla de ADN , Células HeLa , Humanos , Plásmidos , Reacción en Cadena de la Polimerasa

RESUMEN

For nondestructive analysis of chemical processes in living mammalian cells, here we show a new reporter gene assay for detecting Ras-Raf-1 interactions based on protein splicing of transcription factors with DnaE inteins. The protein splicing induces connection of a DNA-binding protein (modified LexA; mLexA) with a transcription activation domain of a herpes simplex virus protein (VP16AD). Ras is connected with N-terminal DnaE and mLexA, while Raf-1 is connected with C-terminal DnaE and VP16AD. Upon stimulation with EGF, the interaction between Ras and Raf-1 triggers folding of the DnaEs, thereby inducing protein splicing to form mLexA-VP16AD fusion protein and transcription of a reporter gene, firefly luciferase. The extent of Ras-Raf-1 interaction is quantified by measuring the luciferase activity. By using the protein-splicing elements and the reporter gene, the Ras-Raf-1 interaction close to cell membranes can be evaluated.

Asunto(s)

Genes Reporteros , Inteínas , Luminiscencia , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Receptores ErbB/genética , Proteína Oncogénica p21(ras)/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo

RESUMEN

Increases in reactive oxygen species (ROS) have been implicated in age-related diseases, including cancer. The serine/threonine kinase protein kinase D1 (PKD1) is a stress-responsive kinase and sensor for reactive oxygen species, which can initiate cell survival through NF-kappaB signaling. We have previously shown that in response to ROS, PKD1 is activated at the mitochondria. However, the initial signaling events leading to localization of PKD1 to the mitochondria are unknown. Here, we show that formation of mitochondrial diacylglycerol (DAG) and its binding to PKD1 is the means by which PKD1 is localized to the mitochondria in response to ROS. Interestingly, DAG to which PKD1 is recruited in this pathway is formed downstream of phospholipase D1 (PLD1) and a lipase-inactive PLD1 or inhibition of PLD1 by pharmacological inhibitors blocked PKD1 activation under oxidative stress. To date it has been viewed that monosaturated and saturated DAG formed via PLD1 have no signaling function. However, our data describe a role for PLD1-induced DAG as a competent second messenger at the mitochondria that relays ROS to PKD1-mediated mitochondria-to-nucleus signaling.

Asunto(s)

Diglicéridos/metabolismo , Mitocondrias/enzimología , Proteína Quinasa C/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Sitios de Unión , Biocatálisis , Activación Enzimática , Células HeLa , Humanos , Modelos Biológicos , Proteínas Mutantes/metabolismo , Mutación/genética , Estrés Oxidativo , Fosfolipasa D/metabolismo , Fosfotirosina/metabolismo , Unión Proteica , Proteína Quinasa C/química , Estructura Terciaria de Proteína , Transporte de Proteínas , Familia-src Quinasas/metabolismo

RESUMEN

Genetically encoded bioluminescent indicators for intracellular Ca2+ are described here with CaM-M13 interaction-induced complementation of split Renilla luciferase. The Ca2+-induced interaction between CaM and M13 leads to complementation of the N- and C-terminal halves of split Renilla luciferase in living cells. This intramolecular interaction results in the spontaneous and simultaneous emission of bioluminescence split Renilla luciferase. This is how intracellular Ca2+ is illuminated with the intramolecular complementation of split Renilla luciferase. The Ca2+-dependent spontaneous and simultaneous emission of bioluminescence promises to reveal Ca2+ dynamics in living cells, and also in vivo using the present indicators.

Asunto(s)

Calcio/análisis , Luciferasas de Renilla , Calmodulina , Línea Celular Tumoral , Humanos , Proteínas Luminiscentes , Técnicas de Sonda Molecular , Proteínas Recombinantes de Fusión

RESUMEN

Chiral surfaces attract increasing interest due to their vital role in a variety of scientific fields, such as chiral separation and heterogeneous enantioselective catalysis. The most urgent issue in research on such two-dimensional chirality is a lack of methodologies that recognize molecular chirality on a surface. Here we show that the chiral molecular tips enable for the first time discrimination of enantiomers on a single-molecule basis. The chiral selectivity is attributed to favorable chemical interactions that the molecular tips form with only one of two enantiomers. The stereoselective observation reveals spatial distribution of the enantiomers on a surface at the molecular level. The chiral molecular tips open a way for control of organization of enantiomers toward the advanced functionality of these chiral surfaces through knowledge on pivotal roles of chirality on molecular assemblies as shown here.

RESUMEN

Specific targeting of disease cells has the potential of far-reaching applications, such as diagnostic imaging and therapies of diseases. Here we describe a novel method for selective targeting of a type of cells among various cell types. Activated macrophages are disease cells related to initiation and progression of atherosclerosis. We developed a molecular probe of which uptake into cells is synergistically activated by scavenger receptor class A type I (SR-AI) and matrix metalloproteinase-9 (MMP-9), which are the marker receptor and the marker protease of atherosclerosis, respectively. We demonstrated that the present targeting probe is selectively incorporated into activated macrophages expressing both SR-AI and the activated form of MMP-9 but not into resting macrophages having only SR-AI. The present approach may provide a powerful tool for cell-specific imaging and therapies.

Asunto(s)

Aterosclerosis , Activación de Macrófagos/efectos de los fármacos , Macrófagos , Metaloproteinasa 9 de la Matriz/metabolismo , Sondas Moleculares/farmacología , Receptores Depuradores de Clase A/metabolismo , Animales , Apolipoproteínas B/química , Apolipoproteínas B/farmacología , Aterosclerosis/diagnóstico , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cricetinae , Cricetulus , Sinergismo Farmacológico , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/farmacología , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Macrófagos/metabolismo , Metaloproteinasa 9 de la Matriz/biosíntesis , Metaloproteinasa 9 de la Matriz/genética , Ratones , Sondas Moleculares/química , Unión Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/farmacología , Receptores Depuradores de Clase A/biosíntesis , Receptores Depuradores de Clase A/genética , Transfección

RESUMEN

Bioactive small molecules, including steroids, activate multiple signaling pathways in mammalian cells. However, current technologies cannot illuminate such multiple effects of a ligand in mammalian cells. Here, we demonstrate integrated-molecule-format multicolor systems simultaneously visualizing bifacial activities of a ligand, where estrogen receptor alpha (ERalpha) was exemplified to demonstrate the present technology. First, we developed a single-molecule-format probe emitting red bioluminescence for imaging interaction between the phosphorylated ligand binding domain of ERalpha (ER LBD) and the Src homology-2 (SH2) domain of Src. The SH2 domain-linked ER LBD was sandwiched between dissected N- and C-terminal fragments of Pyrophorus plagiophthalamus (click beetle) luciferase emitting red bioluminescence. Second, another single-molecule-format bio-luminescent probe emitting green bioluminescence was constructed to visualize intramolecular interaction between ER LBD and LXXLL motifs. Mammalian cells carrying the two probes emit red and/or green light in response to agonistic and antagonistic activities of a ligand, which correspond to its genomic and nongenomic activities, respectively. Third, the two probes were assembled to make an single-molecule-format multicolor indicator, in which all of the components for ligand sensing and multiple-light emission were integrated. The probe emitted characteristic light spectra in response to various agonists and antagonists. This is the first example where (i) protein phosphorylation was recognized with a single bioluminescent probe and (ii) bifacial activities of a ligand, either agonistic or antagonistic, were simultaneously visualized with multiple colors.

Asunto(s)

Células , Receptor alfa de Estrógeno , Luciferasas/química , Sustancias Luminiscentes/química , Mediciones Luminiscentes/métodos , Dominios Homologos src/fisiología , Familia-src Quinasas , Animales , Sitios de Unión , Células/citología , Células/metabolismo , Escarabajos/enzimología , Receptor alfa de Estrógeno/análisis , Receptor alfa de Estrógeno/metabolismo , Ligandos , Fosforilación , Sensibilidad y Especificidad , Familia-src Quinasas/análisis , Familia-src Quinasas/metabolismo

RESUMEN

Brain-derived neurotrophic factor (BDNF) is a polypeptide that is secreted from neurons. Although there is mounting evidence that BDNF regulates neuronal development and synaptic plasticity, BDNF secretion has remained unclear due to lack of appropriate methods for the analysis of its dynamics. To visualize BDNF secretion from neurons, here we have developed a cell-based fluorescent indicator for BDNF. We showed that the present cell-based fluorescent indicator, named "Bescell", has high selectivity to BDNF and detects picomolar concentrations of BDNF (detection limit of 60 pM). Bescell has visualized endogenous BDNF secreted from hippocampal neurons. It thus provides a powerful tool for the analysis of BDNF secretion from living neurons.

Asunto(s)

Técnicas Biosensibles/métodos , Factor Neurotrófico Derivado del Encéfalo , Transferencia Resonante de Energía de Fluorescencia/métodos , Hipocampo , Sustancias Luminiscentes/química , Neuronas , Animales , Sitios de Unión , Factor Neurotrófico Derivado del Encéfalo/análisis , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Células Cultivadas , Clonación Molecular , Cricetinae , Cricetulus , ADN Complementario/genética , Femenino , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Neuronas/citología , Neuronas/metabolismo , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Factores de Tiempo