RESUMEN
The eukaryotic genome is organized within cells as chromatin. For proper information output, higher-order chromatin structures can be regulated dynamically. How such structures form and behave in various cellular processes remains unclear. Here, by combining super-resolution imaging (photoactivated localization microscopy [PALM]) and single-nucleosome tracking, we developed a nuclear imaging system to visualize the higher-order structures along with their dynamics in live mammalian cells. We demonstrated that nucleosomes form compact domains with a peak diameter of â¼160 nm and move coherently in live cells. The heterochromatin-rich regions showed more domains and less movement. With cell differentiation, the domains became more apparent, with reduced dynamics. Furthermore, various perturbation experiments indicated that they are organized by a combination of factors, including cohesin and nucleosome-nucleosome interactions. Notably, we observed the domains during mitosis, suggesting that they act as building blocks of chromosomes and may serve as information units throughout the cell cycle.
Asunto(s)
Ensamble y Desensamble de Cromatina , Heterocromatina/metabolismo , Microscopía por Video/métodos , Mitosis , Nucleosomas/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas Humanos , Células HCT116 , Células HeLa , Heterocromatina/química , Humanos , Ratones , Movimiento (Física) , Conformación de Ácido Nucleico , Nucleosomas/química , Conformación Proteica , Interferencia de ARN , Relación Estructura-Actividad , Factores de Tiempo , Transcripción Genética , Transfección , CohesinasRESUMEN
RAS is an important cell signaling molecule, regulating the activities of various effector proteins, including the kinase c-RAF (RAF). Despite the critical function of RAS signaling, the activation kinetics have not been analyzed experimentally in living cells for any of the RAS effectors. Here, we analyzed the kinetics of RAF activation on the plasma membrane in living HeLa cells after stimulation with EGF to activate RAS. RAF is recruited by the active form of RAS (RAS-GTP) from the cytoplasm to the plasma membrane through two RAS-binding sites (the RAS-binding domain and the cysteine-rich domain (CRD)) and is activated by its phosphorylation by still undetermined kinases on the plasma membrane. Using single-molecule imaging, we measured the dissociation time courses of GFP-tagged molecules of wild type RAF and fragments or mutants of RAF containing one or two of the three functional domains (the RAS-binding domain, the CRD, and the catalytic domain) to determine their interaction with membrane components. Each molecule showed a unique dissociation time course, indicating that both its interaction with RAS-GTP and its phosphorylation by the kinases are rate-limiting steps in RAF activation. Based on our experimental results, we propose a kinetic model for the activation of RAF. The model suggests the importance of the interaction between RAS-GTP and CRD for the effective activation of RAF, which is triggered by rapid RAS-GTP-induced conformational changes in RAF and the subsequent presentation of RAF to the kinase. The model also suggests necessary properties of the kinases that activate RAF.
Asunto(s)
Membrana Celular/enzimología , Modelos Biológicos , Complejos Multienzimáticos/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas ras/metabolismo , Activación Enzimática/fisiología , Células HeLa , Humanos , Cinética , Microscopía Fluorescente , Complejos Multienzimáticos/genética , Mutación , Proteínas Proto-Oncogénicas c-raf/genética , Proteínas ras/genéticaRESUMEN
Liquid droplets formed inside the cell by liquid-liquid phase separation maintain membrane-less condensates/bodies (or compartments). These droplets are important for concentrating certain molecules and facilitating spatiotemporal regulation of cellular functions. 1,6-hexanediol (1,6-HD), an aliphatic alcohol, inhibits weak hydrophobic protein-protein/protein-RNA interactions required for the droplet formation (droplet melting activity) and is used here to elucidate the formation process of cytoplasmic/nuclear condensates/bodies. However, the effect of 1,6-HD on chromatin in living cells remains unclear. We found that 1,6-HD drastically suppresses chromatin motion and hyper-condenses chromatin in human cells by using live-cell single-nucleosome imaging, which detects changes in the state of chromatin. These effects were enhanced in a dose-dependent manner. Chromatin was "frozen" by 5%, or higher, concentrations of 1,6-HD. 1,6-HD greatly facilitated cation-dependent chromatin condensation in vitro. This 1,6-HD action is distinct from its melting activity of liquid droplets. Alcohols, such as 1,6-HD, appear to remove water molecules around chromatin and locally condense chromatin. Therefore, liquid droplet results obtained using 1,6-HD should be carefully interpreted or reconsidered when these droplets are associated with chromatin.
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Cromatina/efectos de los fármacos , Cromatina/metabolismo , Glicoles/farmacología , Núcleo Celular/efectos de los fármacos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Cultivadas , Cromatina/genética , Proteínas de Unión al ADN , Relación Dosis-Respuesta a Droga , Técnica del Anticuerpo Fluorescente , HumanosRESUMEN
Eukaryotic chromatin is a complex of nucleic acids and proteins that is central to interpreting the information coded in the genome. Chromatin is rather irregularly folded inside the nucleus in a fluid-like state that exhibits dynamic local movement. The highly dynamic nature of chromatin has become increasingly appreciated, particularly in DNA-templated processes including transcription, because this dynamic property ensures a degree of DNA accessibility, even in compacted chromatin. Many proteins globally constrain local chromatin movements, which seem to be driven essentially by thermal fluctuation in living cells. For instance, loss of the cohesin complex, which can capture chromatin fibers, leads to an increase in chromatin motion. Another constraining factor of chromatin motion is the transcription machinery. Although the previously held view is that transcription requires open and highly dynamic chromatin, a number of studies are now pointing to a more nuanced role of transcription in constraining chromatin movement: dynamic clustering of active RNA polymerase II and other transcription factors can serve as a hub that transiently bridges active DNA regions to be transcribed, thereby loosely networking chromatin and constraining chromatin motion. In contrast, outside heterochromatin, the transcriptionally less active regions might be less constrained, more dynamic and accessible, implying a high competency state for rapid and efficient recruitment of protein factors. This new view on the interplay of local chromatin motion and transcription reflects traditional models of the transcription factories and, more recently, liquid droplets of transcription factors, providing new insight into chromatin function.
Asunto(s)
Cromatina/química , Cromatina/metabolismo , Sustancias Macromoleculares/química , Sustancias Macromoleculares/metabolismo , Conformación Molecular , Movimiento (Física) , Eucariontes , Regulación de la Expresión Génica , Transcripción GenéticaRESUMEN
BACKGROUND: The structures of proteins are intimately related to their functions. Significant efforts have been dedicated to the structural investigation of proteins, mainly those of purified proteins in in vitro environments. Proteins function in living cells and thus protein structures must be regulated by interactions with various molecules, some of which participate in reaction networks, depending on the states, conditions, or actions of the cell. Therefore, it is very important to understand the structural behavior of proteins in living cells. METHODS: Single-molecule Förster resonance energy transfer (smFRET) measurements were conducted using the alternative laser excitation (ALEX) technique. smFRET distributions of cytosolic Rapidly Accelerated Fibrosarcoma (RAF) proteins in living HeLa cells were obtained with exclusion of the negative effects of photobleached fluorophores and incompletely labeled proteins on smFRET. RESULTS: smFRET histograms of wildtype (wt) RAF in live cells exhibited two major peaks, whereas that of the S621A mutant, which has been thought to have an expanded structure, was almost single-peaked. A population shift involving the peaks for wt RAF was detected upon epidermal growth factor stimulation. Spontaneous transitions between the conformational states corresponding to the two peaks were also detected using the FRET-two-channel kernel-based density distribution estimator method in comparison to static double-stranded DNA samples. CONCLUSIONS: Cytosolic CRAF has at least three conformational states; in addition to the closed and open forms, the fully-open form was distinctly specified. Based on the results, we propose a speculative structural model for CRAF. GENERAL SIGNIFICANCE: Structural distribution and changes to proteins in live cells as a result of intracellular interactions were successfully identified. smFRET using ALEX is applicable to any other cytosolic proteins.
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Citosol/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Mutación , Proteínas Proto-Oncogénicas c-raf/química , Factor de Crecimiento Epidérmico/metabolismo , Colorantes Fluorescentes/química , Células HeLa , Humanos , Microscopía Confocal , Plásmidos/metabolismo , Conformación Proteica , Análisis de la Célula IndividualRESUMEN
The dysregulation of Ras-RAF signaling is associated with many types of human cancer. However, the kinetic and dynamic features of the mutual molecular recognition of Ras and RAF remain unknown. Here, we developed a technique for imaging single-pair fluorescence resonance energy transfer in living cells, and coupled this technique to single-molecule kinetic analysis to investigate how C-RAF (a subtype of RAF) molecules distinguish the active form of Ras (RasGTP) from the inactive form (RasGDP). Functional fragments of C-RAF containing the Ras-binding domains did not detect the switch in Ras activity in living cells as efficiently as did C-RAF. Single-molecule analysis showed that RasGDP associates with closed-conformation C-RAF, whereas the association of C-RAF with RasGTP immediately triggers the open RAF conformation, which induces an effective interaction between C-RAF and RasGTP. Spontaneous conformational changes from closed C-RAF to the open form rarely occur in quiescent cells. The conformational change in C-RAF is so important to Ras-RAF molecular recognition that C-RAF mutants lacking the conformational change cannot distinguish between RasGDP and RasGTP. The manipulation of the conformation of an effector molecule is a newly identified function of RasGTP.
Asunto(s)
Proteínas Proto-Oncogénicas c-raf/química , Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas ras/metabolismo , Sitios de Unión , Membrana Celular/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Espacio Intracelular/metabolismo , Cinética , Microscopía Confocal , Modelos Biológicos , Mutación , Conformación Proteica , Proteínas Proto-Oncogénicas c-raf/genética , Transfección , Grabación en VideoRESUMEN
Oligonucleotide-templated reactions are attracting attention as a method for RNA detection in living cells. Previously, a reduction-triggered fluorescence probe has been reported that is based on azide reduction to switch fluorescence on. In this article, we report a more advanced probe, a reduction-triggered fluorescent amplification probe that is capable of amplifying a target signal. Azidomethyl fluorescein was newly synthesized and introduced into a probe. Azido-masked fluorescein on the probe showed a strong turn-on fluorescence signal upon oligonucleotide-templated Staudinger reduction. The catalytic reaction of the probe offered a turnover number of 50 as fluorescence readout within 4 h. Finally, probes were introduced into human leukemia HL-60 cells by use of streptolysin O pore-forming peptide. We successfully detected and quantitated the 28S rRNA and beta-Actin mRNA signal above the background by flow cytometry. In addition, the same RNA targets were imaged by fluorescence microscopy. The data suggest that a reduction-triggered amplification probe may be a powerful tool in analyzing the localization, transcription, or processing of RNA species in living eukaryotic cells.
Asunto(s)
Colorantes Fluorescentes/química , Imagen Molecular/métodos , ARN/metabolismo , Azidas/química , Supervivencia Celular , Cumarinas/química , ADN/química , Diseño de Fármacos , Citometría de Flujo , Fluoresceína/síntesis química , Fluoresceína/química , Colorantes Fluorescentes/síntesis química , Células HL-60 , Humanos , Cinética , Microscopía Fluorescente , Oxidación-Reducción , ARN Mensajero/metabolismo , ARN Ribosómico 28S/metabolismo , Espectrometría de FluorescenciaRESUMEN
This chapter focuses on single-molecule imaging (SMI) in living cells using green fluorescent protein (GFP) or its related fluorescent protein tags (GFPs). Use of GFPs is a convenient technique to achieve molecular imaging of most proteins in living cells. However, because of difficulties in preparing samples suitable for SMI and the instability of fluorescence signals, special care is required for SMI using GFPs in living cells. Techniques for vector preparation, protein expression, sample preparation, microscopy, and image processing for SMI of GFPs in living cells are discussed in this chapter, along with examples of imaging applications. Double labeling of single molecules and single-pair fluorescent resonance energy transfer (spFRET) are possible in living cells using GFP and YFP as fluorescent tags. The limitations of SMI using GFPs are also discussed.
Asunto(s)
Células/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Microscopía Fluorescente/métodos , Animales , Células CHO , Técnicas de Cultivo de Célula , Cricetinae , Cricetulus , Transferencia Resonante de Energía de Fluorescencia/métodos , Expresión Génica , Vectores Genéticos , Proteínas Fluorescentes Verdes/genética , Células HeLa , Humanos , Microscopía Fluorescente/instrumentación , Dispositivos Ópticos , Proteínas Proto-Oncogénicas c-raf/genética , Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
Although chromatin organization and dynamics play a critical role in gene transcription, how they interplay remains unclear. To approach this issue, we investigated genome-wide chromatin behavior under various transcriptional conditions in living human cells using single-nucleosome imaging. While transcription by RNA polymerase II (RNAPII) is generally thought to need more open and dynamic chromatin, surprisingly, we found that active RNAPII globally constrains chromatin movements. RNAPII inhibition or its rapid depletion released the chromatin constraints and increased chromatin dynamics. Perturbation experiments of P-TEFb clusters, which are associated with active RNAPII, had similar results. Furthermore, chromatin mobility also increased in resting G0 cells and UV-irradiated cells, which are transcriptionally less active. Our results demonstrated that chromatin is globally stabilized by loose connections through active RNAPII, which is compatible with models of classical transcription factories or liquid droplet formation of transcription-related factors. Together with our computational modeling, we propose the existence of loose chromatin domain networks for various intra-/interchromosomal contacts via active RNAPII clusters/droplets.
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Cromatina/metabolismo , Histonas/metabolismo , Procesamiento de Imagen Asistido por Computador/métodos , Nucleosomas/metabolismo , ARN Polimerasa II/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Transcripción Genética , Células Cultivadas , Cromatina/genética , Simulación por Computador , Genoma Humano , Histonas/genética , Humanos , Microscopía Fluorescente , Nucleosomas/genética , ARN Polimerasa II/genética , Epitelio Pigmentado de la Retina/citologíaRESUMEN
Condensins are key players in mitotic chromosome condensation. Using an elegant combination of state-of-the-art imaging techniques, Walther et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201801048) counted the number of Condensins, examined their behaviors on human mitotic chromosomes, and integrated the quantitative data to propose a new mechanistic model for chromosome condensation.
Asunto(s)
Adenosina Trifosfatasas/genética , Segregación Cromosómica/genética , Proteínas de Unión al ADN/genética , Mitosis/genética , Complejos Multiproteicos/genética , Proteínas de Ciclo Celular/genética , Cromosomas/genética , HumanosRESUMEN
In eukaryotic cells, highly condensed inactive/silenced chromatin has long been called "heterochromatin." However, recent research suggests that such regions are in fact not fully transcriptionally silent and that there exists only a moderate access barrier to heterochromatin. To further investigate this issue, it is critical to elucidate the physical properties of heterochromatin such as its total density in live cells. Here, using orientation-independent differential interference contrast (OI-DIC) microscopy, which is capable of mapping optical path differences, we investigated the density of the total materials in pericentric foci, a representative heterochromatin model, in live mouse NIH3T3 cells. We demonstrated that the total density of heterochromatin (208 mg/ml) was only 1.53-fold higher than that of the surrounding euchromatic regions (136 mg/ml) while the DNA density of heterochromatin was 5.5- to 7.5-fold higher. We observed similar minor differences in density in typical facultative heterochromatin, the inactive human X chromosomes. This surprisingly small difference may be due to that nonnucleosomal materials (proteins/RNAs) (â¼120 mg/ml) are dominant in both chromatin regions. Monte Carlo simulation suggested that nonnucleosomal materials contribute to creating a moderate access barrier to heterochromatin, allowing minimal protein access to functional regions. Our OI-DIC imaging offers new insight into the live cellular environments.
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Heterocromatina/fisiología , Microscopía Fluorescente/métodos , Imagen Óptica/métodos , Animales , Recuento de Células , Cromatina/fisiología , Simulación por Computador , Histonas/metabolismo , Humanos , Ratones , Microscopía/métodos , Células 3T3 NIH , Gravedad EspecíficaRESUMEN
Son of sevenless (SOS) is a guanine nucleotide exchange factor that regulates cell behavior by activating the small GTPase RAS. Recent in vitro studies have suggested that an interaction between SOS and the GTP-bound active form of RAS generates a positive feedback loop that propagates RAS activation. However, it remains unclear how the multiple domains of SOS contribute to the regulation of the feedback loop in living cells. Here, we observed single molecules of SOS in living cells to analyze the kinetics and dynamics of SOS behavior. The results indicate that the histone fold and Grb2-binding domains of SOS concertedly produce an intermediate state of SOS on the cell surface. The fraction of the intermediated state was reduced in positive feedback mutants, suggesting that the feedback loop functions during the intermediate state. Translocation of RAF, recognizing the active form of RAS, to the cell surface was almost abolished in the positive feedback mutants. Thus, the concerted functions of multiple membrane-associating domains of SOS governed the positive feedback loop, which is crucial for cell fate decision regulated by RAS.
RESUMEN
Eukaryotic chromatin is a negatively charged long polymer composed of genomic DNA, histones, and various proteins. The charged property causes the chromatin structure to be dynamically changed. These dynamic changes are critical for genome functions such as gene expression because they directly govern the degree of DNA accessibility. Although the chromatin structure is not yet fully understood, currently increasing evidence suggests that chromatin has a dynamic liquid-like structure based on the 10-nm fiber but not the 30-nm fiber. This liquid-like property can drive the process of 'scanning and targeting genomic DNA,' which contributes to various genome functions including gene expression and DNA replication, repair, and recombination. Here, we discuss the liquid-like behavior of chromatin and its physical and biological relevance.
Asunto(s)
Cromatina/genética , ADN/genética , Regulación de la Expresión Génica/genética , Conformación de Ácido Nucleico , Cromatina/química , ADN/química , Reparación del ADN , Replicación del ADN/genética , Recombinación GenéticaRESUMEN
The phosphorylation cascade in the extracellular signal-regulated kinase (ERK) pathway is a versatile reaction network motif that can potentially act as a switch, oscillator or memory. Nevertheless, there is accumulating evidence that the phosphorylation response is mostly linear to extracellular signals in mammalian cells. Here we find that subsequent nuclear translocation gives rise to a switch-like increase in nuclear ERK concentration in response to signal input. The switch-like response disappears in the presence of ERK inhibitor, suggesting the existence of autoregulatory mechanisms for ERK nuclear translocation involved in conversion from a graded to a switch-like response. In vitro reconstruction of ERK nuclear translocation indicates that ERK-mediated phosphorylation of nucleoporins regulates ERK translocation. A mathematical model and knockdown experiments suggest a contribution of nucleoporins to regulation of the ERK nuclear translocation response. Taken together, this study provides evidence that nuclear translocation with autoregulatory mechanisms acts as a switch in ERK signalling.
Asunto(s)
Núcleo Celular/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Animales , Quinasas MAP Reguladas por Señal Extracelular/genética , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Células PC12 , Fosforilación/genética , Fosforilación/fisiología , Transporte de Proteínas/genética , Transporte de Proteínas/fisiología , Interferencia de ARN , RatasRESUMEN
Analyses of mice lacking glycosyltransferase have suggested that their pathological phenotypes are not attributable to the overall change of the sugar modification, but instead the result of changes of the glycan structures on a specific 'target' glycoprotein. Therefore, detecting or monitoring the glycosylation status of a specific protein in living cells is important, but no such methods are currently available. Here we demonstrate the detection of glycoforms of a specific glycoprotein using the fluorescence resonance energy transfer technique. Using model proteins, we detect characteristic fluorescence resonance energy transfer signals from the specific glycoform-bearing target glycoprotein. We also show that, upon insulin removal, sialylated glycoforms of green fluorescent protein-tagged GLUT4 seem to be internalized more slowly than non-sialylated GLUT4. This novel analytical imaging tool allows studying the roles of specific glycan modifications of a protein of interest.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas/química , Proteínas/metabolismo , Animales , Células CHO , Cricetinae , Transportador de Glucosa de Tipo 4/química , Transportador de Glucosa de Tipo 4/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Glicosilación , Proteínas Fluorescentes Verdes , Células HeLa , HumanosRESUMEN
In this review, we describe technology and use of single-fluorophore imaging and detection in living cells with regard to application in systems biology and medicine. Because all biological reactions occur under aqueous conditions, the realization of single-fluorophore imaging using an optical microscope has led to the direct observation of biological molecules at work. Today, we can observe single molecules in individual living cells and even higher multicellular organisms. Using single-molecule imaging, we can determine the absolute values of kinetic and dynamic parameters of molecular reactions as a whole and during fluctuations and distribution. In addition, identification of the coordinate of single molecules has enabled super-localization techniques to virtually improve spatial resolution of optical microscopy. Single-molecule detection that depends on point detection instead of imaging is also useful in detecting concentrations, diffusive movements, and molecular interactions in living cells, especially in the cytoplasm. The precise and absolute values of positional, kinetic, and dynamic parameters that are determined by single-molecule imaging and detection in living cells constitute valuable data on unitary biological reactions, because they are obtained without destroying the integrity of complex cellular systems. Moreover, most parameters that are determined by single-molecule measurements can be substituted directly into equations that describe kinetic and dynamic models in systems biology and medicine.
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Biología de Sistemas , Receptores ErbB/metabolismo , Colorantes Fluorescentes/química , Células HeLa , Humanos , Cinética , Microscopía Fluorescente , Modelos Biológicos , Simulación de Dinámica Molecular , Nanotecnología , FotonesRESUMEN
Nerve growth factor (NGF) is a secreted neurotrophin involved in the differentiation, growth, and maintenance of neurons. Here, we have used single-molecule imaging to characterize the behavior of Cy3-tagged NGF after binding to receptor complexes on the surfaces of PC12 cells. We show that NGF-receptor complexes have two distinct diffusive states, characterized as mobile and immobile phase. The transition between the two diffusive states occurred reversibly with duration times determined by a single rate limiting process. The abrupt transition to the immobile phase often occurred simultaneously with the clustering of NGF-receptor complexes. Immobilization depended on the phosphorylation of the TrkA NGF-receptor. Using dual-color imaging, it was demonstrated that the membrane recruitment of the intercellular signaling protein occurs with NGF-receptor complexes in the immobile phase indicating signal transduction occurs during this phase. Thus, NGF signaling is performed through a repetitive random process to induce transient formation of signaling complexes.