RESUMEN
In eukaryotic nuclei, chromatin loops mediated through cohesin are critical structures that regulate gene expression and DNA replication. Here, we demonstrate a new method to see endogenous genomic loci using synthetic zinc-finger proteins harboring repeat epitope tags (ZF probes) for signal amplification via binding of tag-specific intracellular antibodies, or frankenbodies, fused with fluorescent proteins. We achieve this in two steps: First, we develop an anti-FLAG frankenbody that can bind FLAG-tagged proteins in diverse live-cell environments. The anti-FLAG frankenbody complements the anti-HA frankenbody, enabling two-color signal amplification from FLAG- and HA-tagged proteins. Second, we develop a pair of cell-permeable ZF probes that specifically bind two endogenous chromatin loci predicted to be involved in chromatin looping. By coupling our anti-FLAG and anti-HA frankenbodies with FLAG- and HA-tagged ZF probes, we simultaneously see the dynamics of the two loci in single living cells. This shows a close association between the two loci in the majority of cells, but the loci markedly separate from the triggered degradation of the cohesin subunit RAD21. Our ability to image two endogenous genomic loci simultaneously in single living cells provides a proof of principle that ZF probes coupled with frankenbodies are useful new tools for exploring genome dynamics in multiple colors.
Asunto(s)
Núcleo Celular , Cromatina , Cromatina/genética , Epítopos , Genómica , ZincRESUMEN
When T cells recognize a peptide-major histocompatibility complex on antigen-presenting cells (APCs), T cell receptor microclusters (TCR-MCs) are generated and move to the center of the T cell-APC interface to form the central supramolecular activation cluster (cSMAC). cSMAC formation depends on stimulation strength and regulates T cell activation. We demonstrate that the dynein motor complex colocalized and coimmunoprecipitated with the TCR complex and that TCR-MCs moved along microtubules (MTs) toward the center of the immune synapse in a dynein-dependent manner to form cSMAC. MTs are located in close proximity to the plasma membrane at the activation site. TCR-MC velocity and cSMAC formation were impaired by dynein or MT inhibitors or by ablation of dynein expression. T cells with impaired cSMAC formation exhibited enhanced cellular activation including protein phosphorylation and interleukin-2 production. These results indicate that cSMAC formation by TCR-MC movement depends on dynein and MTs, and the movement regulates T cell activation.
Asunto(s)
Dineínas/inmunología , Sinapsis Inmunológicas/inmunología , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Animales , Membrana Celular/inmunología , Membrana Celular/metabolismo , Sinapsis Inmunológicas/ultraestructura , Ratones , Microscopía Electrónica , Unión Proteica , Transporte de Proteínas , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismoRESUMEN
In eukaryotic cells, post-translational histone modifications have an important role in gene regulation. Starting with early work on histone acetylation, a variety of residue-specific modifications have now been linked to RNA polymerase II (RNAP2) activity, but it remains unclear if these markers are active regulators of transcription or just passive byproducts. This is because studies have traditionally relied on fixed cell populations, meaning temporal resolution is limited to minutes at best, and correlated factors may not actually be present in the same cell at the same time. Complementary approaches are therefore needed to probe the dynamic interplay of histone modifications and RNAP2 with higher temporal resolution in single living cells. Here we address this problem by developing a system to track residue-specific histone modifications and RNAP2 phosphorylation in living cells by fluorescence microscopy. This increases temporal resolution to the tens-of-seconds range. Our single-cell analysis reveals histone H3 lysine-27 acetylation at a gene locus can alter downstream transcription kinetics by as much as 50%, affecting two temporally separate events. First acetylation enhances the search kinetics of transcriptional activators, and later the acetylation accelerates the transition of RNAP2 from initiation to elongation. Signatures of the latter can be found genome-wide using chromatin immunoprecipitation followed by sequencing. We argue that this regulation leads to a robust and potentially tunable transcriptional response.
Asunto(s)
Histonas/química , Histonas/metabolismo , ARN Polimerasa II/metabolismo , Análisis de la Célula Individual , Transcripción Genética , Acetilación , Animales , Línea Celular Tumoral , Supervivencia Celular , Inmunoprecipitación de Cromatina , Activación Enzimática , Genoma/genética , Cinética , Lisina/metabolismo , Ratones , Microscopía Fluorescente , Fosforilación , Factores de Tiempo , Elongación de la Transcripción Genética , Iniciación de la Transcripción GenéticaRESUMEN
T cell activation is positively and negatively regulated by a pair of costimulatory receptors, CD28 and CTLA-4, respectively. Because these receptors share common ligands, CD80 and CD86, the expression and behavior of CTLA-4 is critical for T cell costimulation regulation. However, in vivo blocking of CD28-mediated costimulation by CTLA-4 and its mechanisms still remain elusive. Here, we demonstrate the dynamic behavior of CTLA-4 in its real-time competition with CD28 at the central-supramolecular activation cluster (cSMAC), resulting in the dislocalization of protein kinase C-θ and CARMA1 scaffolding protein. CTLA-4 translocation to the T cell receptor microclusters and the cSMAC is tightly regulated by its ectodomain size, and its accumulation at the cSMAC is required for its inhibitory function. The CTLA-4-mediated suppression was demonstrated by the in vitro anergy induction in regulatory T cells constitutively expressing CTLA-4. These results show the dynamic mechanism of CTLA-4-mediated T cell suppression at the cSMAC.
Asunto(s)
Antígenos CD/fisiología , Activación de Linfocitos , Linfocitos T/inmunología , Animales , Proteínas Adaptadoras de Señalización CARD/fisiología , Antígenos CD28/fisiología , Complejo CD3/fisiología , Antígeno CTLA-4 , Células Cultivadas , Tolerancia Inmunológica , Isoenzimas/fisiología , Ratones , Proteína Quinasa C/fisiología , Proteína Quinasa C-theta , Linfocitos T Reguladores/fisiologíaRESUMEN
T cell activation is mediated by microclusters (MCs) containing T cell receptors (TCRs), kinases, and adaptors. Although TCR MCs translocate to form a central supramolecular activation cluster (cSMAC) of the immunological synapse at the interface of a T cell and an antigen-presenting cell, the role of MC translocation in T cell signaling remains unclear. Here, we found that the accumulation of MCs at cSMAC was important for T cell costimulation. Costimulatory receptor CD28 was initially recruited coordinately with TCR to MCs, and its signals were mediated through the assembly with the kinase PKCtheta. The accumulation of MCs at the cSMAC was accompanied by the segregation of CD28 from the TCR, which resulted in the translocation of both CD28 and PKCtheta to a spatially unique subregion of cSMAC. Thus, costimulation is mediated by the generation of a unique costimulatory compartment in the cSMAC via the dynamic regulation of MC translocation.
Asunto(s)
Antígenos CD28/metabolismo , Proteína Quinasa C/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Linfocitos T/inmunología , Animales , Células Cultivadas , Células Dendríticas/inmunología , Ratones , Ratones Noqueados , Ratones Transgénicos , Linfocitos T/metabolismoRESUMEN
RNA microarray analyses revealed that nuclear actin activated many human transcription factor genes including OCT4, which is required for gene reprogramming. Oct4 is known to be activated by nuclear actin in Xenopus oocytes. Our findings imply that this process of OCT4 activation is conserved in vertebrates and among cell types and could be used for gene reprogramming of human cells.
Asunto(s)
Actinas/metabolismo , Núcleo Celular/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Activación Transcripcional , Animales , Células HeLa , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Transcripción Genética/genéticaRESUMEN
Vesnarinone is a synthetic quinolinone derivative used in the treatment of cardiac failure and cancer. It is also known to cause agranulocytosis as a side effect, which restricts its use, although the mechanism underlying agranulocytosis is not well understood. Here, we show that vesnarinone binds to valosin-containing protein (VCP), which interacts with polyubiquitinated proteins and is essential for the degradation of IκBα to activate nuclear factor (NF)κB. We show that vesnarinone impairs the degradation of IκBα, and that the impairment of the degradation of IκBα is the result of the inhibition of the interaction between VCP and the 26S proteasome by vesnarinone. These results suggest that vesnarinone suppresses NFκB activation by inhibiting the VCP-dependent degradation of polyubiquitinated IκBα, resulting in the suppression of tumor necrosis factor-α mRNA expression.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Proteínas de Ciclo Celular/antagonistas & inhibidores , Quinolinas/farmacología , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/biosíntesis , Factor de Necrosis Tumoral alfa/genética , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Células HEK293 , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Inhibidor NF-kappaB alfa , FN-kappa B/genética , FN-kappa B/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Pirazinas , ARN Mensajero/genética , Factor de Necrosis Tumoral alfa/metabolismo , Proteína que Contiene ValosinaRESUMEN
Zinc is an essential trace element required for enzymatic activity and for maintaining the conformation of many transcription factors; thus, zinc homeostasis is tightly regulated. Although zinc affects several signaling molecules and may act as a neurotransmitter, it remains unknown whether zinc acts as an intracellular second messenger capable of transducing extracellular stimuli into intracellular signaling events. In this study, we report that the cross-linking of the high affinity immunoglobin E receptor (Fcepsilon receptor I [FcepsilonRI]) induced a release of free zinc from the perinuclear area, including the endoplasmic reticulum in mast cells, a phenomenon we call the zinc wave. The zinc wave was dependent on calcium influx and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase activation. The results suggest that the zinc wave is involved in intracellular signaling events, at least in part by modulating the duration and strength of FcepsilonRI-mediated signaling. Collectively, our findings indicate that zinc is a novel intracellular second messenger.
Asunto(s)
Líquido Intracelular/fisiología , Sistemas de Mensajero Secundario/fisiología , Zinc/fisiología , Animales , Células Cultivadas , Líquido Intracelular/enzimología , MAP Quinasa Quinasa Quinasa 3/metabolismo , Mastocitos/enzimología , Mastocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Receptores de IgE/metabolismo , Receptores de IgE/fisiologíaRESUMEN
In eukaryotic nuclei, most genes are transcribed by RNA polymerase II (RNAP2), whose regulation is a key to understanding the genome and cell function. RNAP2 has a long heptapeptide repeat (Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7), and Ser2 is phosphorylated on an elongation form. To detect RNAP2 Ser2 phosphorylation (RNAP2 Ser2ph) in living cells, we developed a genetically encoded modification-specific intracellular antibody (mintbody) probe. The RNAP2 Ser2ph-mintbody exhibited numerous foci, possibly representing transcription "factories," and foci were diminished during mitosis and in a Ser2 kinase inhibitor. An in vitro binding assay using phosphopeptides confirmed the mintbody's specificity. RNAP2 Ser2ph-mintbody foci were colocalized with proteins associated with elongating RNAP2 compared with factors involved in the initiation. These results support the view that mintbody localization represents the sites of RNAP2 Ser2ph in living cells. RNAP2 Ser2ph-mintbody foci showed constrained diffusional motion like chromatin, but they were more mobile than DNA replication domains and p300-enriched foci, suggesting that the elongating RNAP2 complexes are separated from more confined chromatin domains.
Asunto(s)
Imagen Molecular , ARN Polimerasa II/metabolismo , Sondas ARN/metabolismo , Transcripción Genética , Núcleo Celular/metabolismo , Supervivencia Celular , Células HeLa , Humanos , Interfase , Fosforilación , Fosfoserina/metabolismoRESUMEN
The nucleolus is the site of ribosome assembly and formed through liquid-liquid phase separation. Multiple ribosomal DNA (rDNA) arrays are bundled in the nucleolus, but the underlying mechanism and significance are unknown. In the present study, we performed high-content screening followed by image profiling with the wndchrm machine learning algorithm. We revealed that cells lacking a specific 60S ribosomal protein set exhibited common nucleolar disintegration. The depletion of RPL5 (also known as uL18), the liquid-liquid phase separation facilitator, was most effective, and resulted in an enlarged and un-separated sub-nucleolar compartment. Single-molecule tracking analysis revealed less-constrained mobility of its components. rDNA arrays were also unbundled. These results were recapitulated by a coarse-grained molecular dynamics model. Transcription and processing of ribosomal RNA were repressed in these aberrant nucleoli. Consistently, the nucleoli were disordered in peripheral blood cells from a Diamond-Blackfan anemia patient harboring a heterozygous, large deletion in RPL5 Our combinatorial analyses newly define the role of RPL5 in rDNA array bundling and the biophysical properties of the nucleolus, which may contribute to the etiology of ribosomopathy.
Asunto(s)
Nucléolo Celular , Proteínas Ribosómicas , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , ADN Ribosómico/genética , ADN Ribosómico/metabolismo , Humanos , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismoRESUMEN
mRNA transport and local translation in dendrites play key roles in use-dependent synaptic modification and in higher-order brain functions. RNG105, an RNA-binding protein, has previously been identified as a component of RNA granules that mediate dendritic mRNA localization and local translation. Here, we demonstrate that RNG105 knock-out in mice reduces the dendritic localization of mRNAs for Na+/K+ ATPase (NKA) subunit isoforms (i.e., α3, FXYD1, FXYD6, and FXYD7). The loss of dendritic mRNA localization is accompanied by the loss of function of NKA in dendrites without affecting the NKA function in the soma. Furthermore, we show that RNG105 deficiency affects the formation and maintenance of synapses and neuronal networks. These phenotypes are partly explained by an inhibition of NKA, which is known to influence synaptic functions as well as susceptibility to neurotoxicity. The present study first demonstrates the in vivo role of RNG105 in the dendritic localization of mRNAs and uncovers a novel link between dendritic mRNA localization and the development and maintenance of functional networks.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Dendritas/metabolismo , Red Nerviosa/metabolismo , ARN Mensajero/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Sinapsis/metabolismo , Análisis de Varianza , Animales , Transporte Biológico/fisiología , Proteínas de Ciclo Celular/genética , Células Cultivadas , Corteza Cerebral/metabolismo , Dendritas/genética , Inmunohistoquímica , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones , Ratones Noqueados , Neuronas/metabolismo , Interferencia de ARN , ARN Mensajero/genética , ATPasa Intercambiadora de Sodio-Potasio/genéticaRESUMEN
RNA granules mediate the transport and local translation of their mRNA cargoes, which regulate cellular processes such as stress response and neuronal synaptic plasticity. RNA granules contain specific RNA-binding proteins, including RNA granule protein 105 (RNG105), which is likely to participate in the transport and translation of mRNAs. In the present report, an RNG105 paralog, RNG140 is described. A homolog of RNG105/RNG140 is found in insects, echinoderms, and urochordates, whereas vertebrates have both of the two genes. RNG140 and RNG105 are similar in that both bind to mRNAs and inhibit translation in vitro, induce the formation of RNA granules, are most highly expressed in the brain, and are localized to dendritic RNA granules, part of which are accumulated at postsynapses. However, they differ in several characteristics; RNG105 is highly expressed in embryonic brains, whereas RNG140 is highly expressed in adult brains. Furthermore, the granules where RNG105 or RNG140 is localized are distinct RNA granules in both cultured cells and neuronal dendrites. Thus, RNG140 is an RNA-binding protein that shows different expression and localization patterns from RNG105. Knockdown experiments in cultured neurons also are performed, which demonstrate that suppression of RNG140 or RNG105 reduces dendrite length and spine density. Knockdown effects of RNG140 were not rescued by RNG105, and vise versa, suggesting distinct roles of RNG105 and RNG140. These results suggest that RNG140 has roles in the maintenance of the dendritic structure in the adult vertebrate brain through localizing to a kind of RNA granules that are distinct from RNG105-containing granules.
Asunto(s)
Encéfalo/metabolismo , Gránulos Citoplasmáticos/metabolismo , Proteínas del Tejido Nervioso/fisiología , Neuronas/metabolismo , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Proteínas de Pez Cebra/fisiología , Secuencia de Aminoácidos , Animales , Dendritas/metabolismo , Humanos , Ratones , Microscopía Fluorescente/métodos , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Unión Proteica , Estructura Terciaria de Proteína , Interferencia de ARN , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/fisiología , Ratas , Homología de Secuencia de Aminoácido , Proteínas de Pez Cebra/químicaRESUMEN
We describe a simple illumination method of fluorescence microscopy for molecular imaging. Illumination by a highly inclined and thin beam increases image intensity and decreases background intensity, yielding a signal/background ratio about eightfold greater than that of epi-illumination. A high ratio yielded clear single-molecule images and three-dimensional images using cultured mammalian cells, enabling one to visualize and quantify molecular dynamics, interactions and kinetics in cells for molecular systems biology.
Asunto(s)
Biopolímeros/metabolismo , Aumento de la Imagen/métodos , Iluminación/métodos , Microscopía Fluorescente/métodos , Mapeo de Interacción de Proteínas , Biología de Sistemas/métodosRESUMEN
The microtubule-organizing centre (MTOC) is repositioned to the centre of the contacted cell surface, the immunological synapse, during T cell activation. However, our understanding of its molecular mechanism remains limited. Here, we found that the microtubule plus-end tracking cytoplasmic linker protein 170 (CLIP-170) plays a novel role in MTOC repositioning using fluorescence imaging. Inhibition of CLIP-170 phosphorylation impaired both MTOC repositioning and interleukin-2 (IL-2) expression. T cell stimulation induced some fraction of dynein to colocalise with CLIP-170 and undergo plus-end tracking. Concurrently, it increased dynein in minus-end-directed movement. It also increased dynein relocation to the centre of the contact surface. Dynein not colocalised with CLIP-170 showed both an immobile state and minus-end-directed movement at a velocity in good agreement with the velocity of MTOC repositioning, which suggests that dynein at the immunological synapse may pull the microtubules and the MTOC. Although CLIP-170 is phosphorylated by AMP-activated protein kinase (AMPK) irrespective of stimulation, phosphorylated CLIP-170 is essential for dynein recruitment to plus-end tracking and for dynein relocation. This indicates that dynein relocation results from coexistence of plus-end- and minus-end-directed translocation. In conclusion, CLIP-170 plays an indispensable role in MTOC repositioning and full activation of T cells by regulating dynein localisation.
Asunto(s)
Dineínas/metabolismo , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Proteínas Asociadas a Microtúbulos/genética , Centro Organizador de los Microtúbulos/inmunología , Centro Organizador de los Microtúbulos/metabolismo , Proteínas de Neoplasias/genética , Linfocitos T/inmunología , Linfocitos T/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Humanos , Sinapsis Inmunológicas , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Transporte de ProteínasRESUMEN
T cell receptors (TCR) are activated by a specific antigen and interact with other signaling molecules, such as kinases and adaptors. Aiming at analyzing precisely the dynamic process of T cell signaling, we used a combined system of a planar bilayer and TIRF microscopy. This system allowed us to observe the T cell activation process from the initial cell-bilayer contact (time 0). Our observation revealed that microclusters with TCR were generated at the initial contact to gather into central supramolecular cluster, the immunological synapse, which was believed to be responsible for T cell receptor signaling. Furthermore the microclusters were generated continuously at the periphery even at the sustained state and they migrated toward the central cluster. These results suggested the important role of microclusters in T cell activation.
Asunto(s)
Diagnóstico por Imagen/métodos , Técnicas de Diagnóstico Molecular/métodos , Receptores de Antígenos de Linfocitos T/fisiología , Transducción de Señal/fisiología , Animales , Técnicas In Vitro , Membrana Dobles de Lípidos , Microscopía FluorescenteRESUMEN
The dynamic properties of molecules in living cells are attracting increasing interest. We propose a new method, moving subtrajectory analysis using single-molecule tracking, and demonstrate its utility in the spatiotemporal quantification of not only dynamics but also the kinetics of interactions using single-color images. Combining this technique with three-color simultaneous single-molecule imaging, we quantified the dynamics and kinetics of molecules in spatial relation to T cell receptor (TCR) microclusters, which trigger TCR signaling. CD3ε, a component of the TCR/CD3 complex, and CD45, a phosphatase positively and negatively regulating signaling, were each found in two mobility states: faster (associated) and slower (dissociated) states. Dynamics analysis suggests that the microclusters are loosely composed of heterogeneous nanoregions, possibly surrounded by a weak barrier. Kinetics analysis quantified the association and dissociation rates of interactions with the microclusters. The associations of both CD3ε and CD45 were single-step processes. In contrast, their dissociations were each composed of two components, indicating transient and stable associated states. Inside the microclusters, the association was accelerated, and the stable association was increased. Only CD45 showed acceleration of association at the microcluster boundary, suggesting specific affinity on the boundary. Thus, this method is an innovative and versatile tool for spatiotemporal quantification.
Asunto(s)
Activación de Linfocitos , Microscopía Fluorescente/métodos , Imagen Individual de Molécula/métodos , Linfocitos T/inmunología , Complejo CD3/análisis , Humanos , Células Jurkat , Antígenos Comunes de Leucocito/análisis , Receptores de Antígenos de Linfocitos T/análisis , Análisis Espacio-TemporalRESUMEN
Activation of NF-κB transcription factor is strictly regulated to prevent excessive inflammatory responses leading to immunopathology. However, it still remains unclear how NF-κB activation is negatively controlled. The PDZ-LIM domain-containing protein PDLIM2 is a nuclear ubiquitin E3 ligase targeting the p65 subunit of NF-κB for degradation, thus terminating NF-κB-mediated inflammation. Using yeast two-hybrid screening, we sought to isolate PDLIM2-interacting proteins that are critical for suppressing NF-κB signaling. Here we identified MKRN2, a RING finger domain-containing protein that belongs to the makorin ring finger protein gene family, as a novel p65 ubiquitin E3 ligase. MKRN2 bound to p65 and promoted the polyubiquitination and proteasome-dependent degradation of p65 through the MKRN2 RING finger domain, thereby suppressing p65-mediated NF-κB transactivation. Notably, MKRN2 and PDLIM2 synergistically promote polyubiquitination and degradation of p65. Consistently, MKRN2 knockdown in dendritic cells resulted in larger amounts of nuclear p65 and augmented production of proinflammatory cytokines in responses to innate stimuli. These results delineate a novel role of MKRN2 in negatively regulating NF-κB-mediated inflammatory responses, cooperatively with PDLIM2.
Asunto(s)
Inflamación/metabolismo , Inflamación/patología , Subunidades de Proteína/metabolismo , Ribonucleoproteínas/metabolismo , Factor de Transcripción ReIA/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Línea Celular , Humanos , Proteínas con Dominio LIM/metabolismo , Ratones , Poliubiquitina/metabolismo , Unión Proteica , Proteolisis , Dominios RING Finger , Ribonucleoproteínas/química , Ribonucleoproteínas/deficiencia , Transducción de Señal , UbiquitinaciónRESUMEN
Inhibitory PAS domain protein (IPAS) is a dual function protein acting as a transcriptional repressor and as a pro-apoptotic protein. Simultaneous dual-color single-molecule imaging of EGFP-IPAS coexpressed with Mit-TagRFP-T in living HeLa cells revealed that fraction of EGFP-IPAS was arrested in the nucleus and on mitochondria. Transiently expressed Cerulean-IPAS in HEK293T cells was present in nuclear speckles when coexpressed with Citrine-HIF-1α or Citrine-HLF. Fluorescence lifetime imaging microscopy (FLIM) analysis of Citrine-IPAS-Cerulean in living CHO-K1 cells clarified the presence of intramolecular FRET. Reduced lifetimes of the donor were partially restored by coexpression of HIF-1α or Bcl-xL, binding proteins of IPAS in the nucleus and mitochondria, respectively. This alteration in lifetimes demonstrates that conformational changes occurred in IPAS by their binding.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Proteína bcl-X/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/química , Sitios de Unión , Células CHO , Cricetulus , Células HEK293 , Células HeLa , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/química , Proteínas Represoras , Proteína bcl-X/químicaRESUMEN
Local translation in neuronal dendrites is an important basis for long-term synaptic plasticity, and RNA granules in the dendrites are involved in the local translation. Here, we identify RNG105 (RNA granule protein 105), a novel RNA-binding protein, as a component of the RNA granules in dendrites of hippocampal neurons. The RNG105-localizing RNA granules contain mRNAs, the translational products of which play key roles in synaptic plasticity. RNG105 has an ability to repress translation both in vitro and in vivo, consistent with the finding that the RNA granule is translationally arrested in the basal conditions. Dissociation of RNG105 from the RNA granules is induced by BDNF, a growth factor responsible for synaptic plasticity. The RNG105 dissociation is coincident with the induction of local translation near the granules. These findings suggest that RNG105 is a translational repressor in the RNA granules and provide insight into the link between RNG105 dynamics and local translational regulation.
Asunto(s)
Encéfalo/citología , Regulación de la Expresión Génica/fisiología , Neuronas/metabolismo , Biosíntesis de Proteínas , Proteínas de Unión al ARN/fisiología , ARN/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Encéfalo/metabolismo , Factor Neurotrófico Derivado del Encéfalo/farmacología , Proteínas de Ciclo Celular , Células Cultivadas , Clonación Molecular/métodos , Proteínas de Unión al ADN/metabolismo , Dendritas/metabolismo , Diagnóstico por Imagen/métodos , Homólogo 4 de la Proteína Discs Large , Expresión Génica/fisiología , Proteínas Fluorescentes Verdes/biosíntesis , Humanos , Inmunohistoquímica/métodos , Inmunoprecipitación/métodos , Hibridación in Situ/métodos , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Metionina/metabolismo , Microscopía Inmunoelectrónica/métodos , Proteínas Asociadas a Microtúbulos/metabolismo , Neuronas/ultraestructura , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/ultraestructura , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Proteína S6 Ribosómica/metabolismo , Isótopos de Azufre/metabolismo , Testículo/metabolismo , Factores de Tiempo , Factores de Transcripción/metabolismo , Transfección/métodos , Xenopus , Proteínas de Xenopus/genética , Proteínas de Xenopus/fisiologíaRESUMEN
The nano-kinetics movement of a single DNA molecule was visualized by a newly developed video-microscope system with an optical fiber, called a "pin-fiber video scope". The stretching and shrinking motion was clearly observed, and its kinetics was analyzed by numerical calculations. The new video-microscope system has a potential to analyze the nano-kinetics of a molecule.