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1.
Proc Natl Acad Sci U S A ; 114(10): E1875-E1884, 2017 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-28223507

RESUMO

RNA-protein interactions are essential for proper gene expression regulation, particularly in neurons with unique spatial constraints. Currently, these interactions are defined biochemically, but a method is needed to evaluate them quantitatively within morphological context. Colocalization of two-color labels using wide-field microscopy is a method to infer these interactions. However, because of chromatic aberrations in the objective lens, this approach lacks the resolution to determine whether two molecules are physically in contact or simply nearby by chance. Here, we developed a robust super registration methodology that corrected the chromatic aberration across the entire image field to within 10 nm, which is capable of determining whether two molecules are physically interacting or simply in proximity by random chance. We applied this approach to image single-molecule FISH in combination with immunofluorescence (smFISH-IF) and determined whether the association between an mRNA and binding protein(s) within a neuron was significant or accidental. We evaluated several mRNA-binding proteins identified from RNA pulldown assays to determine which of these exhibit bona fide interactions. Surprisingly, many known mRNA-binding proteins did not bind the mRNA in situ, indicating that adventitious interactions are significant using existing technology. This method provides an ability to evaluate two-color registration compatible with the scale of molecular interactions.


Assuntos
Neurônios/química , Proteínas/isolamento & purificação , RNA Mensageiro/isolamento & purificação , Proteínas de Ligação a RNA/isolamento & purificação , Corantes Fluorescentes/química , Regulação da Expressão Gênica/genética , Hibridização in Situ Fluorescente/métodos , Neurônios/ultraestrutura , Proteínas/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Imagem Individual de Molécula/métodos
2.
Microsc Microanal ; 22(Suppl 3): 2064-2065, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27516727

RESUMO

A challenge in any imaging laboratory, especially one that uses modern techniques, is to achieve a sustainable and productive balance between using open source and commercial software to perform quantitative image acquisition, analysis and visualization. In addition to considering the expense of software licensing, one must consider factors such as the quality and usefulness of the software's support, training and documentation. Also, one must consider the reproducibility with which multiple people generate results using the same software to perform the same analysis, how one may distribute their methods to the community using the software and the potential for achieving automation to improve productivity.

3.
Genes Dev ; 26(17): 1885-90, 2012 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-22948660

RESUMO

Directed cell motility is at the basis of biological phenomena such as development, wound healing, and metastasis. It has been shown that substrate attachments mediate motility by coupling the cell's cytoskeleton with force generation. However, it has been unclear how the persistence of cell directionality is facilitated. We show that mRNA localization plays an important role in this process, but the mechanism of action is still unknown. In this study, we show that the zipcode-binding protein 1 transports ß-actin mRNA to the focal adhesion compartment, where it dwells for minutes, suggesting a means for associating its localization with motility through the formation of stable connections between adhesions and newly synthesized actin filaments. In order to demonstrate this, we developed an approach for assessing the functional consequences of ß-actin mRNA and protein localization by tethering the mRNA to a specific location-in this case, the focal adhesion complex. This approach will have a significant impact on cell biology because it is now possible to forcibly direct any mRNA and its cognate protein to specific locations in the cell. This will reveal the importance of localized protein translation on various cellular processes.


Assuntos
Actinas/metabolismo , Compartimento Celular , Movimento Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Adesões Focais/fisiologia , RNA Mensageiro/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Glicoproteínas/genética , Glicoproteínas/metabolismo , Camundongos , Camundongos Knockout , Proteínas de Ligação a RNA
4.
Nat Protoc ; 7(2): 408-19, 2012 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-22301778

RESUMO

Fluorescent in situ hybridization (FISH) allows the quantification of single mRNAs in budding yeast using fluorescently labeled single-stranded DNA probes, a wide-field epifluorescence microscope and a spot-detection algorithm. Fixed yeast cells are attached to coverslips and hybridized with a mixture of FISH probes, each conjugated to several fluorescent dyes. Images of cells are acquired in 3D and maximally projected for single-molecule analysis. Diffraction-limited labeled mRNAs are observed as bright fluorescent spots and can be quantified using a spot-detection algorithm. FISH preserves the spatial distribution of cellular RNA distribution within the cell and the stochastic fluctuations in individual cells that can lead to phenotypic differences within a clonal population. This information, however, is lost if the RNA content is measured on a population of cells by using reverse transcriptase PCR, microarrays or high-throughput sequencing. The FISH procedure and image acquisition described here can be completed in 3 d.


Assuntos
Hibridização in Situ Fluorescente/métodos , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Algoritmos , Elementos Antissenso (Genética)
5.
Cancer Res ; 70(13): 5348-57, 2010 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-20570902

RESUMO

Novel imaging of active transcription sites in interphase nuclei of intestinal epithelial cells in situ showed that key genes associated with Wnt and Notch signaling were dynamically regulated as the cells underwent normal maturation during their migration along the mouse crypt-villus axis (CVA). However, oscillating patterns of activation of these genes were displaced along this axis in the histologically normal intestinal mucosa of Apc(1638N/+) mice before tumor development. Gene expression profiling then showed that the normal reprogramming of cells along the CVA was dampened in the Apc(1638N/+) mice, with an overrepresentation of c-myc target genes among those loci affected in the mutant mice. Moreover, in the Apc(1638N/+) mice, there was a perturbed pattern of expression of lineage-specific markers along the CVA consistent with transcription site repression of the Math1 gene, and genes encoding enzymes of every step of the tricarboxylic acid cycle were downregulated in the crypt of Apc(1638N/+) mice compared with WT, but not in the villus. These changes may alter energy metabolism and generate a pseudohypoxic state, suggested by elevated expression of Hif1alpha and its target genes. Thus, although intestinal tumors develop in Apc(1638N/+) mice on focal loss or inactivation of the WT allele, our results show that in the Apc(1638N/+) mouse, inheritance of only a single WT Apc allele perturbs the dynamic and complex reprogramming underlying normal cell maturation, which links epithelial function and homeostasis with architectural organization of the intestine.


Assuntos
Genes APC , Neoplasias Intestinais/genética , Intestinos/fisiologia , Alelos , Animais , Linhagem da Célula , Perfilação da Expressão Gênica , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/fisiologia , Neoplasias Intestinais/metabolismo , Neoplasias Intestinais/patologia , Intestinos/citologia , Camundongos , Sítio de Iniciação de Transcrição
6.
J Cell Physiol ; 218(3): 638-42, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19034928

RESUMO

In stimulating maturation of colonic carcinoma cells, the short chain fatty acid butyrate, and 1alpha,25-dihydroxyvitamin D(3), were shown to attenuate transcription of the cyclin D1 gene, giving rise to truncated transcripts of this locus. Moreover, a sequence which is highly conserved in the human, mouse, rat, and dog genome was found in the 4 kb long intron 3 of the human cyclin D1 gene, and is capable of forming a hairpin structure similar to that of microRNA precursors. The expression of this sequence is also decreased by the attenuation. Thus, the transcriptional attenuation at the cyclin D1 locus not only down-regulates the expression of this key gene in mucosal cell maturation and tumorigenesis, but may also abrogate the generation of a molecule that encompasses this conserved sequence in cyclin D1 intron 3.


Assuntos
Butiratos/farmacologia , Colecalciferol/farmacologia , Neoplasias do Colo/genética , Ciclina D1/genética , Transcrição Gênica/efeitos dos fármacos , Animais , Sequência de Bases , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Hibridização in Situ Fluorescente , Íntrons/genética , Camundongos , Dados de Sequência Molecular , Conformação de Ácido Nucleico
7.
Nat Protoc ; 3(11): 1809-14, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18974739

RESUMO

Power output of light bulbs changes over time and the total energy delivered will depend on the optical beam path of the microscope, filter sets and objectives used, thus making comparison between experiments performed on different microscopes complicated. Using a thermocoupled power meter, it is possible to measure the exact amount of light applied to a specimen in fluorescence microscopy, regardless of the light source, as the light power measured can be translated into a power density at the sample. This widely used and simple tool forms the basis of a new degree of calibration precision and comparability of results among experiments and setups. Here we describe an easy-to-follow protocol that allows researchers to precisely estimate excitation intensities in the object plane, using commercially available opto-mechanical components. The total duration of this protocol for one objective and six filter cubes is 75 min including start-up time for the lamp.


Assuntos
Luz , Microscopia/métodos , Calibragem , Microscopia/instrumentação , Microscopia/normas
8.
J Cell Biochem ; 102(3): 609-17, 2007 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-17849428

RESUMO

We have previously developed technology for multiplexing probes for the detection of transcription of many genes simultaneously within single cells. This has allowed us to determine the spatial localization of multiple genes with respect to each other in the nucleus, and ultimately the expression profile of the cell with respect to surrounding cells in a tissue. Six parameters of transcriptional organization in individual cells from culture and tissue were used to characterize significant differences in intracellular and intercellular expression patterns while preserving cellular morphology and histological context. We found that, unlike yeast, mammalian expression is excluded from the periphery and in addition, a subtle but complex organization underlies the transcriptional activity of these cells, both intra- and intercellularly. The approach has sufficient spatial resolution to be applied to the detection of chromosomal translocations or the identification of cancer cells.


Assuntos
Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Biologia Molecular/métodos , Transcrição Gênica , Linhagem Celular Tumoral , Cromatina/metabolismo , Análise por Conglomerados , Humanos , Hibridização in Situ Fluorescente , Microscopia , Modelos Biológicos , Modelos Genéticos , Translocação Genética
9.
Nat Struct Mol Biol ; 14(9): 796-806, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17676063

RESUMO

We imaged transcription in living cells using a locus-specific reporter system, which allowed precise, single-cell kinetic measurements of promoter binding, initiation and elongation. Photobleaching of fluorescent RNA polymerase II revealed several kinetically distinct populations of the enzyme interacting with a specific gene. Photobleaching and photoactivation of fluorescent MS2 proteins used to label nascent messenger RNAs provided sensitive elongation measurements. A mechanistic kinetic model that fits our data was validated using specific inhibitors. Polymerases elongated at 4.3 kilobases min(-1), much faster than previously documented, and entered a paused state for unexpectedly long times. Transcription onset was inefficient, with only 1% of polymerase-gene interactions leading to completion of an mRNA. Our systems approach, quantifying both polymerase and mRNA kinetics on a defined DNA template in vivo with high temporal resolution, opens new avenues for studying regulation of transcriptional processes in vivo.


Assuntos
RNA Polimerase II/genética , Transcrição Gênica , Sequência de Bases , Linhagem Celular Tumoral , Primers do DNA , Humanos , Hibridização in Situ Fluorescente , Cinética , Fosforilação , Fotoquímica , RNA Polimerase II/metabolismo , Transcrição Gênica/efeitos dos fármacos
10.
J Cell Biol ; 175(1): 67-76, 2006 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-17030983

RESUMO

The role of mRNA localization is presumably to effect cell asymmetry by synthesizing proteins in specific cellular compartments. However, protein synthesis has never been directly demonstrated at the sites of mRNA localization. To address this, we developed a live cell method for imaging translation of beta-actin mRNA. Constructs coding for beta-actin, containing tetracysteine motifs, were transfected into C2C12 cells, and sites of nascent polypeptide chains were detected using the biarsenial dyes FlAsH and ReAsH, a technique we call translation site imaging. These sites colocalized with beta-actin mRNA at the leading edge of motile myoblasts, confirming that they were translating. beta-Actin mRNA lacking the sequence (zipcode) that localizes the mRNA to the cell periphery, eliminated the translation there. A pulse-chase experiment on living cells showed that the recently synthesized protein correlated spatially with the sites of its translation. Additionally, localization of beta-actin mRNA and translation activity was enhanced at cell contacts and facilitated the formation of intercellular junctions.


Assuntos
Actinas/biossíntese , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/metabolismo , Actinas/genética , Animais , Caderinas/metabolismo , Compartimento Celular , Linhagem Celular , Hibridização in Situ Fluorescente , Indicadores e Reagentes/análise , Junções Intercelulares/metabolismo , Camundongos , Microscopia de Fluorescência , Polirribossomos/fisiologia , RNA Mensageiro/análise , Proteínas Recombinantes de Fusão/análise
11.
Curr Biol ; 16(10): 1018-25, 2006 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-16713960

RESUMO

It has not been possible to view the transcriptional activity of a single gene within a living eukaryotic cell. It is therefore unclear how long and how frequently a gene is actively transcribed, how this is modulated during differentiation, and how transcriptional events are dynamically coordinated in cell populations. By means of an in vivo RNA detection technique , we have directly visualized transcription of an endogenous developmental gene. We found discrete "pulses" of gene activity that turn on and off at irregular intervals. Surprisingly, the length and height of these pulses were consistent throughout development. However, there was strong developmental variation in the proportion of cells recruited to the expressing pool. Cells were more likely to re-express than to initiate new expression, indicating that we directly observe a transcriptional memory. In addition, we used a clustering algorithm to reveal synchronous transcription initiation in neighboring cells. This study represents the first direct visualization of transcriptional pulsing in eukaryotes. Discontinuity of transcription may allow greater flexibility in the gene-expression decisions of a cell.


Assuntos
Dictyostelium/metabolismo , Genes Controladores do Desenvolvimento , Transcrição Gênica , Animais , Diferenciação Celular/fisiologia , Dictyostelium/citologia , Células Eucarióticas/citologia , Células Eucarióticas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Cinética , RNA Mensageiro/biossíntese
12.
Nat Methods ; 2(9): 663-5, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16118636

RESUMO

We developed a robust multiplex fluorescent in situ hybridization (FISH) technique in archival formalin-fixed, paraffin-embedded (FFPE) human tissue sections while preserving the microanatomical context. This identifies single-cell gene expression patterns by probing multiple, unique nascent RNA transcripts and yields predictive quantitative gene expression signatures.


Assuntos
Biomarcadores Tumorais/metabolismo , Perfilação da Expressão Gênica/métodos , Hibridização in Situ Fluorescente/métodos , Proteínas de Neoplasias/metabolismo , Inclusão em Parafina/métodos , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Humanos , Masculino , Distribuição Tecidual , Células Tumorais Cultivadas
13.
Science ; 304(5678): 1797-800, 2004 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-15205532

RESUMO

Understanding gene expression requires the ability to follow the fate of individual molecules. Here we use a cellular system for monitoring messenger RNA (mRNA)expression to characterize the movement in real time of single mRNA-protein complexes (mRNPs) in the nucleus of living mammalian cells. This mobility was not directed but was governed by simple diffusion. Some mRNPs were partially corralled throughout the nonhomogenous nuclear environment, but no accumulation at subnuclear domains was observed. Following energy deprivation, energy-independent motion of mRNPs was observed in a highly ATP-dependent nuclear environment; movements were constrained to chromatin-poor domains and excluded by newly formed chromatin barriers. This observation resolves a controversy, showing that the energetic requirements of nuclear mRNP trafficking are consistent with a diffusional model.


Assuntos
Núcleo Celular/metabolismo , Proteínas/metabolismo , RNA Mensageiro/metabolismo , Transporte Ativo do Núcleo Celular , Trifosfato de Adenosina/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular Tumoral , Cromatina/metabolismo , Citoplasma/metabolismo , Difusão , Metabolismo Energético , Recuperação de Fluorescência Após Fotodegradação , Globinas/genética , Globinas/metabolismo , Proteínas de Fluorescência Verde , Humanos , Hibridização in Situ Fluorescente , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Peroxissomos/metabolismo , Biossíntese de Proteínas , Proteínas/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transcrição Gênica , Transfecção
14.
Cell Microbiol ; 5(9): 613-24, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12925131

RESUMO

The association of PRP1, a Paramecium parafusin orthologue, with Toxoplasma gondii micronemes, now confirmed by immunoelectron microscopy, has here been studied in relation to exocytosis and cell invasion. PRP1 becomes labelled in vivo by inorganic 32P and is dephosphorylated when ethanol is used to stimulate Ca2+-dependent exocytosis of the micronemes. The ethanol Ca2+-stimulated exocytosis is accompanied by translocation of PRP1 and microneme content protein (MIC3) from the apical end of the parasite. Immunoblotting showed that PRP1 is redistributed inside the parasite, while microneme content is secreted. To study whether similar changes occur during cell invasion, quantitative microscopy was performed during secretion, invasion and exit (egress) from the host cell. Time-course experiments showed that fluorescence intensities of PRP1 and MIC3 immediately after invasion were reduced 10-fold compared to preinvasion levels, indicating that PRP1 translocation and microneme secretion accompanies invasion. MIC3 regained fluorescence intensity and apical distribution after 15 min, while PRP1 recovered after 1 h. Intensity of both proteins then increased throughout the parasite division period until host cell lysis, suggesting the need to secrete microneme proteins to egress. These studies suggest that PRP1 associated with the secretory vesicle scaffold serves an important role in Ca2+-regulated exocytosis and cell invasion.


Assuntos
Exocitose/fisiologia , Fosfoglucomutase , Fosfoproteínas/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Toxoplasma/patogenicidade , Animais , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/parasitologia , Glucose/análogos & derivados , Glucose/metabolismo , Humanos , Fosfoproteínas/genética , Fosforilação , Proteínas de Protozoários/genética , Proteínas Recombinantes de Fusão/metabolismo , Toxoplasma/ultraestrutura
15.
J Neurosci ; 23(16): 6627-37, 2003 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-12878704

RESUMO

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by deletion and/or mutation of the survival motor neuron protein Gene (SMN1) that results in the expression of a truncated protein lacking the C terminal exon-7. Whereas SMN has been shown to be an important component of diverse ribonucleoprotein (RNP) complexes, its function in neurons is unknown. We hypothesize that the active transport of SMN may be important for neurite outgrowth and that disruption of exon-7 could impair its normal intracellular trafficking. SMN was localized in granules that were associated with cytoskeletal filament systems and distributed throughout neurites and growth cones. Live cell imaging of enhanced green fluorescent protein (EGFP)-SMN granules revealed rapid, bidirectional and cytoskeletal-dependent movements. Exon-7 was necessary for localization of SMN into the cytoplasm but was not sufficient for granule formation and transport. A cytoplasmic targeting signal within exon-7 was identified that could completely redistribute the nuclear protein D-box binding factor 1 into the cytoplasm. Neurons transfected with SMN lacking exon-7 had significantly shorter neurites, a defect that could be rescued by redirecting the exon-7 deletion mutant into neurites by a targeting sequence from growth-associated protein-43. These findings provide the first demonstration of cytoskeletal-based active transport of SMN in neuronal processes and the function of exon-7 in cytoplasmic localization. Such observations provide motivation to investigate possible transport defects or inefficiency of SMN associated RNPs in motor neuron axons in SMA.


Assuntos
Citoplasma/metabolismo , Atrofia Muscular Espinal/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Citoesqueleto de Actina/fisiologia , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Embrião de Galinha , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Grânulos Citoplasmáticos/metabolismo , Éxons/fisiologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Genes Reporter , Cones de Crescimento/metabolismo , Humanos , Microtúbulos/fisiologia , Proteínas do Tecido Nervoso/genética , Neuritos/metabolismo , Neurônios/citologia , Transporte Proteico/fisiologia , Proteínas de Ligação a RNA , Ratos , Proteínas do Complexo SMN , Deleção de Sequência , Proteína 1 de Sobrevivência do Neurônio Motor
16.
Dev Cell ; 4(6): 941-52, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12791277

RESUMO

Although it is generally accepted that nuclear architecture is an important determinant of nuclear activity, it is not clear whether cytoplasmic events, such as transcript localization and cell polarity, are affected by this architecture. Characterization of the nuclear architecture of the single-cell alga Chlamydomonas reinhardtii revealed a polarized nucleus, with nuclear pore complexes preferentially concentrated at the posterior side of the nucleus. Nuclear asymmetry was greatly exaggerated during the upregulation of genes encoding flagellar proteins, when nuclear pore complexes (NPCs) were observed to hyperpolarize to the posterior side of the nucleus while heterochromatin polarized to the anterior side. Interestingly, prior to deflagellation, the beta2-tubulin gene was preferentially located in the posterior region of the nucleus, and following deflagellation, beta2-tubulin transcripts accumulated posteriorly in polysome-rich cytoplasmic regions adjacent to the highest concentration of NPCs, suggesting a connection between nuclear architecture and cytoplasmic transcript localization.


Assuntos
Chlamydomonas reinhardtii/genética , Citoplasma/metabolismo , Poro Nuclear/metabolismo , RNA Mensageiro/genética , Tubulina (Proteína)/genética , Animais , Núcleo Celular/ultraestrutura , Polaridade Celular , Chlamydomonas reinhardtii/metabolismo , Chlamydomonas reinhardtii/fisiologia , Citoplasma/química , Citoplasma/ultraestrutura , Flagelos/fisiologia , Heterocromatina/metabolismo , Heterocromatina/ultraestrutura , Membrana Nuclear/ultraestrutura , Poro Nuclear/ultraestrutura , RNA Mensageiro/metabolismo , Tubulina (Proteína)/ultraestrutura
17.
J Neurosci ; 23(8): 3251-61, 2003 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-12716932

RESUMO

RNA binding proteins may be important mediators of the activity-dependent transport of mRNAs to dendritic spines of activated synapses. We used fluorescence microscopy and digital imaging techniques applied to both fixed and live cultured hippocampal neurons to visualize the localization of the mRNA binding protein, zipcode binding protein 1 (ZBP1), and its dynamic movements in response to KCl-induced depolarization at high spatial and temporal resolution. With the use of immunofluorescence, image deconvolution, and three-dimensional reconstruction, ZBP1 was localized in the form of granules that were distributed in dendrites, spines, and subsynaptic sites. KCl depolarization increased the dendritic localization of ZBP1 that was not attributed to an increase in ZBP1 expression. Live cell imaging of single cells before and after perfusion of KCl revealed the rapid and directed efflux of ZBP1 granules from the cell body into dendrites in a proximo-distal gradient. High-speed imaging of enhanced green fluorescence protein-ZBP1 granules revealed rapid anterograde and retrograde movements in dendrites as well as dynamic movements in dendritic spines. A population of ZBP1 granules colocalized with beta-actin mRNA, and their spatial association in dendrites was increased by KCl depolarization. The NMDA receptor antagonist AP-5 impaired the dendritic localization of ZBP1 and beta-actin mRNA and inhibited the KCl-induced transport of ZBP1. The activity-dependent trafficking of ZBP1 and its dynamic movements within dendritic spines provide new evidence to implicate RNA binding proteins as regulators of mRNA transport to activated synapses in response to synaptic activity.


Assuntos
Actinas/metabolismo , Extensões da Superfície Celular/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , Proteínas de Ligação a RNA/metabolismo , Actinas/biossíntese , Actinas/genética , Animais , Astrócitos/citologia , Células Cultivadas , Técnicas de Cocultura , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/ultraestrutura , Dendritos/metabolismo , Antagonistas de Aminoácidos Excitatórios/farmacologia , Proteínas de Fluorescência Verde , Hipocampo/citologia , Hipocampo/embriologia , Internet , Proteínas Luminescentes/genética , Microscopia de Fluorescência/métodos , Neurônios/citologia , Cloreto de Potássio/farmacologia , Transporte Proteico/fisiologia , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Ratos , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Gravação em Vídeo
18.
Curr Biol ; 13(2): 161-167, 2003 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-12546792

RESUMO

Cytoplasmic mRNA movements ultimately determine the spatial distribution of protein synthesis. Although some mRNAs are compartmentalized in cytoplasmic regions, most mRNAs, such as housekeeping mRNAs or the poly-adenylated mRNA population, are believed to be distributed throughout the cytoplasm. The general mechanism by which all mRNAs may move, and how this may be related to localization, is unknown. Here, we report a method to visualize single mRNA molecules in living mammalian cells, and we report that, regardless of any specific cytoplasmic distribution, individual mRNA molecules exhibit rapid and directional movements on microtubules. Importantly, the beta-actin mRNA zipcode increased both the frequency and length of these movements, providing a common mechanistic basis for both localized and nonlocalized mRNAs. Disruption of the cytoskeleton with drugs showed that microtubules and microfilaments are involved in the types of mRNA movements we have observed, which included complete immobility and corralled and nonrestricted diffusion. Individual mRNA molecules switched frequently among these movements, suggesting that mRNAs undergo continuous cycles of anchoring, diffusion, and active transport.


Assuntos
RNA Mensageiro/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/genética , Animais , Células COS , Citoplasma/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde , Óperon Lac , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microtúbulos/metabolismo , Modelos Biológicos , Modelos Estatísticos , Movimento , Biossíntese de Proteínas , Proteínas/genética , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
19.
Cancer Res ; 62(21): 6006-10, 2002 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-12414619

RESUMO

Mutations in the adenomatous polyposis coli (APC) gene, which initiate almost all human colon cancers, directly target the proto-oncogene, c-myc, by elevating beta-catenin/T-cell factor (TCF) signaling. We have shown that agents ascribed chemopreventive activity for colon cancer in fact also stimulate beta-catenin/TCF activity in vitro. Their effects on c-myc transcription were assayed using a novel variant of fluorescence in situ hybridization that detects c-myc transcription sites in intact nuclei. Increased transcriptional initiation of c-myc induced by the short-chain fatty acid, butyrate, consistent with elevated beta-catenin/TCF activity, was efficiently abrogated by a block to transcriptional elongation, resulting in decreased c-myc expression. 1alpha,25-Dihydroxyvitamin D(3) also induced transcriptional blockage. In contrast, the nonsteroidal anti-inflammatory drug, sulindac, increased c-myc expression, an effect attributable at least in part to its failure to induce transcriptional blockage. We have described a novel approach for evaluating the effects of chemopreventive agents on the expression of a gene critical in colonic tumorigenesis.


Assuntos
Anticarcinógenos/farmacologia , Neoplasias do Colo/genética , Neoplasias do Colo/prevenção & controle , Genes myc/efeitos dos fármacos , Butiratos/farmacologia , Sondas de DNA , Di-Hidroxicolecalciferóis/farmacologia , Interações Medicamentosas , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Inativação Gênica/efeitos dos fármacos , Genes myc/genética , Predisposição Genética para Doença , Humanos , Proto-Oncogene Mas , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sulindaco/farmacologia , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética
20.
Science ; 297(5582): 836-40, 2002 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-12161654

RESUMO

A key goal of biology is to relate the expression of specific genes to a particular cellular phenotype. However, current assays for gene expression destroy the structural context. By combining advances in computational fluorescence microscopy with multiplex probe design, we devised technology in which the expression of many genes can be visualized simultaneously inside single cells with high spatial and temporal resolution. Analysis of 11 genes in serum-stimulated cultured cells revealed unique patterns of gene expression within individual cells. Using the nucleus as the substrate for parallel gene analysis, we provide a platform for the fusion of genomics and cell biology: "cellular genomics."


Assuntos
Células/citologia , Células/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Microscopia de Fluorescência/métodos , Adenocarcinoma/genética , Núcleo Celular/genética , Neoplasias do Colo/genética , Cor , Sondas de DNA , Fibroblastos , Perfilação da Expressão Gênica/instrumentação , Genes , Genômica/instrumentação , Genômica/métodos , Humanos , Microscopia de Fluorescência/instrumentação , Razão de Chances , RNA/genética , RNA/metabolismo , Sensibilidade e Especificidade , Fatores de Tempo , Transcrição Gênica , Células Tumorais Cultivadas
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