Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 6 de 6
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Front Cell Dev Biol ; 11: 1133994, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37305680

RESUMO

Introduction: Despite continued technological improvements, measurement errors always reduce or distort the information that any real experiment can provide to quantify cellular dynamics. This problem is particularly serious for cell signaling studies to quantify heterogeneity in single-cell gene regulation, where important RNA and protein copy numbers are themselves subject to the inherently random fluctuations of biochemical reactions. Until now, it has not been clear how measurement noise should be managed in addition to other experiment design variables (e.g., sampling size, measurement times, or perturbation levels) to ensure that collected data will provide useful insights on signaling or gene expression mechanisms of interest. Methods: We propose a computational framework that takes explicit consideration of measurement errors to analyze single-cell observations, and we derive Fisher Information Matrix (FIM)-based criteria to quantify the information value of distorted experiments. Results and Discussion: We apply this framework to analyze multiple models in the context of simulated and experimental single-cell data for a reporter gene controlled by an HIV promoter. We show that the proposed approach quantitatively predicts how different types of measurement distortions affect the accuracy and precision of model identification, and we demonstrate that the effects of these distortions can be mitigated through explicit consideration during model inference. We conclude that this reformulation of the FIM could be used effectively to design single-cell experiments to optimally harvest fluctuation information while mitigating the effects of image distortion.

2.
Bio Protoc ; 12(15)2022 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-36082371

RESUMO

In eukaryotic cells, RNA Polymerase II (RNAP2) is the enzyme in charge of transcribing mRNA from DNA. RNAP2 possesses an extended carboxy-terminal domain (CTD) that gets dynamically phosphorylated as RNAP2 progresses through the transcription cycle, therefore regulating each step of transcription from recruitment to termination. Although RNAP2 residue-specific phosphorylation has been characterized in fixed cells by immunoprecipitation-based assays, or in live cells by using tandem gene arrays, these assays can mask heterogeneity and limit temporal and spatial resolution. Our protocol employs multi-colored complementary fluorescent antibody-based (Fab) probes to specifically detect the CTD of the RNAP2 (CTD-RNAP2), and its phosphorylated form at the serine 5 residue (Ser5ph-RNAP2) at a single-copy HIV-1 reporter gene. Together with high-resolution fluorescence microscopy, single-molecule tracking analysis, and rigorous computational modeling, our system allows us to visualize, quantify, and predict endogenous RNAP2 phosphorylation dynamics and mRNA synthesis at a single-copy gene, in living cells, and throughout the transcription cycle. Graphical abstract: Schematic of the steps for visualizing, quantifying, and predicting RNAP2 phosphorylation at a single-copy gene.

3.
Nat Commun ; 12(1): 3158, 2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-34039974

RESUMO

The carboxyl-terminal domain of RNA polymerase II (RNAP2) is phosphorylated during transcription in eukaryotic cells. While residue-specific phosphorylation has been mapped with exquisite spatial resolution along the 1D genome in a population of fixed cells using immunoprecipitation-based assays, the timing, kinetics, and spatial organization of phosphorylation along a single-copy gene have not yet been measured in living cells. Here, we achieve this by combining multi-color, single-molecule microscopy with fluorescent antibody-based probes that specifically bind to different phosphorylated forms of endogenous RNAP2 in living cells. Applying this methodology to a single-copy HIV-1 reporter gene provides live-cell evidence for heterogeneity in the distribution of RNAP2 along the length of the gene as well as Serine 5 phosphorylated RNAP2 clusters that remain separated in both space and time from nascent mRNA synthesis. Computational models determine that 5 to 40 RNAP2 cluster around the promoter during a typical transcriptional burst, with most phosphorylated at Serine 5 within 6 seconds of arrival and roughly half escaping the promoter in ~1.5 minutes. Taken together, our data provide live-cell support for the notion of efficient transcription clusters that transiently form around promoters and contain high concentrations of RNAP2 phosphorylated at Serine 5.


Assuntos
Microscopia Intravital/métodos , RNA Polimerase II/metabolismo , Imagem Individual de Molécula/métodos , Transcrição Gênica , Genes Reporter/genética , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Microscopia de Fluorescência , Fosforilação , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , Serina/metabolismo , Análise Espaço-Temporal , Imagem com Lapso de Tempo
4.
J Biol Chem ; 294(2): 593-607, 2019 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-30446621

RESUMO

Monocarboxylate transporters (MCTs) mediate the proton-coupled exchange of high-energy metabolites, including lactate and pyruvate, between cells and tissues. The transport activity of MCT1, MCT2, and MCT4 can be facilitated by the extracellular carbonic anhydrase IV (CAIV) via a noncatalytic mechanism. Combining physiological measurements in HEK-293 cells and Xenopus oocytes with pulldown experiments, we analyzed the direct interaction between CAIV and the two MCT chaperones basigin (CD147) and embigin (GP70). Our results show that facilitation of MCT transport activity requires direct binding of CAIV to the transporters chaperones. We found that this binding is mediated by the highly conserved His-88 residue in CAIV, which is also the central residue of the enzyme's intramolecular proton shuttle, and a charged amino acid residue in the Ig1 domain of the chaperone. Although the position of the CAIV-binding site in the chaperone was conserved, the amino acid residue itself varied among different species. In human CD147, binding of CAIV was mediated by the negatively charged Glu-73 and in rat CD147 by the positively charged Lys-73. In rat GP70, we identified the positively charged Arg-130 as the binding site. Further analysis of the CAIV-binding site revealed that the His-88 in CAIV can either act as H donor or H acceptor for the hydrogen bond, depending on the charge of the binding residue in the chaperone. Our results suggest that the CAIV-mediated increase in MCT transport activity requires direct binding between CAIV-His-88 and a charged amino acid in the extracellular domain of the transporter's chaperone.


Assuntos
Basigina/metabolismo , Anidrase Carbônica IV/metabolismo , Glicoproteínas/metabolismo , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Mapas de Interação de Proteínas , Sequência de Aminoácidos , Animais , Basigina/química , Células HEK293 , Humanos , Proteínas de Membrana , Modelos Moleculares , Domínios Proteicos , Ratos , Alinhamento de Sequência , Simportadores/metabolismo , Xenopus
5.
Sci Rep ; 7(1): 4900, 2017 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-28687765

RESUMO

Epilepsy is a chronic neurological disorder that affects approximately 50 million people worldwide. Ketogenic diet (KD) can be a very effective treatment for intractable epilepsy. Potential mechanisms of action for KD have been proposed, including the re-balance among excitatory and inhibitory neurotransmission and decrease in the glycolytic rate in brain cells. KD has been shown to have an effect on the expression pattern of monocarboxylate transporters (MCT), however, it is unknown whether MCT transport activity is affected by KD and linked to the reduction of seizures during KD. Therefore, we studied the influence of KD on MCT transport activity and the role of MCTs during epileptiform activity. Our results showed a decrease in the epileptiform activity in cortical slices from mice fed on KD and in the presence of beta-hydroxybutyrate. KD increased transport capacity for ketone bodies and lactate in cortical astrocytes by raising the MCT1 expression level. Inhibition of MCT1 and MCT2 in control conditions decreases epileptiform activity, while in KD it induced an increase in epileptiform activity. Our results suggest that MCTs not only play an important role in the transport of ketone bodies, but also in the modulation of brain energy metabolism under normal and ketogenic conditions.


Assuntos
Dieta Cetogênica , Epilepsia/dietoterapia , Corpos Cetônicos/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Convulsões/dietoterapia , Simportadores/genética , Ácido 3-Hidroxibutírico/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Modelos Animais de Doenças , Epilepsia/genética , Epilepsia/metabolismo , Epilepsia/fisiopatologia , Regulação da Expressão Gênica , Glicólise/efeitos dos fármacos , Glicólise/genética , Ácido Láctico/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microtomia , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Transportadores de Ácidos Monocarboxílicos/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Cultura Primária de Células , Convulsões/genética , Convulsões/metabolismo , Convulsões/fisiopatologia , Transdução de Sinais , Simportadores/antagonistas & inibidores , Simportadores/metabolismo , Tiofenos/farmacologia , Técnicas de Cultura de Tecidos , Uracila/análogos & derivados , Uracila/farmacologia
6.
PLoS One ; 8(4): e59364, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23565149

RESUMO

We report an experimental study of mouse sperm motility that shows chief aspects characteristic of neurons: the anesthetic (produced by tetracaine) and excitatory (produced by either caffeine or calcium) effects and their antagonic action. While tetracaine inhibits sperm motility and caffeine has an excitatory action, the combination of these two substances balance the effects, producing a motility quite similar to that of control cells. We also study the effects of these agents (anesthetic and excitatory) on the melting points of pure lipid liposomes constituted by 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and dipalmitoyl phosphatidic acid (DPPA). Tetracaine induces a large fluidization of the membrane, shifting the liposomes melting transition temperature to much lower values. The effect of caffeine is null, but its addition to tetracaine-doped liposomes greatly screen the fluidization effect. A high calcium concentration stiffens pure lipid membranes and strongly reduces the effect of tetracaine. Molecular Dynamics Simulations are performed to further understand our experimental findings at the molecular level. We find a strong correlation between the effect of antagonic molecules that could explain how the mechanical properties suitable for normal cell functioning are affected and recovered.


Assuntos
Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Bicamadas Lipídicas , Lipídeos de Membrana/metabolismo , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Animais , Cafeína/farmacologia , Interações Hidrofóbicas e Hidrofílicas , Lipossomos/química , Lipossomos/metabolismo , Masculino , Lipídeos de Membrana/química , Camundongos , Conformação Molecular , Simulação de Dinâmica Molecular , Motilidade dos Espermatozoides/fisiologia , Temperatura , Tetracaína/farmacologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...