RESUMO
Neuronal differentiation-the development of neurons from neural stem cells-involves neurite outgrowth and is a key process during the development and regeneration of neural functions. In addition to various chemical signaling mechanisms, it has been suggested that thermal stimuli induce neuronal differentiation. However, the function of physiological subcellular thermogenesis during neuronal differentiation remains unknown. Here we create methods to manipulate and observe local intracellular temperature, and investigate the effects of noninvasive temperature changes on neuronal differentiation using neuron-like PC12 cells. Using quantitative heating with an infrared laser, we find an increase in local temperature (especially in the nucleus) facilitates neurite outgrowth. Intracellular thermometry reveals that neuronal differentiation is accompanied by intracellular thermogenesis associated with transcription and translation. Suppression of intracellular temperature increase during neuronal differentiation inhibits neurite outgrowth. Furthermore, spontaneous intracellular temperature elevation is involved in neurite outgrowth of primary mouse cortical neurons. These results offer a model for understanding neuronal differentiation induced by intracellular thermal signaling.
Assuntos
Diferenciação Celular , Neurônios , Transdução de Sinais , Temperatura , Animais , Células PC12 , Neurônios/fisiologia , Neurônios/citologia , Camundongos , Ratos , Crescimento Neuronal , Neurogênese/fisiologia , Neuritos/metabolismo , Neuritos/fisiologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/fisiologia , Termometria/métodos , Termogênese/fisiologiaRESUMO
Intracellular temperature affects a wide range of cellular functions in living organisms. However, it remains unclear whether temperature in individual animal cells is controlled autonomously as a response to fluctuations in environmental temperature. Using two distinct intracellular thermometers, we find that the intracellular temperature of steady-state Drosophila S2 cells is maintained in a manner dependent on Δ9-fatty acid desaturase DESAT1, which introduces a double bond at the Δ9 position of the acyl moiety of acyl-CoA. The DESAT1-mediated increase of intracellular temperature is caused by the enhancement of F1Fo-ATPase-dependent mitochondrial respiration, which is coupled with thermogenesis. We also reveal that F1Fo-ATPase-dependent mitochondrial respiration is potentiated by cold exposure through the remodeling of mitochondrial cristae structures via DESAT1-dependent unsaturation of mitochondrial phospholipid acyl chains. Based on these findings, we propose a cell-autonomous mechanism for intracellular temperature control during environmental temperature changes.
Assuntos
Ácidos Graxos Dessaturases , Fosfolipídeos , Adenosina Trifosfatases , Animais , Drosophila , Estearoil-CoA Dessaturase , TemperaturaRESUMO
Recent studies suggest that adult humans have active brown or beige adipocytes, the activation of which might be a therapeutic strategy for the treatment of diverse metabolic diseases. Here we show that the protein kinase ASK1 regulates brown and beige adipocytes function. In brown or white adipocytes, the PKA-ASK1-p38 axis is activated in response to cAMP signalling and contributes to the cell-autonomous induction of genes, including Ucp1. Global and fat-specific ASK1 deficiency leads to impaired metabolic responses, including thermogenesis and oxygen consumption, at the cell and whole-body levels, respectively. Our data thus indicate that the ASK1 signalling axis is a regulator of brown and beige adipocyte gene expression and function.
Assuntos
Adipócitos Marrons/metabolismo , Tecido Adiposo Marrom/metabolismo , MAP Quinase Quinase Quinase 5/metabolismo , Transdução de Sinais , Termogênese/genética , Células 3T3-L1 , Adipócitos Marrons/citologia , Adipócitos Brancos/citologia , Adipócitos Brancos/metabolismo , Tecido Adiposo Marrom/citologia , Tecido Adiposo Branco/citologia , Tecido Adiposo Branco/metabolismo , Animais , Diferenciação Celular , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Regulação da Expressão Gênica , Células HEK293 , Humanos , Canais Iônicos/genética , Canais Iônicos/metabolismo , MAP Quinase Quinase Quinase 5/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Consumo de Oxigênio/genética , Proteína Desacopladora 1 , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Dysregulation of oxidative phosphorylation is associated with increased mitochondrial reactive oxygen species production and some of the most prevalent human diseases including obesity, cancer, diabetes, neurodegeneration, and heart disease. Chemical 'mitochondrial uncouplers' are lipophilic weak acids that transport protons into the mitochondrial matrix via a pathway that is independent of ATP synthase, thereby uncoupling nutrient oxidation from ATP production. Mitochondrial uncouplers also lessen the proton motive force across the mitochondrial inner membrane and thereby increase the rate of mitochondrial respiration while decreasing production of reactive oxygen species. Thus, mitochondrial uncouplers are valuable chemical tools that enable the measurement of maximal mitochondrial respiration and they have been used therapeutically to decrease mitochondrial reactive oxygen species production. However, the most widely used protonophore uncouplers such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and 2,4-dinitrophenol have off-target activity at other membranes that lead to a range of undesired effects including plasma membrane depolarization, mitochondrial inhibition, and cytotoxicity. These unwanted properties interfere with the measurement of mitochondrial function and result in a narrow therapeutic index that limits their usefulness in the clinic. To identify new mitochondrial uncouplers that lack off-target activity at the plasma membrane we screened a small molecule chemical library. Herein we report the identification and validation of a novel mitochondrial protonophore uncoupler (2-fluorophenyl){6-[(2-fluorophenyl)amino](1,2,5-oxadiazolo[3,4-e]pyrazin-5-yl)}amine, named BAM15, that does not depolarize the plasma membrane. Compared to FCCP, an uncoupler of equal potency, BAM15 treatment of cultured cells stimulates a higher maximum rate of mitochondrial respiration and is less cytotoxic. Furthermore, BAM15 is bioactive in vivo and dose-dependently protects mice from acute renal ischemic-reperfusion injury. From a technical standpoint, BAM15 represents an effective new tool that allows the study of mitochondrial function in the absence of off-target effects that can confound data interpretation. From a therapeutic perspective, BAM15-mediated protection from ischemia-reperfusion injury and its reduced toxicity will hopefully reignite interest in pharmacological uncoupling for the treatment of the myriad of diseases that are associated with altered mitochondrial function.
RESUMO
Visualization and monitoring of endogenous mRNA in the cytoplasm of living cells promises a significant comprehension of refined post-transcriptional regulation. Fluorescently labeled linear antisense oligonucleotides can bind to natural mRNA in a sequence-specific way and, therefore, provide a powerful tool in probing endogenous mRNA. Here, we investigated the feasibility of using linear antisense probes to monitor the variable and dynamic expression of endogenous cytoplasmic mRNAs. Two linear antisense 2'-O-methyl RNA probes, which have different interactive fluorophores at the 5'-end of one probe and at the 3'-end of the other, were used to allow fluorescence resonance energy transfer (FRET) upon hybridization to the target mRNA. By characterizing the formation of the probe-mRNA hybrids in living cells, we found that the probe composition and concentration are crucial parameters in the visualization of endogenous mRNA with high specificity. Furthermore, rapid hybridization (within 1 min) of the linear antisense probe enabled us to visualize dynamic processes of endogenous c-fos mRNA, such as fast elevation of levels after gene induction and the localization of c-fos mRNA in stress granules in response to cellular stress. Thus, our approach provides a basis for real time monitoring of endogenous cytoplasmic mRNA in living cells.
Assuntos
Citoplasma/química , Transferência Ressonante de Energia de Fluorescência , Sondas RNA/química , RNA Antissenso/química , RNA Mensageiro/análise , Animais , Células COS , Chlorocebus aethiops , Grânulos Citoplasmáticos/química , Corantes Fluorescentes , Cinética , Microscopia de Fluorescência , Proteínas Proto-Oncogênicas c-fos/análise , Proteínas Proto-Oncogênicas c-fos/genética , Estabilidade de RNA , RNA Mensageiro/biossínteseRESUMO
GroEL is an Escherichia coli chaperonin that is composed of two heptameric rings stacked back-to-back. GroEL assists protein folding with its cochaperonin GroES in an ATP-dependent manner in vitro and in vivo. However, it is still unclear whether GroES binds to both rings of GroEL simultaneously under physiological conditions. In this study, we monitored the GroEL-GroES interaction in the reaction cycle using fluorescence resonance energy transfer. We found that nearly equivalent amounts of symmetric GroEL-(GroES)(2) (football-shaped) complex and asymmetric GroEL-GroES (bullet-shaped) complex coexist during the functional reaction cycle. We also found that D398A, an ATP hydrolysis defective mutant of GroEL, forms a football-shaped complex with ATP bound to the two rings. Furthermore, we showed that ADP prevents the association of ATP to the trans-ring of GroEL, and as a consequence, the second GroES cannot bind to GroEL. Considering the concentrations of ADP and ATP in E. coli, ADP is expected to have a small effect on the inhibition of GroES binding to the trans-ring of GroEL in vivo. These results suggest that we should reconsider the chaperonin-mediated protein-folding mechanism that involves the football-shaped complex.
Assuntos
Chaperonina 10/química , Chaperonina 60/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Complexos Multiproteicos/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Substituição de Aminoácidos , Chaperonina 10/genética , Chaperonina 10/metabolismo , Chaperonina 60/genética , Chaperonina 60/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação de Sentido Incorreto , Ligação Proteica/fisiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The selection of effective antisense target sites on a given mRNA molecule is a major problem in the detection of target mRNA in oligonucleotide arrays. In general, antisense oligodeoxynucleotides (asODNs) of about 10-20 nucleotides (nt) in length are used. However, the demand for predicting the sequence of potent asODNs much longer than those mentioned above has been increasing. Here, we prepared 40-nt asODNs directed against fluorescence-labeled green fluorescent protein (GFP) mRNA and quantified their hybridization efficiencies by fluorescence microscopy. We found that the hybridization efficiency depended on the TC content or the minimum free energy of the asODNs. On the basis of these findings, a semiempirical parameter called accessibility score was introduced to predict the potency of asODNs. The results of this study aided in the development of an effective two-step procedure for determining mRNA accessibility, namely, the computer-aided selection of asODN binding sites using an accessibility score followed by an experimental procedure for measuring the hybridization efficiencies between the selected asODNs and the target mRNA by fluorescence microscopy.
Assuntos
Oligodesoxirribonucleotídeos Antissenso/análise , Oligodesoxirribonucleotídeos Antissenso/genética , Sequência de Bases , Sítios de Ligação/genética , Biotecnologia , Genes fos , Proteínas de Fluorescência Verde/genética , Técnicas In Vitro , Microscopia de Fluorescência , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , RNA Mensageiro/química , RNA Mensageiro/genética , Proteínas Recombinantes/genética , Análise de Sequência de DNA/métodos , TermodinâmicaRESUMO
Real time imaging of mRNA in living cell is powerful tool in investigating the role of mRNA. Here we developed the method for visualizing specific mRNAs in living cell. The proposed method contains hybridization of antisense nucleic acids with mRNA transcripted inside cell. As antisense nucleic acid, 2'O-methyl RNA and Peptide Nucleic Acid (PNA) were chosen because these artificial nucleic acids are nuclease resistant and have high affinity to mRNA. Two antisense probes labelled with different fluorescent dyes were designed and prepared. When two antisense probes were hybridized to target human c-fos mRNA, two fluorescent dyes became very close and fluorescence resonance energy transfer (FRET) occurred, resulting in changes in fluorescence spectra. Such FRET signals were detected when 2'O-methyl RNA and Peptide Nucleic Acid were used as antisense probes. The biggest signal was detected when 2'O-methyl RNA as donor probe and PNA as acceptor probe were used. The timecourse study indicated two antisense probes can hybridize fast enough to subject to real time imaging.