RESUMO
Transcript degradation is a key step in gene regulation. In eukaryotes, mRNA decay is generally initiated by removal of the poly(A) tail mediated by the Ccr4-Caf1-Not complex. Deadenylated transcripts are then rapidly degraded, primarily via the decapping-dependent pathway. Components of this pathway can be localized into highly dynamic cytoplasmic foci, the mRNA processing (P)-bodies. We have undertaken confocal fluorescence microscopy to monitor P-bodies in Aspergillus nidulans. As in other organisms a dynamic shift in P-body formation occurs in response to diverse physiological signals. Significantly, both this cellular response and the signalled degradation of specific transcripts are dependent on the nuclease activity of Caf1 but not Ccr4. P-body formation is disrupted in A. nidulans strains deleted for Edc3, an enhancer of decapping, or CutA, which encodes a nucleotidyltransferase that triggers mRNA decapping by the addition of a CUCU tag to the poly(A) tail. As with DeltacutA, Deltaedc3 led to reduced rates of transcript degradation. These data link P-bodies to both the optimization and regulation of transcript degradation.
Assuntos
Aspergillus nidulans/fisiologia , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Estabilidade de RNA , RNA Fúngico/metabolismo , Aspergillus nidulans/metabolismo , Deleção de Genes , Microscopia Confocal , Microscopia de Fluorescência , Estresse FisiológicoRESUMO
The development and application of single cell optical imaging has identified dynamic and oscillatory signalling processes in individual cells. This requires single cell analyses since the processes may otherwise be masked by the population average. These oscillations range in timing from seconds/minutes (e.g. calcium) to minutes/hours (e.g. NF-kappaB, Notch/Wnt and p53) and hours/days (e.g. circadian clock and cell cycle). Quantitative live cell measurement of the protein processes underlying these complex networks will allow characterisation of the core mechanisms that drive these signalling pathways and control cell function. Ultimately, such studies can be applied to develop predictive models of whole tissues and organisms.
Assuntos
Proteínas/metabolismo , NF-kappa B/metabolismo , Transdução de SinaisRESUMO
A critical step of neuronal terminal differentiation is the permanent withdrawal from the cell cycle that requires the silencing of genes that drive mitosis. Here, we describe that the alpha isoform of the heterochromatin protein 1 (HP1) protein family exerts such silencing on several E2F-targeted genes. Among the different isoforms, HP1alpha levels progressively increase throughout differentiation and take over HP1gamma binding on E2F sites in mature neurons. When overexpressed, only HP1alpha is able to ensure a timed repression of E2F genes. Specific inhibition of HP1alpha expression drives neuronal progenitors either towards death or cell cycle progression, yet preventing the expression of the neuronal marker microtubule-associated protein 2. Furthermore, we provide evidence that this mechanism occurs in cerebellar granule neurons in vivo, during the postnatal development of the cerebellum. Finally, our results suggest that E2F-targeted genes are packaged into higher-order chromatin structures in mature neurons relative to neuroblasts, likely reflecting a transition from a 'repressed' versus 'silenced' status of these genes. Together, these data present new epigenetic regulations orchestrated by HP1 isoforms, critical for permanent cell cycle exit during neuronal differentiation.
Assuntos
Diferenciação Celular , Proteínas Cromossômicas não Histona/fisiologia , Fatores de Transcrição E2F/fisiologia , Inativação Gênica , Neurônios/citologia , Animais , Sequência de Bases , Linhagem da Célula , Células Cultivadas , Homólogo 5 da Proteína Cromobox , Citometria de Fluxo , Camundongos , RNA Interferente PequenoRESUMO
An artificial mutant Ala25Ser precursor cystatin C was created to help elucidate the cause of intracellular mis-localisation of the biochemically related variant B (Ala25Thr) precursor cystatin C to the mitochondria. Homozygotes of variant B precursor cystatin C were reported to carry an increased susceptibility to developing the exudative form of AMD. Ala25Ser precursor cystatin C shows a dual distribution to the Golgi apparatus and to the mitochondria. This localisation is thus intermediary between that of wild-type cystatin C (targeted to ER/Golgi compartment) and that of variant B precursor cystatin C. Furthermore, the level of secretion of Ala25Ser cystatin C by RPE cells is intermediary between wild type and variant B cystatin C. Ala25Ser precursor cystatin C thus represents a biochemical intermediate between the wild type and the AMD-associated cystatin C and as such, is a novel tool for the investigation of the mechanism of intracellular mis-localisation of variant B cystatin C. Our findings further support the hypothesis that substitution of the alanine residue in the penultimate position of precursor cystatin C signal sequence with a less hydrophobic amino acid residue, such as threonine (as in variant B cystatin C) or serine is sufficient to impair the intracellular trafficking and processing of the protein.
Assuntos
Cistatinas/genética , Degeneração Macular/genética , Cistatina C , Predisposição Genética para Doença , Complexo de Golgi/metabolismo , Humanos , Mitocôndrias/metabolismoRESUMO
Osteonectin is a glycoprotein that modulates several aspects of cellular behaviour including proliferation and adhesion. The retinal pigment epithelium forms a continuous monolayer of polarised cells immediately bellow the neuroretina, and is integral to the homeostasis of photoreceptor cells. While osteonectin is expressed by normal retinal pigment epithelium in situ, its expression is significantly increased in retinal pigment epithelial cells associated with several common retinal diseases. This pattern of expression implies an important role for osteonectin in the biology of retinal pigment epithelial cells. However, the trafficking, processing, and eventual fate of osteonectin in these cells is not clear at present. Although the theoretical report of a leader sequence within the osteonectin open reading frame and its extracellular presence in some tissues indirectly support secretion of the protein, there is no direct experimental demonstration of the secretion route to date. As a first step towards understanding the role of osteonectin in retinal pigment epithelium, we studied the intracellular distribution and trafficking of the protein in living cells. Here, we present experimental evidence that a precursor osteonectin fusion protein is targeted to the endoplasmic reticulum/Golgi pathway, with a likely basal secretion in retinal pigment epithelial cells. In addition, we show that the precursor osteonectin protein having the leader sequence masked fails to undergo secretion leading to cell death, a phenotype which may be of relevance not only for retinal pathology, but also for other diseases such as the bone disorder known as pseudoachondroplasia that is associated with a lack of osteonectin secretion.
Assuntos
Complexo de Golgi/metabolismo , Osteonectina/metabolismo , Epitélio Pigmentado Ocular/metabolismo , Acondroplasia/metabolismo , Adesão Celular/fisiologia , Morte Celular , Linhagem Celular , Polaridade Celular/fisiologia , Proliferação de Células , Humanos , Células Fotorreceptoras/citologia , Células Fotorreceptoras/metabolismo , Epitélio Pigmentado Ocular/citologia , Sinais Direcionadores de Proteínas/genética , Transporte Proteico/genética , Transporte Proteico/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Pituitary function has been shown to be regulated by an increasing number of intrapituitary factors, including cytokines. Here we show that the important cytokine TNF-alpha activates prolactin gene transcription in pituitary GH3 cells stably expressing luciferase under control of 5 kb of the human prolactin promoter. Similar regulation of the endogenous rat prolactin gene by TNF-alpha in GH3 cells was confirmed using real-time PCR. Luminescence microscopy revealed heterogeneous dynamic response patterns of promoter activity in individual cells. In GH3 cells treated with TNF-alpha, Western blot analysis showed rapid inhibitory protein kappaB (IkappaBalpha) degradation and phosphorylation of p65. Confocal microscopy of cells expressing fluorescence-labeled p65 and IkappaBalpha fusion proteins showed transient cytoplasmic-nuclear translocation and subsequent oscillations in p65 localization and confirmed IkappaBalpha degradation. This was associated with increased nuclear factor kappaB (NF-kappaB)-mediated transcription from an NF-kappaB-responsive luciferase reporter construct. Disruption of NF-kappaB signaling by expression of dominant-negative variants of IkappaB kinases or truncated IkappaBalpha abolished TNF-alpha activation of the prolactin promoter, suggesting that this effect was mediated by NF-kappaB. TNF-alpha signaling was found to interact with other endocrine signals to regulate prolactin gene expression and is likely to be a major paracrine modulator of lactotroph function.
Assuntos
NF-kappa B/metabolismo , Hipófise/metabolismo , Prolactina/metabolismo , Regiões Promotoras Genéticas/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Linhagem Celular Tumoral , Regulação da Expressão Gênica/fisiologia , Genes Reporter , Humanos , Hipófise/citologia , Prolactina/genética , Regiões Promotoras Genéticas/genética , Ratos , Transdução de Sinais/fisiologia , TransfecçãoRESUMO
Prenatal airways from diverse species are capable of spontaneous peristaltic contractions in each trimester. The function of this smooth muscle activity is unknown. We demonstrate that peristalsis of the embryonic airway originates from a sided pacemaker focus, is stimulated in a calcium-dependent fashion by the pulmonary morphogen fibroblast growth factor-10 (FGF-10), and appears coupled to lung growth. Airway peristalsis may be crucial for lung development (thereby providing a physiologic role for airway smooth muscle) and play a hitherto unanticipated role in reported transgenic mutant lung phenotypes.
Assuntos
Relógios Biológicos/fisiologia , Pulmão/embriologia , Morfogênese/fisiologia , Contração Muscular/fisiologia , Músculo Liso/fisiologia , Mecânica Respiratória/fisiologia , Animais , Cálcio/metabolismo , Fator 10 de Crescimento de Fibroblastos , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Imuno-Histoquímica , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-DawleyRESUMO
Cystatin C is abundantly expressed by the retinal pigment epithelium (RPE) of the eye. Targeting of cystatin C to the Golgi apparatus and processing through the secretory pathway of RPE cells are dependent upon a 26-amino acid signal sequence of precursor cystatin C. A variant with an alanine (A) to threonine (T) mutation in the penultimate amino acid of the signal sequence (A25T) was recently correlated with increased risk of developing exudative age-related macular degeneration. The biochemical consequence of the A25T mutation upon targeting of the protein is reported here. Targeting and trafficking of full-length mutant (A25T) precursor cystatin C-enhanced green fluorescent protein fusion protein were studied in living, cultured retinal pigment epithelial and HeLa cells. Confocal microscopy studies were substantiated by immunodetection. In striking contrast to wild-type precursor cystatin C fusion protein conspicuously targeted to the Golgi apparatus, the threonine variant was associated principally with mitochondria. Some diffuse fluorescence was also observed throughout the cytoplasm and nucleus (but not nucleoli). Secretion of fusion protein derived from the threonine variant was reduced by approximately 50% compared with that of the wild-type cystatin C fusion protein. Expression of the variant fusion protein did not appear to impair expression or secretion of endogenous cystatin C.