RESUMO
Cellular senescence correlates with changes in the transcriptome. To obtain a complete view on senescence-associated transcription networks and pathways, we assessed by deep RNA sequencing the transcriptomes of five of the most commonly used laboratory strains of human fibroblasts during their transition into senescence. In a number of cases, we verified the RNA-seq data by real-time PCR. By determining cellular protein levels we observed that the age-related expression of most but not all genes is regulated at the transcriptional level. We found that 78% of the age-affected differentially expressed genes were commonly regulated in the same direction (either up- or down-regulated) in all five fibroblast strains, indicating a strong conservation of age-associated changes in the transcriptome. KEGG pathway analyses confirmed up-regulation of the senescence-associated secretory phenotype and down-regulation of DNA synthesis/repair and most cell cycle pathways common in all five cell strains. Newly identified senescence-induced pathways include up-regulation of endocytotic/phagocytic pathways and down-regulation of the mRNA metabolism and the mRNA splicing pathways. Our results provide an unprecedented comprehensive and deep view into the individual and common transcriptome and pathway changes during the transition into of senescence of five human fibroblast cell strains.
Assuntos
Senescência Celular/genética , Sequência Conservada , Fibroblastos/citologia , Fibroblastos/metabolismo , Análise de Sequência de RNA , Proliferação de Células , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Progress has been made in characterizing the secretases involved in endoproteolytic processing of the beta-amyloid precursor protein - the precursor of the amyloid beta-peptide (Abeta), which is the main constituent of amyloid plaques that form in the brains of patients with Alzheimer's disease. It is now thought that Abeta is pivotal in the pathogenesis of Alzheimer's disease, and that reducing brain Abeta levels may help to treat or prevent the disease. Two essential factors for the proteolytic generation of Abeta have been identified, beta-secretase and the presenilins, which might aid the design of drugs against this disease.
Assuntos
Doença de Alzheimer/enzimologia , Endopeptidases/fisiologia , Secretases da Proteína Precursora do Amiloide , Precursor de Proteína beta-Amiloide/fisiologia , Animais , Ácido Aspártico Endopeptidases , HumanosRESUMO
beta-Secretase (BACE) is a transmembrane aspartyl protease, which generates the N terminus of Alzheimer's disease amyloid beta-peptide. Here, we report that BACE can be phosphorylated within its cytoplasmic domain at serine residue 498 by casein kinase 1. Phosphorylation exclusively occurs after full maturation of BACE by propeptide cleavage and complex N-glycosylation. Phosphorylation/dephosphorylation affects the subcellular localization of BACE. BACE wild type and an S498D mutant that mimics phosphorylated BACE are predominantly located within juxtanuclear Golgi compartments and endosomes, whereas nonphosphorylatable BACE S498A accumulates in peripheral EEA1-positive endosomes. Antibody uptake assays revealed that reinternalization of BACE from the cell surface is independent of its phosphorylation state. After reinternalization, BACE wild type as well as BACE S498D are efficiently retrieved from early endosomal compartments and further targeted to later endosomal compartments and/or the trans-Golgi network. In contrast, nonphosphorylatable BACE S498A is retained within early endosomes. Our results therefore demonstrate regulated trafficking of BACE within the secretory and endocytic pathway.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Secretases da Proteína Precursora do Amiloide , Animais , Caseína Quinases , Linhagem Celular , Citoplasma/metabolismo , Endocitose , Endopeptidases , Humanos , Fosforilação , Proteínas Quinases/metabolismo , Transporte Proteico , Frações Subcelulares/enzimologiaRESUMO
Neurons transport newly synthesized membrane proteins along axons by microtubule-mediated fast axonal transport. Membrane proteins destined for different axonal subdomains are thought to be transported in different transport carriers. To analyze this differential transport in living neurons, we tagged the amyloid precursor protein (APP) and synaptophysin (p38) with green fluorescent protein (GFP) variants. The resulting fusion proteins, APP-yellow fluorescent protein (YFP), p38-enhanced GFP, and p38-enhanced cyan fluorescent protein, were expressed in hippocampal neurons, and the cells were imaged by video microscopy. APP-YFP was transported in elongated tubules that moved extremely fast (on average 4.5 micrometer/s) and over long distances. In contrast, p38-enhanced GFP-transporting structures were more vesicular and moved four times slower (0.9 micrometer/s) and over shorter distances only. Two-color video microscopy showed that the two proteins were sorted to different carriers that moved with different characteristics along axons of doubly transfected neurons. Antisense treatment using oligonucleotides against the kinesin heavy chain slowed down the long, continuous movement of APP-YFP tubules and increased frequency of directional changes. These results demonstrate for the first time directly the sorting and transport of two axonal membrane proteins into different carriers. Moreover, the extremely fast-moving tubules represent a previously unidentified type of axonal carrier.
Assuntos
Transporte Axonal/fisiologia , Hipocampo/metabolismo , Proteínas de Membrana/metabolismo , Neurônios/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Proteínas de Bactérias/genética , Células Cultivadas , Imunofluorescência , Proteínas de Fluorescência Verde , Hipocampo/citologia , Cinesinas/química , Proteínas Luminescentes/genética , Proteínas de Membrana/genética , Microscopia de Vídeo , Microtúbulos/metabolismo , Neurônios/citologia , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/farmacologia , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sinaptofisina/genética , Sinaptofisina/metabolismoRESUMO
Transfection of primary neurons in culture has proven to be experimentally challenging in the past. To overcome these limitations, we present a detailed transfection protocol for hippocampal neurons based on DNA/Ca(2+)-phosphate coprecipitation. The main advantages being (1) the speed and convenience, (2) the remarkable efficiency of transfection for mature neurons, and (3) consistent health of the neurons upon transfection allowing subsequent manipulations. The strength of this protocol is convincingly demonstrated by the fact that the expressed protein can be detected biochemically on Western blots.
Assuntos
Hipocampo/citologia , Hipocampo/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Animais , Técnicas de Cultura de Células , Proteínas de Fluorescência Verde , Indicadores e Reagentes/metabolismo , Proteínas Luminescentes , Ratos , TransfecçãoRESUMO
Dendritic mRNA transport and local translation at individual potentiated synapses may represent an elegant way to form synaptic memory. Recently, we characterized Staufen, a double-stranded RNA-binding protein, in rat hippocampal neurons and showed its presence in large RNA-containing granules, which colocalize with microtubules in dendrites. In this paper, we transiently transfect hippocampal neurons with human Staufen-green fluorescent protein (GFP) and find fluorescent granules in the somatodendritic domain of these cells. Human Stau-GFP granules show the same cellular distribution and size and also contain RNA, as already shown for the endogenous Stau particles. In time-lapse videomicroscopy, we show the bidirectional movement of these Staufen-GFP-labeled granules from the cell body into dendrites and vice versa. The average speed of these particles was 6.4 microm/min with a maximum velocity of 24. 3 microm/min. Moreover, we demonstrate that the observed assembly into granules and their subsequent dendritic movement is microtubule dependent. Taken together, we have characterized a novel, nonvesicular, microtubule-dependent transport pathway involving RNA-containing granules with Staufen as a core component. This is the first demonstration in living neurons of movement of an essential protein constituent of the mRNA transport machinery.
Assuntos
Dendritos/metabolismo , Hipocampo/citologia , Microtúbulos/metabolismo , Organelas/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Células Cultivadas , Proteínas do Citoesqueleto , Dendritos/efeitos dos fármacos , Proteínas de Fluorescência Verde , Hipocampo/embriologia , Humanos , Cinética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Microscopia de Vídeo , Microtúbulos/efeitos dos fármacos , Movimento/efeitos dos fármacos , Nocodazol/farmacologia , Distribuição Normal , Organelas/efeitos dos fármacos , Organelas/genética , Proteínas de Ligação a RNA/genética , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
Recent development of in vivo microscopy techniques, including green fluorescent proteins, has allowed the visualization of a wide range of dynamic processes in living cells. For quantitative and visual interpretation of such processes, new concepts for time-resolved image analysis and continuous time-space visualization are required. Here, we describe a versatile and fully automated approach consisting of four techniques, namely highly sensitive object detection, fuzzy logic-based dynamic object tracking, computer graphical visualization, and measurement in time-space. Systematic model simulations were performed to evaluate the reliability of the automated object detection and tracking method. To demonstrate potential applications, the method was applied to the analysis of secretory membrane traffic and the functional dynamics of nuclear compartments enriched in pre-mRNA splicing factors.
Assuntos
Fenômenos Fisiológicos Celulares , Microscopia/métodos , Modelos Biológicos , Animais , Biologia/métodos , Chlorocebus aethiops , Cromograninas/análise , Cromograninas/genética , Simulação por Computador , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Proteínas Recombinantes de Fusão/análise , Fatores de Tempo , Transfecção , Células VeroAssuntos
Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência/métodos , Proteínas/metabolismo , Células 3T3 , Animais , Membrana Celular/metabolismo , Chlorocebus aethiops , Cricetinae , Cães , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Processamento de Imagem Assistida por Computador , Camundongos , Microscopia de Fluorescência/instrumentação , Microscopia de Vídeo , Transporte Proteico , Transfecção , Células Vero , Rede trans-Golgi/metabolismoRESUMO
Human chromogranin B (hCgB), a soluble marker protein of neuroendocrine secretory granules, was fused to green fluorescent protein (GFP). Two GFP-mutants with different folding properties, S65T and EGFP, were used to produce two recombinant proteins, hCgB-GFP(S65T) and hCgB-EGFP, respectively. After transient expression only hCgB-EGFP elicited green fluorescence in the neuroendocrine cell line PC12. Pulse-chase experiments with [35S]sulfate followed by subcellular fractionation showed that hCgB-EGFP was sorted with high efficiency to immature secretory granules (ISG). Confocal microscopy revealed that fluorescent hCgB-EGFP colocalized largely with synaptotagmin, a membrane marker of secretory granules and synaptic-like microvesicles, and significantly with endogenous rat chromogranin B (rCgB), a soluble marker of secretory granules. Upon stimulation of transfected cells with 5 mM Ba2+ or by depolarization with 50 mM K+ hCgB-EGFP underwent regulated exocytosis. The dynamics of green fluorescent secretory granules beneath the plasma membrane (PM) of living PC12 cells were visualized by confocal microscopy. The majority of these vesicles did not move within 8.5 sec as if they were docked. In contrast, in NGF-induced cells most of the secretory granules beneath the somatic PM moved within the same time period whereas only little movement was observed in the neurites. These findings indicate that in differentiated PC12 cells the majority of the docking zones are not in the soma but are distributed along the neurites. In conclusion, the fusion protein hCgB-EGFP provides a powerful tool to study in real time vesicular traffic in the regulated pathway of protein secretion.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Proteínas Luminescentes/metabolismo , Sistemas Neurossecretores/ultraestrutura , Animais , Compostos de Bário/farmacologia , Biomarcadores/análise , Cromograninas/análise , Cromograninas/efeitos dos fármacos , Cromograninas/metabolismo , Fluorescência , Expressão Gênica , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/efeitos dos fármacos , Proteínas Luminescentes/genética , Microscopia Confocal , Fatores de Crescimento Neural/farmacologia , Células PC12 , Proteínas/metabolismo , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Projetos de Pesquisa , Fatores de TempoRESUMO
Biosynthetic transport from the trans-Golgi network (TGN) to the plasma membrane (PM) is mediated by secretory vesicles. We analyzed secretory vesicle transport in real time using a GFP-tagged secretory protein, hCgB-GFP, consisting of human chromogranin B (hCgB) and green fluorescent protein (GFP). The fusion protein was expressed transiently in Vero cells or in a stable clone after induction with butyrate. After arrest of the biosynthetic protein transport at 20 degrees C, fluorescent hCgB-GFP colocalized with TGN38, a marker of the TGN. Subsequent release of the secretion block at 37 degrees C led to the formation of green fluorescent vesicles. Confocal analysis revealed that these vesicles were devoid of TGN38 and of Texas Red-coupled transferrin and cathepsin D, markers of the endosomal/lysosomal pathway. As determined by fluorometry and metabolic labelling hCgB-GFP was secreted from the TGN to the PM with a t(1/2) of 20-30 minutes. Video-microscope analysis of green fluorescent vesicles showed brief periods of rapid directed movement with maximal velocities of 1 microm/second. Vesicle movement occurred in all directions, centrifugal, centripetal and circumferential, and 50% of the vesicles analyzed reversed their direction of movement at least once within an observation period of 45 seconds. In the presence of nocodazole the movement of fluorescent vesicles ceased. Concomitantly, secretion of hCgB-GFP was slowed but not completely blocked. We suggest that microtubules (MT) facilitate the delivery of secretory vesicles to the PM by a stochastic transport, thereby increasing the probability for a vesicle/target membrane encounter.
Assuntos
Grânulos Citoplasmáticos/fisiologia , Microtúbulos/fisiologia , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Membrana Celular/fisiologia , Chlorocebus aethiops , Cromograninas/metabolismo , Endossomos/fisiologia , Complexo de Golgi/fisiologia , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/metabolismo , Lisossomos/fisiologia , Microtúbulos/efeitos dos fármacos , Movimento , Nocodazol/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Células VeroRESUMO
Green fluorescent protein fused to human chromogranin B or neuropeptide Y was expressed in PC12 cells and caused bright, punctate fluorescence. The fluorescent points colocalized with the endogenous secretory granule marker dopamine beta-hydroxylase. Stimulation of live PC12 cells with elevated [K+], or of permeabilized PC12 cells with Ca2+, led to Ca2+-dependent loss of fluorescence from neurites. Ca2+ stimulated secretion of both fusion proteins equally well. In living cells, single fluorescent granules were imaged by evanescent-wave fluorescence microscopy. Granules were seen to migrate; to stop, as if trapped by plasmalemmal docking sites; and then to disappear abruptly, as if through exocytosis. Evidently, GFP fused to secreted peptides is a fluorescent marker for dense-core secretory granules and may be used for time-resolved microscopy of single granules.
Assuntos
Cálcio/fisiologia , Proteínas Luminescentes , Peptídeos/metabolismo , Animais , Degranulação Celular , Cromograninas/metabolismo , Dopamina beta-Hidroxilase/metabolismo , Proteínas de Fluorescência Verde , Humanos , Microscopia de Fluorescência/métodos , Neuritos/metabolismo , Neuropeptídeo Y/metabolismo , Células PC12 , Ratos , Proteínas Recombinantes de Fusão , Gravação em VídeoRESUMO
The green fluorescent protein (GFP) of Aequorea victoria is a unique in vivo reporter for monitoring dynamic processes in cells or organisms. As a fusion tag GFP can be used to localize proteins, to follow their movement or to study the dynamics of the subcellular compartments to which these proteins are targeted. Recent studies where GFP technology has revealed new insights regarding physiological activities of living cells are discussed.
Assuntos
Proteínas Luminescentes , Animais , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Dados de Sequência MolecularRESUMO
We have expressed green fluorescent protein (GFP) from A. victoria in the secretory pathway of HeLa cells by fusing it to the C-terminus of a secretory protein, chromogranin B. Under normal culture conditions at 37 degrees C maturation of GFP to the fluorescent form was not detectable. However, fluorescent GFP was observed when biosynthetic protein transport was arrested at the intermediate compartment or the trans-Golgi network by temperature blocks (15 degrees C and 20 degrees C, respectively). Reversal of the temperature blocks allowed the visualization of secretion of fluorescent GFP and offers the possibility to analyse transport in the secretory pathway in living cells.