RESUMO
Understanding the dynamic relationship between components of a system or pathway at the individual cell level is a current challenge. To address this, we developed an approach that allows simultaneous tracking of several endogenous proteins of choice within individual living human cells. The approach is based on fluorescent tagging of proteins at their native locus by directed gene targeting. A fluorescent tag-encoding DNA is introduced as a new exon into the intronic region of the gene of interest, resulting in expression of a full-length fluorescently tagged protein. We used this approach to establish human cell lines simultaneously expressing two components of a major antioxidant defense system, thioredoxin 1 (Trx) and thioredoxin reductase 1 (TrxR1), labeled with CFP and YFP, respectively. We find that the distributions of both proteins between nuclear and cytoplasmic compartments were highly variable between cells. However, the two proteins did not vary independently of each other: protein levels of Trx and TrxR1 in both the whole cell and the nucleus were substantially correlated. We further find that in response to a stress-inducing drug (CPT), both Trx and TrxR1 accumulated in the nuclei in a manner that was highly temporally correlated. This accumulation considerably reduced cell-to-cell variability in nuclear content of both proteins, suggesting a uniform response of the thioredoxin system to stress. These results indicate that Trx and TrxR1 act in concert in response to stress in regard to both time course and variability. Thus, our approach provides an efficient tool for studying dynamic relationship between components of systems of interest at a single-cell level.
Assuntos
Proteínas/metabolismo , Linhagem Celular , Separação Celular , Citometria de Fluxo , Corantes Fluorescentes , Humanos , Proteínas/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Recent advances allow tracking the levels and locations of a thousand proteins in individual living human cells over time using a library of annotated reporter cell clones (LARC). This library was created by Cohen et al. to study the proteome dynamics of a human lung carcinoma cell-line treated with an anti-cancer drug. Here, we report the Dynamic Proteomics database for the proteins studied by Cohen et al. Each cell-line clone in LARC has a protein tagged with yellow fluorescent protein, expressed from its endogenous chromosomal location, under its natural regulation. The Dynamic Proteomics interface facilitates searches for genes of interest, downloads of protein fluorescent movies and alignments of dynamics following drug addition. Each protein in the database is displayed with its annotation, cDNA sequence, fluorescent images and movies obtained by the time-lapse microscopy. The protein dynamics in the database represents a quantitative trace of the protein fluorescence levels in nucleus and cytoplasm produced by image analysis of movies over time. Furthermore, a sequence analysis provides a search and comparison of up to 50 input DNA sequences with all cDNAs in the library. The raw movies may be useful as a benchmark for developing image analysis tools for individual-cell dynamic-proteomics. The database is available at http://www.dynamicproteomics.net/.
Assuntos
Biologia Computacional/métodos , Bases de Dados Genéticas , Bases de Dados de Ácidos Nucleicos , Bases de Dados de Proteínas , Proteômica/métodos , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Biologia Computacional/tendências , Ensaios de Seleção de Medicamentos Antitumorais , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacologia , Biblioteca Gênica , Humanos , Armazenamento e Recuperação da Informação/métodos , Internet , Estrutura Terciária de Proteína , SoftwareRESUMO
Signal-transduction cascades are usually studied on cell averages, masking variability between individual cells. To address this, we studied in individual cells the dynamic response of ERK2, a well-characterized MAPK signaling protein, which enters the nucleus upon stimulation. Using fluorescent tagging at the endogenous chromosomal locus, we found that cells show wide basal variation in ERK2 nuclear levels. Upon EGF stimulation, cells show (1) a fold-change response, where peak nuclear accumulation of ERK2 is proportional to basal level in each cell; and (2) exact adaptation in nuclear levels of ERK2, returning to original basal level of each cell. The timing of ERK2 dynamics is more precise between cells than its amplitude. We further found that in some cells ERK2 exhibits a second pulse of nuclear entry, smaller than the first. The present study suggests that this signaling system compensates for natural biological noise: despite large variation in nuclear basal levels, ERK2's fold dynamics is similar between cells.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Expressão Gênica/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Humanos , Proteínas Luminescentes/análise , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismoRESUMO
In the present study, we demonstrated that the antiapoptotic function of Bcl-2 in mast cells is significantly dependent on its association with the heat shock protein 90beta (Hsp90beta). Dissociation of these 2 proteins inhibits the antiapoptotic activity of Bcl-2 by initiating the release of cytochrome c from mitochondria into cytosol and increasing the activity of caspase 3 and caspase 7, resulting in mast-cell apoptosis. The antiapoptotic activity of Bcl-2 was greatly affected by knocking-out specifically Hsp90beta using the RNA interference approach. Thus, for the first time, it has been shown that Hsp90beta might modulate the antiapoptotic activity of Bcl-2 at least in mast cells. These findings could have implications for a novel strategy of regulating apoptosis in patients with mastocytosis and other mast cell-associated diseases.
Assuntos
Apoptose/fisiologia , Proteínas de Choque Térmico HSP90/fisiologia , Mastócitos/citologia , Mastócitos/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Animais , Células da Medula Óssea , Caspase 3 , Caspases/metabolismo , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/fisiologia , Mitocôndrias/ultraestrutura , Ratos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Apoptosis or programmed cell death plays an important role in a wide variety of physiological processes. Apoptosis is regulated by proteins of the Bcl-2 family consisting of both anti-apoptotic and pro-apoptotic factors. The direct involvement of the Bcl-2 protein family in the process of mast cell apoptosis has not been clarified. We have used a single-chain antibody (scFv) raised against Bcl-2 derived from human phage-display antibody library. The addition of TAT sequence, which is responsible for translocation through the membrane, endows the anti-Bcl-2-scFv with the ability to penetrate living cells. The association of anti-Bcl-2-scFv-TAT with intracellular Bcl-2 leads to neutralization of Bcl-2 and eradication of its anti-apoptotic activity in two types of mast cells and in a human breast cancer cell line. Moreover, we found by mass spectrometry and co-immunoprecipitation assay that heat shock protein 90b (Hsp90b) forms a complex with Bcl-2 in mast cells. Thus, understanding the network of interactions between Bcl-2 and non-Bcl-2 family members might help in development of more specific drugs and cancer therapy.
Assuntos
Apoptose/fisiologia , Mastócitos/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Células Cultivadas , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Mastócitos/citologiaRESUMO
Apoptosis or programmed cell death plays an important role in a wide variety of physiologic processes and is regulated by proteins of the Bcl-2 family consisting of both antiapoptotic and proapoptotic factors. The direct involvement of the Bcl-2 protein family in the process of mast cell apoptosis has not been clarified. In the present work we have used a single-chain antibody (scFv) raised against Bcl-2 derived from a semisynthetic human phage-display antibody library. The addition of TAT sequence, which is responsible for translocation through the membrane, endows the anti-Bcl-2-scFv with the ability to penetrate living cells. Moreover, it specifically neutralizes Bcl-2 intracellularly by binding to the BH1 domain and eradicates its antiapoptotic activity in 2 types of mast cells and in a human breast cancer cell line.