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Generation of double-labeled reporter cell lines for studying co-dynamics of endogenous proteins in individual human cells.

Issaeva I, Cohen AA, Eden E, Cohen-Saidon C, Danon T, Cohen L, Alon U - PLoS ONE (2010)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel. irina.issaeva@weizmann.ac.il

ABSTRACT
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.

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Nuclear levels of both tagged and wild-type Trx and TrxR1 increase after H2O2 exposure.A) Nuclear fluorescence profile of Trx-CFP, TrxR1-YFP and Cherry-tagged proteins following H2O2 addition. Data obtained from time-lapse movies of Trx-CFP/TrxR1-YFP cells. Error bars denote standard error. B) Nuclear accumulation of tagged and wild-type Trx and TrxR1 upon H2O2 treatment. Average nuclear protein levels measured before (0h) and after (6h) H2O2 addition. Blue bars, data from fixed H1299 cells immunostained for Trx and TrxR1. Red bars, data from time-lapse movies of live Trx-CFP/TrxR1-YFP cells. C) Fluorescent images of live Trx-CFP/TrxR1-YFP cells and of fixed H1299 cells immunostained for Trx and TrxR1 before (0h) and after (6h) H2O2 addition. Images of Trx-CFP/TrxR1-YFP cells represent snapshots from the time-lapse movie. DAPI staining is used to mark nuclear boundaries in the H1299 cells. Scale bars denote 20 microns.
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pone-0013524-g006: Nuclear levels of both tagged and wild-type Trx and TrxR1 increase after H2O2 exposure.A) Nuclear fluorescence profile of Trx-CFP, TrxR1-YFP and Cherry-tagged proteins following H2O2 addition. Data obtained from time-lapse movies of Trx-CFP/TrxR1-YFP cells. Error bars denote standard error. B) Nuclear accumulation of tagged and wild-type Trx and TrxR1 upon H2O2 treatment. Average nuclear protein levels measured before (0h) and after (6h) H2O2 addition. Blue bars, data from fixed H1299 cells immunostained for Trx and TrxR1. Red bars, data from time-lapse movies of live Trx-CFP/TrxR1-YFP cells. C) Fluorescent images of live Trx-CFP/TrxR1-YFP cells and of fixed H1299 cells immunostained for Trx and TrxR1 before (0h) and after (6h) H2O2 addition. Images of Trx-CFP/TrxR1-YFP cells represent snapshots from the time-lapse movie. DAPI staining is used to mark nuclear boundaries in the H1299 cells. Scale bars denote 20 microns.

Mentions: We also treated the cells with H2O2, a classical inducer of oxidative stress. Using time-lapse microscopy we observed a considerable rise in nuclear levels of both Trx-CFP and TrxR1-YFP following H2O2 addition (Figure 6A). Nuclear accumulation was also observed in the H1299 cells fixed at 6 h after H2O2 exposure and immunostained for the endogenous Trx and TrxR1 proteins (Figure 6B, C).


Generation of double-labeled reporter cell lines for studying co-dynamics of endogenous proteins in individual human cells.

Issaeva I, Cohen AA, Eden E, Cohen-Saidon C, Danon T, Cohen L, Alon U - PLoS ONE (2010)

Nuclear levels of both tagged and wild-type Trx and TrxR1 increase after H2O2 exposure.A) Nuclear fluorescence profile of Trx-CFP, TrxR1-YFP and Cherry-tagged proteins following H2O2 addition. Data obtained from time-lapse movies of Trx-CFP/TrxR1-YFP cells. Error bars denote standard error. B) Nuclear accumulation of tagged and wild-type Trx and TrxR1 upon H2O2 treatment. Average nuclear protein levels measured before (0h) and after (6h) H2O2 addition. Blue bars, data from fixed H1299 cells immunostained for Trx and TrxR1. Red bars, data from time-lapse movies of live Trx-CFP/TrxR1-YFP cells. C) Fluorescent images of live Trx-CFP/TrxR1-YFP cells and of fixed H1299 cells immunostained for Trx and TrxR1 before (0h) and after (6h) H2O2 addition. Images of Trx-CFP/TrxR1-YFP cells represent snapshots from the time-lapse movie. DAPI staining is used to mark nuclear boundaries in the H1299 cells. Scale bars denote 20 microns.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2958823&req=5

pone-0013524-g006: Nuclear levels of both tagged and wild-type Trx and TrxR1 increase after H2O2 exposure.A) Nuclear fluorescence profile of Trx-CFP, TrxR1-YFP and Cherry-tagged proteins following H2O2 addition. Data obtained from time-lapse movies of Trx-CFP/TrxR1-YFP cells. Error bars denote standard error. B) Nuclear accumulation of tagged and wild-type Trx and TrxR1 upon H2O2 treatment. Average nuclear protein levels measured before (0h) and after (6h) H2O2 addition. Blue bars, data from fixed H1299 cells immunostained for Trx and TrxR1. Red bars, data from time-lapse movies of live Trx-CFP/TrxR1-YFP cells. C) Fluorescent images of live Trx-CFP/TrxR1-YFP cells and of fixed H1299 cells immunostained for Trx and TrxR1 before (0h) and after (6h) H2O2 addition. Images of Trx-CFP/TrxR1-YFP cells represent snapshots from the time-lapse movie. DAPI staining is used to mark nuclear boundaries in the H1299 cells. Scale bars denote 20 microns.
Mentions: We also treated the cells with H2O2, a classical inducer of oxidative stress. Using time-lapse microscopy we observed a considerable rise in nuclear levels of both Trx-CFP and TrxR1-YFP following H2O2 addition (Figure 6A). Nuclear accumulation was also observed in the H1299 cells fixed at 6 h after H2O2 exposure and immunostained for the endogenous Trx and TrxR1 proteins (Figure 6B, C).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel. irina.issaeva@weizmann.ac.il

ABSTRACT
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.

Show MeSH