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A Quantitative Approach to Evaluate the Impact of Fluorescent Labeling on Membrane-Bound HIV-Gag Assembly by Titration of Unlabeled Proteins.

Gunzenhäuser J, Wyss R, Manley S - PLoS ONE (2014)

Bottom Line: Using super-resolution imaging based on photoactivated localization microscopy (PALM) combined with molecular counting we then study the nanoscale morphology of Gag clusters as a function of unlabeled to labeled Gag ratios in single cells.We show that for a given co-transfection ratio, individual cells express a wide range of protein ratios, necessitating a quantitative read-out for the expression of unlabeled Gag.Further, we show that monomerically labeled Gag assembles into membrane-bound clusters that are morphologically indistinguishable from mixtures of unlabeled and labeled Gag.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Experimental Biophysics, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.

ABSTRACT
The assembly process of the human immunodeficiency virus 1 (HIV-1) is driven by the viral polyprotein Gag. Fluorescence imaging of Gag protein fusions is widely performed and has revealed important information on viral assembly. Gag fusion proteins are commonly co-transfected with an unlabeled form of Gag to prevent labeling artifacts such as morphological defects and decreased infectivity. Although viral assembly is widely studied on individual cells, the efficiency of the co-transfection rescue has never been tested at the single cell level. Here, we first develop a methodology to quantify levels of unlabeled to labeled Gag in single cells using a fluorescent reporter protein for unlabeled Gag and fluorescence correlation spectroscopy. Using super-resolution imaging based on photoactivated localization microscopy (PALM) combined with molecular counting we then study the nanoscale morphology of Gag clusters as a function of unlabeled to labeled Gag ratios in single cells. We show that for a given co-transfection ratio, individual cells express a wide range of protein ratios, necessitating a quantitative read-out for the expression of unlabeled Gag. Further, we show that monomerically labeled Gag assembles into membrane-bound clusters that are morphologically indistinguishable from mixtures of unlabeled and labeled Gag.

No MeSH data available.


Related in: MedlinePlus

Quantifying the bulk expression levels of H2B-mPlum and Gag.(A) Quantitative western blot. Lane 1: non-transfected cells. Lanes 2, 3, 4: biological replicates of total cell extracts from Cos7 cells transfected with the pH2B-mPlum/Gag construct. Gag (the diamonds indicate bands corresponding to degradation products that were included in the quantification) and H2B-mPlum were detected using specific anti-Gag and anti-RFP antibodies. Lanes 5, 6, 7, 8, 9: purified Gag (upper panel) and mPlum (lower panel) ranging from 6.25 to 100 ng and 15 to 240 ng respectively. (B) Calibration curves obtained from serial protein dilutions (closed squares, from lanes 5–9) and measured Gag and H2B-mPlum concentration in cell extracts (open square, from lanes 2–4, mean and standard deviation for the triplicate are shown). The bands corresponding to the highest concentrations were omitted in the fit, their intensity being close to saturation.
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pone-0115095-g002: Quantifying the bulk expression levels of H2B-mPlum and Gag.(A) Quantitative western blot. Lane 1: non-transfected cells. Lanes 2, 3, 4: biological replicates of total cell extracts from Cos7 cells transfected with the pH2B-mPlum/Gag construct. Gag (the diamonds indicate bands corresponding to degradation products that were included in the quantification) and H2B-mPlum were detected using specific anti-Gag and anti-RFP antibodies. Lanes 5, 6, 7, 8, 9: purified Gag (upper panel) and mPlum (lower panel) ranging from 6.25 to 100 ng and 15 to 240 ng respectively. (B) Calibration curves obtained from serial protein dilutions (closed squares, from lanes 5–9) and measured Gag and H2B-mPlum concentration in cell extracts (open square, from lanes 2–4, mean and standard deviation for the triplicate are shown). The bands corresponding to the highest concentrations were omitted in the fit, their intensity being close to saturation.

Mentions: The presence of the fluorescent reporter H2B-mPlum provides only a qualitative read-out of unlabeled Gag in single cells (Fig. 1, B–D). If we can quantify the relative expression levels of the reporter protein and Gag, encoded from similar promoters within the same plasmid, it could also provide a quantitative read-out of unlabeled Gag expression. So, we assessed the total expression of H2B-mPlum and Gag by quantitative western blotting (Fig. 2, A and B).


A Quantitative Approach to Evaluate the Impact of Fluorescent Labeling on Membrane-Bound HIV-Gag Assembly by Titration of Unlabeled Proteins.

Gunzenhäuser J, Wyss R, Manley S - PLoS ONE (2014)

Quantifying the bulk expression levels of H2B-mPlum and Gag.(A) Quantitative western blot. Lane 1: non-transfected cells. Lanes 2, 3, 4: biological replicates of total cell extracts from Cos7 cells transfected with the pH2B-mPlum/Gag construct. Gag (the diamonds indicate bands corresponding to degradation products that were included in the quantification) and H2B-mPlum were detected using specific anti-Gag and anti-RFP antibodies. Lanes 5, 6, 7, 8, 9: purified Gag (upper panel) and mPlum (lower panel) ranging from 6.25 to 100 ng and 15 to 240 ng respectively. (B) Calibration curves obtained from serial protein dilutions (closed squares, from lanes 5–9) and measured Gag and H2B-mPlum concentration in cell extracts (open square, from lanes 2–4, mean and standard deviation for the triplicate are shown). The bands corresponding to the highest concentrations were omitted in the fit, their intensity being close to saturation.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4262470&req=5

pone-0115095-g002: Quantifying the bulk expression levels of H2B-mPlum and Gag.(A) Quantitative western blot. Lane 1: non-transfected cells. Lanes 2, 3, 4: biological replicates of total cell extracts from Cos7 cells transfected with the pH2B-mPlum/Gag construct. Gag (the diamonds indicate bands corresponding to degradation products that were included in the quantification) and H2B-mPlum were detected using specific anti-Gag and anti-RFP antibodies. Lanes 5, 6, 7, 8, 9: purified Gag (upper panel) and mPlum (lower panel) ranging from 6.25 to 100 ng and 15 to 240 ng respectively. (B) Calibration curves obtained from serial protein dilutions (closed squares, from lanes 5–9) and measured Gag and H2B-mPlum concentration in cell extracts (open square, from lanes 2–4, mean and standard deviation for the triplicate are shown). The bands corresponding to the highest concentrations were omitted in the fit, their intensity being close to saturation.
Mentions: The presence of the fluorescent reporter H2B-mPlum provides only a qualitative read-out of unlabeled Gag in single cells (Fig. 1, B–D). If we can quantify the relative expression levels of the reporter protein and Gag, encoded from similar promoters within the same plasmid, it could also provide a quantitative read-out of unlabeled Gag expression. So, we assessed the total expression of H2B-mPlum and Gag by quantitative western blotting (Fig. 2, A and B).

Bottom Line: Using super-resolution imaging based on photoactivated localization microscopy (PALM) combined with molecular counting we then study the nanoscale morphology of Gag clusters as a function of unlabeled to labeled Gag ratios in single cells.We show that for a given co-transfection ratio, individual cells express a wide range of protein ratios, necessitating a quantitative read-out for the expression of unlabeled Gag.Further, we show that monomerically labeled Gag assembles into membrane-bound clusters that are morphologically indistinguishable from mixtures of unlabeled and labeled Gag.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Experimental Biophysics, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.

ABSTRACT
The assembly process of the human immunodeficiency virus 1 (HIV-1) is driven by the viral polyprotein Gag. Fluorescence imaging of Gag protein fusions is widely performed and has revealed important information on viral assembly. Gag fusion proteins are commonly co-transfected with an unlabeled form of Gag to prevent labeling artifacts such as morphological defects and decreased infectivity. Although viral assembly is widely studied on individual cells, the efficiency of the co-transfection rescue has never been tested at the single cell level. Here, we first develop a methodology to quantify levels of unlabeled to labeled Gag in single cells using a fluorescent reporter protein for unlabeled Gag and fluorescence correlation spectroscopy. Using super-resolution imaging based on photoactivated localization microscopy (PALM) combined with molecular counting we then study the nanoscale morphology of Gag clusters as a function of unlabeled to labeled Gag ratios in single cells. We show that for a given co-transfection ratio, individual cells express a wide range of protein ratios, necessitating a quantitative read-out for the expression of unlabeled Gag. Further, we show that monomerically labeled Gag assembles into membrane-bound clusters that are morphologically indistinguishable from mixtures of unlabeled and labeled Gag.

No MeSH data available.


Related in: MedlinePlus