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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

Calibration curves obtained with FCS.The molar concentration of (A) mEos2 and (B) mPlum is shown as a function of the integrated cytosolic fluorescence intensity. Counts are given in arbitrary units and refer to the integrated fluorescence intensity of the wide-field image of the cell. Mean and standard deviation from three measurements within one cell are shown. A linear fit (solid line) with a 95% confidence interval (dashed lines) is shown. The extrapolated linear fit of the fluorophore concentration as a function of the integrated fluorescence intensity was used for the determination of protein expression levels.
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pone-0115095-g003: Calibration curves obtained with FCS.The molar concentration of (A) mEos2 and (B) mPlum is shown as a function of the integrated cytosolic fluorescence intensity. Counts are given in arbitrary units and refer to the integrated fluorescence intensity of the wide-field image of the cell. Mean and standard deviation from three measurements within one cell are shown. A linear fit (solid line) with a 95% confidence interval (dashed lines) is shown. The extrapolated linear fit of the fluorophore concentration as a function of the integrated fluorescence intensity was used for the determination of protein expression levels.

Mentions: The western blot approach allowed us to quantify bulk protein concentrations. VLP assembly however, is studied at the single cell level. For single cells the concentration of FPs is reflected by the total integrated fluorescence intensity of the whole cell (for Gag-mEos2) or the nucleus (for unlabeled Gag via the reporter H2B-mPlum). The FPs mPlum and mEos2 used in this study thus provide per se read-outs for single cell expression levels of Gag and Gag-mEos2. The quantitative relationship between fluorescence intensity and protein concentration, however, also depends on the brightness of each FP. To account for this, we used FCS, which permits the experimental determination of molar concentration of FPs in living cells, and in turn can be related to the measured fluorescence intensity. FCS measures spontaneous fluorescence intensity fluctuations in the femtoliter focal volume. The temporal correlation of these fluctuations reflect protein concentrations and mobilities. We performed FCS in living cells to obtain calibration curves relating the protein concentration of mEos2 (Fig. 3A) and mPlum (Fig. 3B) to the mean cytosolic fluorescence intensity (S3 and S4 Figures in S1 File).


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)

Calibration curves obtained with FCS.The molar concentration of (A) mEos2 and (B) mPlum is shown as a function of the integrated cytosolic fluorescence intensity. Counts are given in arbitrary units and refer to the integrated fluorescence intensity of the wide-field image of the cell. Mean and standard deviation from three measurements within one cell are shown. A linear fit (solid line) with a 95% confidence interval (dashed lines) is shown. The extrapolated linear fit of the fluorophore concentration as a function of the integrated fluorescence intensity was used for the determination of protein expression levels.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0115095-g003: Calibration curves obtained with FCS.The molar concentration of (A) mEos2 and (B) mPlum is shown as a function of the integrated cytosolic fluorescence intensity. Counts are given in arbitrary units and refer to the integrated fluorescence intensity of the wide-field image of the cell. Mean and standard deviation from three measurements within one cell are shown. A linear fit (solid line) with a 95% confidence interval (dashed lines) is shown. The extrapolated linear fit of the fluorophore concentration as a function of the integrated fluorescence intensity was used for the determination of protein expression levels.
Mentions: The western blot approach allowed us to quantify bulk protein concentrations. VLP assembly however, is studied at the single cell level. For single cells the concentration of FPs is reflected by the total integrated fluorescence intensity of the whole cell (for Gag-mEos2) or the nucleus (for unlabeled Gag via the reporter H2B-mPlum). The FPs mPlum and mEos2 used in this study thus provide per se read-outs for single cell expression levels of Gag and Gag-mEos2. The quantitative relationship between fluorescence intensity and protein concentration, however, also depends on the brightness of each FP. To account for this, we used FCS, which permits the experimental determination of molar concentration of FPs in living cells, and in turn can be related to the measured fluorescence intensity. FCS measures spontaneous fluorescence intensity fluctuations in the femtoliter focal volume. The temporal correlation of these fluctuations reflect protein concentrations and mobilities. We performed FCS in living cells to obtain calibration curves relating the protein concentration of mEos2 (Fig. 3A) and mPlum (Fig. 3B) to the mean cytosolic fluorescence intensity (S3 and S4 Figures in S1 File).

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