Limits...
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

Variability of protein co-expression ratios at the single cell level for identical co-transfection conditions.(A) Wide-field images of cells co-transfected with a mixture of pH2B-mPlum/Gag and pGag-mEos2 at an equimolar ratio show high variability in H2B-mPlum expression. (B) Measured Gag/Gag-mEos2 co-expression ratios as a function of the co-transfection ratio of plasmids. Each symbol corresponds to the co-expression ratio measured in a single cell. The black circles show the mean values. The linear fit of the mean co-expression level is plotted as a dashed line. The expression levels were extracted from the extrapolated FCS calibration curves shown in Fig. 3. Scale bars correspond to 5 µm.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4262470&req=5

pone-0115095-g004: Variability of protein co-expression ratios at the single cell level for identical co-transfection conditions.(A) Wide-field images of cells co-transfected with a mixture of pH2B-mPlum/Gag and pGag-mEos2 at an equimolar ratio show high variability in H2B-mPlum expression. (B) Measured Gag/Gag-mEos2 co-expression ratios as a function of the co-transfection ratio of plasmids. Each symbol corresponds to the co-expression ratio measured in a single cell. The black circles show the mean values. The linear fit of the mean co-expression level is plotted as a dashed line. The expression levels were extracted from the extrapolated FCS calibration curves shown in Fig. 3. Scale bars correspond to 5 µm.

Mentions: Previous studies have shown that the unlabeled to labeled Gag stoichiometry scales linearly with plasmid stoichiometry upon co-transfection as measured by bulk assays, performed on samples consisting of hundreds of thousands of VLPs produced by thousands of cells [6]. However, the critical biological process of Gag assembly and VLP formation in live cells is studied at the single cell level. Single cell measurements of protein stoichiometry are thus necessary to assess whether linear scaling is also preserved at the single cell level. Using our developed quantification tools, we studied the correlation between the co-transfection and co-expression ratio of Gag/Gag-mEos2 at the single cell level. We co-transfected cells with different plasmid stoichiometries ranging from equimolar concentrations of pGag-mEos2 and pH2B-mPlum/Gag to a fivefold excess in pH2B-mPlum/Gag and measured protein stoichiometry (Fig. 4) using the calibration curves obtained with FCS. We note that the commonly used tenfold excess in unlabeled Gag could not be used here because in those conditions Gag-mEos2 expression was too low for unambiguous identification and quantification of Gag-mEos2 levels in single cells.


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)

Variability of protein co-expression ratios at the single cell level for identical co-transfection conditions.(A) Wide-field images of cells co-transfected with a mixture of pH2B-mPlum/Gag and pGag-mEos2 at an equimolar ratio show high variability in H2B-mPlum expression. (B) Measured Gag/Gag-mEos2 co-expression ratios as a function of the co-transfection ratio of plasmids. Each symbol corresponds to the co-expression ratio measured in a single cell. The black circles show the mean values. The linear fit of the mean co-expression level is plotted as a dashed line. The expression levels were extracted from the extrapolated FCS calibration curves shown in Fig. 3. Scale bars correspond to 5 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0115095-g004: Variability of protein co-expression ratios at the single cell level for identical co-transfection conditions.(A) Wide-field images of cells co-transfected with a mixture of pH2B-mPlum/Gag and pGag-mEos2 at an equimolar ratio show high variability in H2B-mPlum expression. (B) Measured Gag/Gag-mEos2 co-expression ratios as a function of the co-transfection ratio of plasmids. Each symbol corresponds to the co-expression ratio measured in a single cell. The black circles show the mean values. The linear fit of the mean co-expression level is plotted as a dashed line. The expression levels were extracted from the extrapolated FCS calibration curves shown in Fig. 3. Scale bars correspond to 5 µm.
Mentions: Previous studies have shown that the unlabeled to labeled Gag stoichiometry scales linearly with plasmid stoichiometry upon co-transfection as measured by bulk assays, performed on samples consisting of hundreds of thousands of VLPs produced by thousands of cells [6]. However, the critical biological process of Gag assembly and VLP formation in live cells is studied at the single cell level. Single cell measurements of protein stoichiometry are thus necessary to assess whether linear scaling is also preserved at the single cell level. Using our developed quantification tools, we studied the correlation between the co-transfection and co-expression ratio of Gag/Gag-mEos2 at the single cell level. We co-transfected cells with different plasmid stoichiometries ranging from equimolar concentrations of pGag-mEos2 and pH2B-mPlum/Gag to a fivefold excess in pH2B-mPlum/Gag and measured protein stoichiometry (Fig. 4) using the calibration curves obtained with FCS. We note that the commonly used tenfold excess in unlabeled Gag could not be used here because in those conditions Gag-mEos2 expression was too low for unambiguous identification and quantification of Gag-mEos2 levels in single cells.

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