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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

Unlabeled and labeled Gag protein incorporation into forming VLPs.(A) Diffraction limited image and (B) corresponding super-resolution image of a Cos7 cell expressing Gag-mEos2 only. (C) Diffraction limited image and (D) corresponding super-resolution image of a Cos7 cell co-expressing Gag and Gag-mEos2, co-transfected at an equimolar ratio. The presence of Gag is indicated via the fluorescent reporter protein H2B-mPlum (lower left inset). Scale bars, 5 µm. (E) Left, zoom of indicated VLP in (B). Right, zoom of indicated VLP in (D). Scale bars, 100 nm. (F) Gag density in forming VLPs as determined from PALM images and molecular counting. Closed squares indicate the mean, horizontal lines within the boxes median, and boxes indicate 25th and 75th percentiles while vertical bars indicate 5th and 95th percentiles. Cells co-expressing unlabeled and labeled Gag display lower densities, indicating the presence of unlabeled Gag proteins within clusters, invisible to fluorescence detection.
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pone-0115095-g005: Unlabeled and labeled Gag protein incorporation into forming VLPs.(A) Diffraction limited image and (B) corresponding super-resolution image of a Cos7 cell expressing Gag-mEos2 only. (C) Diffraction limited image and (D) corresponding super-resolution image of a Cos7 cell co-expressing Gag and Gag-mEos2, co-transfected at an equimolar ratio. The presence of Gag is indicated via the fluorescent reporter protein H2B-mPlum (lower left inset). Scale bars, 5 µm. (E) Left, zoom of indicated VLP in (B). Right, zoom of indicated VLP in (D). Scale bars, 100 nm. (F) Gag density in forming VLPs as determined from PALM images and molecular counting. Closed squares indicate the mean, horizontal lines within the boxes median, and boxes indicate 25th and 75th percentiles while vertical bars indicate 5th and 95th percentiles. Cells co-expressing unlabeled and labeled Gag display lower densities, indicating the presence of unlabeled Gag proteins within clusters, invisible to fluorescence detection.

Mentions: We used our quantitative read-outs for single-cell expression levels to identify and examine cells effectively expressing relative concentrations of fluorescently labeled to unlabeled Gag over a range of an order of magnitude (Fig. 4B). Changing Gag protein stoichiometry in a cell does not a priori guarantee an incorporation of both types of Gag into VLPs. Thus, we tested whether unlabeled Gag is incorporated by quantitative PALM imaging and molecular counting analysis. Cells expressing Gag-mEos2 alone (Fig. 5, A and B) or Gag and Gag-mEos2 (Fig. 5, C and D) were first imaged in wide-field in the green channel to record diffraction-limited images of forming VLPs (Fig. 5A and C) and in the red/far-red channel to detect and quantify the expression level of H2B-mPlum and thus unlabeled Gag (Fig. 5C, inset). We then acquired data for PALM images by stochastically photoconverting Gag-mEos2 molecules into the red form with 405 laser light. Individual molecules were imaged, bleached, subsequently localized and their positions were rendered to form a composite PALM image (Fig. 5, B and D). Within PALM images we can resolve single clusters of Gag-mEos2 (Fig. 5E). We can moreover estimate the number and density of labeled proteins in nascent VLPs taking into account the photo-physical properties of mEos2 such as the average blinking rate and dark-time [7]. This analysis confirmed that the density of labeled Gag proteins within Gag clusters decreased when unlabeled Gag was co-expressed (Fig. 5F). This indicates incorporation of both labeled and unlabeled Gag proteins into forming VLPs, confirming previous work [9]. However, we did not find a clear correlation between the amount of unlabeled Gag within a single cell and the number of labeled Gag proteins per cluster.


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)

Unlabeled and labeled Gag protein incorporation into forming VLPs.(A) Diffraction limited image and (B) corresponding super-resolution image of a Cos7 cell expressing Gag-mEos2 only. (C) Diffraction limited image and (D) corresponding super-resolution image of a Cos7 cell co-expressing Gag and Gag-mEos2, co-transfected at an equimolar ratio. The presence of Gag is indicated via the fluorescent reporter protein H2B-mPlum (lower left inset). Scale bars, 5 µm. (E) Left, zoom of indicated VLP in (B). Right, zoom of indicated VLP in (D). Scale bars, 100 nm. (F) Gag density in forming VLPs as determined from PALM images and molecular counting. Closed squares indicate the mean, horizontal lines within the boxes median, and boxes indicate 25th and 75th percentiles while vertical bars indicate 5th and 95th percentiles. Cells co-expressing unlabeled and labeled Gag display lower densities, indicating the presence of unlabeled Gag proteins within clusters, invisible to fluorescence detection.
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pone-0115095-g005: Unlabeled and labeled Gag protein incorporation into forming VLPs.(A) Diffraction limited image and (B) corresponding super-resolution image of a Cos7 cell expressing Gag-mEos2 only. (C) Diffraction limited image and (D) corresponding super-resolution image of a Cos7 cell co-expressing Gag and Gag-mEos2, co-transfected at an equimolar ratio. The presence of Gag is indicated via the fluorescent reporter protein H2B-mPlum (lower left inset). Scale bars, 5 µm. (E) Left, zoom of indicated VLP in (B). Right, zoom of indicated VLP in (D). Scale bars, 100 nm. (F) Gag density in forming VLPs as determined from PALM images and molecular counting. Closed squares indicate the mean, horizontal lines within the boxes median, and boxes indicate 25th and 75th percentiles while vertical bars indicate 5th and 95th percentiles. Cells co-expressing unlabeled and labeled Gag display lower densities, indicating the presence of unlabeled Gag proteins within clusters, invisible to fluorescence detection.
Mentions: We used our quantitative read-outs for single-cell expression levels to identify and examine cells effectively expressing relative concentrations of fluorescently labeled to unlabeled Gag over a range of an order of magnitude (Fig. 4B). Changing Gag protein stoichiometry in a cell does not a priori guarantee an incorporation of both types of Gag into VLPs. Thus, we tested whether unlabeled Gag is incorporated by quantitative PALM imaging and molecular counting analysis. Cells expressing Gag-mEos2 alone (Fig. 5, A and B) or Gag and Gag-mEos2 (Fig. 5, C and D) were first imaged in wide-field in the green channel to record diffraction-limited images of forming VLPs (Fig. 5A and C) and in the red/far-red channel to detect and quantify the expression level of H2B-mPlum and thus unlabeled Gag (Fig. 5C, inset). We then acquired data for PALM images by stochastically photoconverting Gag-mEos2 molecules into the red form with 405 laser light. Individual molecules were imaged, bleached, subsequently localized and their positions were rendered to form a composite PALM image (Fig. 5, B and D). Within PALM images we can resolve single clusters of Gag-mEos2 (Fig. 5E). We can moreover estimate the number and density of labeled proteins in nascent VLPs taking into account the photo-physical properties of mEos2 such as the average blinking rate and dark-time [7]. This analysis confirmed that the density of labeled Gag proteins within Gag clusters decreased when unlabeled Gag was co-expressed (Fig. 5F). This indicates incorporation of both labeled and unlabeled Gag proteins into forming VLPs, confirming previous work [9]. However, we did not find a clear correlation between the amount of unlabeled Gag within a single cell and the number of labeled Gag proteins per cluster.

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