Analysis of GPI-Anchored Receptor Distribution and Dynamics in Live Cells by Tag-Mediated Enzymatic Labeling and FRET
The analysis of glycosylphosphatidylinositol (GPI)-anchored receptor distribution and dynamics in live cells is challenging, because their clusters exhibit subdiffraction-limited sizes and are highly dynamic. However, the cellular response depends on the GPI-anchored receptor clusters’ distribution...
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MDPI AG
2020-04-01
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| Series: | Methods and Protocols |
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| Online Access: | https://www.mdpi.com/2409-9279/3/2/33 |
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| author | Maria N. Balatskaya Alexandra I. Baglay Yury P. Rubtsov George V. Sharonov |
| author_facet | Maria N. Balatskaya Alexandra I. Baglay Yury P. Rubtsov George V. Sharonov |
| author_sort | Maria N. Balatskaya |
| collection | DOAJ |
| description | The analysis of glycosylphosphatidylinositol (GPI)-anchored receptor distribution and dynamics in live cells is challenging, because their clusters exhibit subdiffraction-limited sizes and are highly dynamic. However, the cellular response depends on the GPI-anchored receptor clusters’ distribution and dynamics. Here, we compare three approaches to GPI-anchored receptor labeling (with antibodies, fluorescent proteins, and enzymatically modified small peptide tags) and use several variants of Förster resonance energy transfer (FRET) detection by confocal microscopy and flow cytometry in order to obtain insight into the distribution and the ligand-induced dynamics of GPI-anchored receptors. We found that the enzyme-mediated site-specific fluorescence labeling of T-cadherin modified with a short peptide tag (12 residues in length) have several advantages over labeling by fluorescent proteins or antibodies, including (i) the minimized distortion of the protein’s properties, (ii) the possibility to use a cell-impermeable fluorescent substrate that allows for selective labeling of surface-exposed proteins in live cells, and (iii) superior control of the donor to acceptor molar ratio. We successfully detected the FRET of GPI-anchored receptors, T-cadherin, and ephrin-A1, without ligands, and showed in real time that adiponectin induces stable T-cadherin cluster formation. In this paper (which is complementary to our recent research (Balatskaya et al., 2019)), we present the practical aspects of labeling and the heteroFRET measurements of GPI-anchored receptors to study their dynamics on a plasma membrane in live cells. |
| first_indexed | 2024-03-10T20:11:06Z |
| format | Article |
| id | doaj.art-165aeeec454744b4bbc6442b8a5a5637 |
| institution | Directory Open Access Journal |
| issn | 2409-9279 |
| language | English |
| last_indexed | 2024-03-10T20:11:06Z |
| publishDate | 2020-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Methods and Protocols |
| spelling | doaj.art-165aeeec454744b4bbc6442b8a5a56372023-11-19T22:53:44ZengMDPI AGMethods and Protocols2409-92792020-04-01323310.3390/mps3020033Analysis of GPI-Anchored Receptor Distribution and Dynamics in Live Cells by Tag-Mediated Enzymatic Labeling and FRETMaria N. Balatskaya0Alexandra I. Baglay1Yury P. Rubtsov2George V. Sharonov3Faculty of Medicine, Lomonosov Moscow State University, Lomonosovskiy av. 27-1, 119192 Moscow, RussiaFaculty of Medicine, Lomonosov Moscow State University, Lomonosovskiy av. 27-1, 119192 Moscow, RussiaFaculty of Medicine, Lomonosov Moscow State University, Lomonosovskiy av. 27-1, 119192 Moscow, RussiaFaculty of Medicine, Lomonosov Moscow State University, Lomonosovskiy av. 27-1, 119192 Moscow, RussiaThe analysis of glycosylphosphatidylinositol (GPI)-anchored receptor distribution and dynamics in live cells is challenging, because their clusters exhibit subdiffraction-limited sizes and are highly dynamic. However, the cellular response depends on the GPI-anchored receptor clusters’ distribution and dynamics. Here, we compare three approaches to GPI-anchored receptor labeling (with antibodies, fluorescent proteins, and enzymatically modified small peptide tags) and use several variants of Förster resonance energy transfer (FRET) detection by confocal microscopy and flow cytometry in order to obtain insight into the distribution and the ligand-induced dynamics of GPI-anchored receptors. We found that the enzyme-mediated site-specific fluorescence labeling of T-cadherin modified with a short peptide tag (12 residues in length) have several advantages over labeling by fluorescent proteins or antibodies, including (i) the minimized distortion of the protein’s properties, (ii) the possibility to use a cell-impermeable fluorescent substrate that allows for selective labeling of surface-exposed proteins in live cells, and (iii) superior control of the donor to acceptor molar ratio. We successfully detected the FRET of GPI-anchored receptors, T-cadherin, and ephrin-A1, without ligands, and showed in real time that adiponectin induces stable T-cadherin cluster formation. In this paper (which is complementary to our recent research (Balatskaya et al., 2019)), we present the practical aspects of labeling and the heteroFRET measurements of GPI-anchored receptors to study their dynamics on a plasma membrane in live cells.https://www.mdpi.com/2409-9279/3/2/33FRETmembrane proteinsGPI-anchored receptorsprotein labelingfluorescent labelingS6 tag |
| spellingShingle | Maria N. Balatskaya Alexandra I. Baglay Yury P. Rubtsov George V. Sharonov Analysis of GPI-Anchored Receptor Distribution and Dynamics in Live Cells by Tag-Mediated Enzymatic Labeling and FRET Methods and Protocols FRET membrane proteins GPI-anchored receptors protein labeling fluorescent labeling S6 tag |
| title | Analysis of GPI-Anchored Receptor Distribution and Dynamics in Live Cells by Tag-Mediated Enzymatic Labeling and FRET |
| title_full | Analysis of GPI-Anchored Receptor Distribution and Dynamics in Live Cells by Tag-Mediated Enzymatic Labeling and FRET |
| title_fullStr | Analysis of GPI-Anchored Receptor Distribution and Dynamics in Live Cells by Tag-Mediated Enzymatic Labeling and FRET |
| title_full_unstemmed | Analysis of GPI-Anchored Receptor Distribution and Dynamics in Live Cells by Tag-Mediated Enzymatic Labeling and FRET |
| title_short | Analysis of GPI-Anchored Receptor Distribution and Dynamics in Live Cells by Tag-Mediated Enzymatic Labeling and FRET |
| title_sort | analysis of gpi anchored receptor distribution and dynamics in live cells by tag mediated enzymatic labeling and fret |
| topic | FRET membrane proteins GPI-anchored receptors protein labeling fluorescent labeling S6 tag |
| url | https://www.mdpi.com/2409-9279/3/2/33 |
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