Abstract
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.
Highlights
Current knowledge of membrane receptor organization and dynamics in live cells remains far from complete, but it is important for understanding their functional activity
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
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
Summary
Current knowledge of membrane receptor organization and dynamics in live cells remains far from complete, but it is important for understanding their functional activity. This is, in part, due to the resolution limit of 200 nm for conventional optical microscopy, which allows for us to study live cells. It has been shown that the activity of several receptors depends on the oligomeric state in the membrane. The mechanisms of signal transduction by GPI-anchored receptors and the correlation between receptor activity and their structures in a membrane-associated form deserve thorough investigation. Multiple studies have demonstrated that portions of GPI-APs persist in a dimeric or oligomeric form in a steady state [2,3]. The detection of GPI-AP clusters in live cells in real time is a demanding task, because they exhibit subdiffraction-limited sizes and are highly dynamic [7]
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