No 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.

The membrane of platelets contains at least one uncharacterized glycosylphosphatidylinositol (GPI)-anchored protein according to the literature. Moreover, there is not enough knowledge on the receptor of low-density lipoproteins (LDL) mediating rapid Ca2+ signaling in platelets. Coincidentally, expression of a GPI-anchored protein T-cadherin increases LDL-induced Ca2+ signaling in nucleated cells. Here we showed evidence that supports the hypothesis about the presence of T-cadherin on platelets. The presence of T-cadherin on the surface of platelets and megakaryocytes was proven using antibodies whose specificity was tested on several negative and positive control cells by flow cytometry and confocal microscopy. Using phosphatidylinositol-specific phospholipase C, the presence of glycosylphosphatidylinositol anchor in the platelet T-cadherin form as well as in other known forms was confirmed. We showed by immunoblotting that the significant part of T-cadherin was detected in specific membrane domains (detergent Triton X-114 resistant) and the molecular weight of this newly identified protein was greater than that of T-cadherin from nucleated cells. Nevertheless, polymerase chain reaction data confirmed only the presence of isoform-1 of T-cadherin in platelets and megakaryocytes, which was also present in nucleated cells. We observed the redistribution of this newly identified protein after the activation of platelets, but only further work may explain its functional importance. Thus, our data described T-cadherin with some post-translational modifications as a new GPI-anchored protein on human platelets.