Analysis of close associations of uropod-associated proteins in human T-cells using the proximity ligation assay

Abstract

We have shown previously that the raft-associated proteins flotillin-1 and -2 are rapidly recruited to the uropods of chemoattractant-stimulated human neutrophils and T-cells and are involved in cell polarization. Other proteins such as the adhesion receptor PSGL-1, the actin-membrane linker proteins ezrin/radixin/moesin (ERM) and the signaling enzyme phosphatidylinositol-4-phosphate 5-kinase type Iγ90 (PIPKIγ90) also accumulate in the T-cell uropod. Using the in situ proximity ligation assay (PLA) we now have investigated putative close associations of these proteins in human freshly isolated T-cells before and after chemokine addition. The PLA allows in situ subcellular localization of close proximity of endogenous proteins at single-molecule resolution in fixed cells. It allows detection also of weaker and transient complexes that would not be revealed with co-immunoprecipitation approaches. We previously provided evidence for heterodimer formation of tagged flotillin-1 and -2 in T-cells before and after chemokine addition using fluorescence resonance energy transfer (FRET). We now confirm these findings using PLA for the endogenous flotillins in fixed human T-cells. Moreover, in agreement with the literature, our PLA findings confirm a close association of endogenous PSGL-1 and ERM proteins both in resting and chemokine-activated human T-cells. In addition, we provide novel evidence using the PLA for close associations of endogenous activated ERM proteins with PIPKIγ90 and of endogenous flotillins with PSGL-1 in human T-cells, before and after chemokine addition. Our findings suggest that preformed clusters of these proteins coalesce in the uropod upon cell stimulation.

O-glycans direct selectin ligands to lipid rafts on leukocytes

“…Uropods form through membrane interactions with flotillins 1 and 2 and with the actin cytoskeleton (54,55), in part through binding of ERM adaptors to the cytoplasmic domains of membrane glycoproteins (56). PSGL-1 associates with flotillins as measured by coimmunoprecipitation in detergent extracts and by a proximity-ligation assay in intact cells (31,57). However, direct binding of PSGL-1 to flotillins has not been demonstrated.…”

Insights into the Mechanism for Dictating Polarity in Migrating T-Cells

“…Indeed, murine primary T-cells isolated from mice lacking PIP5Kγ661 and human primary T-cells transfected with a kinase-dead mutant of PIP5Kγ661 show elongated tails when migrating on ICAM-1. In this context, a relatively weak in situ interaction of flotillin-2 with PIP5Kγ661 was observed in human T-cells before and after chemokine addition using the proximity ligation assay (PLA), but a strong interaction of PIP5Kγ661 with phosphorylated ERM proteins (P-ERM) (Baumann et al, 2013). These findings thus suggest a role of PIP5Kγ661 mainly in T-cell deadhesion rather than in cell polarization (Mathis et al, 2013;Wernimont et al, 2010a).…”

“…In resting T-cells, approximately 50% of the ERM proteins are phosphorylated (Shaffer et al, 2009). ERM proteins have also been implicated in activating Rho via interacting with the Rho-GEF Dbl (diffuse B-cell lymphoma) (Affentranger et al, 2011;Baumann et al, 2013;Lee et al, 2004;Parameswaran and Gupta, 2013;Sánchez-Madrid and Serrador, 2009;Serrador et al, 1997). Later, the remaining C-terminally P-ERM accumulate in the uropod of chemokine-stimulated T-cells, recruiting transmembrane adhesion receptors such as PSGL-1 to this site and colocalizing with other uropod proteins such as the lipid raft-associated flotillins (Section 8.2) and PIP5Kγ661 (Section 5.2).…”

“…Splenic cells isolated from mice lacking LOK show an approximately 50% reduction in ERM phosphorylation (Belkina et al, 2009). We showed recently that PIP5Kγ661 may interact closely with activated ERM proteins in the uropod using the PLA (Baumann et al, 2013). We showed recently that PIP5Kγ661 may interact closely with activated ERM proteins in the uropod using the PLA (Baumann et al, 2013).…”

“…Affentranger et al (2011), Baumann et al (2012Baumann et al ( , 2013, Martinelli et al (2013), Mathis et al (2013) Ganglioside GM1a Component of uropodlocated rafts Gómez-Mouton et al (2001) Microtubules, MTOC Regulation of Rho activity, transport of vesicles to the uropod Ratner et al (1997), Takesono et al (2010), Tooley et al (2009) Myosin II (activated) a Uropod contraction and retraction Campello et al (2006), Shulman et al (2009) Organelles (mitochondria, ER, Golgi, and lysosomes)…”