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

Baumann, Tommy; Affentranger, Sarah; Niggli, Verena (2013). Analysis of close associations of uropod-associated proteins in human T-cells using the proximity ligation assay. PeerJ, 1, e186. PeerJ, Ltd 10.7717/peerj.186

[img]
Preview
Text (PDF)
186.pdf - Published Version
Available under License Creative Commons: Attribution (CC-BY).

Download (10MB) | Preview

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.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Service Sector > Institute of Pathology > Inflammatory Pathology
04 Faculty of Medicine > Service Sector > Institute of Pathology

UniBE Contributor:

Baumann, Tommy; Affentranger, Sarah and Niggli, Verena

Subjects:

500 Science > 570 Life sciences; biology
600 Technology > 610 Medicine & health

ISSN:

2167-8359

Publisher:

PeerJ, Ltd

Language:

English

Submitter:

Andrea Arnold

Date Deposited:

07 Apr 2014 09:26

Last Modified:

18 Dec 2014 03:05

Publisher DOI:

10.7717/peerj.186

PubMed ID:

24167781

Uncontrolled Keywords:

Uropod, Ezrin/radixin/moesin, T-cell, Flotillin, PSGL-1, Proximity ligation assay

BORIS DOI:

10.7892/boris.46053

URI:

https://boris.unibe.ch/id/eprint/46053

Actions (login required)

Edit item Edit item
Provide Feedback