Evidence for chemokine-mediated coalescence of preformed flotillin hetero-oligomers in human T-cells

Baumann, Tommy; Affentranger, Sarah; Niggli, Verena (2012). Evidence for chemokine-mediated coalescence of preformed flotillin hetero-oligomers in human T-cells. Journal of biological chemistry, 287(47), pp. 39664-72. Bethesda, Md.: American Society for Biochemistry and Molecular Biology 10.1074/jbc.M112.412742

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We have shown previously that endogenous flotillin-1 and -2, closely related proteins implicated in scaffolding of membrane microdomains, are rapidly recruited to the uropods of chemoattractant-stimulated human neutrophils and T-cells and are involved in cell polarization. Coexpressed flotillin-1 and -2, but not singly expressed proteins, are also targeted to the uropod of T-cells and neutrophils. Biochemical studies suggest formation of flotillin homo- and hetero-oligomers in other cell types, but so far knowledge is lacking on in situ flotillin organization in leukocytes. We have now analyzed flotillin organization in human T-cells using fluorescence resonance energy transfer (FRET). Coexpressed C-terminally tagged flotillin-1-mCherry and flotillin-2-enhanced green fluorescent protein (EGFP) show significant FRET when analyzed in intact human T-cells in the absence and presence of chemokine. In contrast, little FRET was observed between coexpressed flotillin-1-mCherry and flotillin-1-EGFP before or after chemokine addition, indicating predominant formation of heterodimers and/or -oligomers. Interestingly coexpression of untagged flotillin-2 strongly enhanced FRET between differently tagged flotillin-1 molecules in resting and chemokine-stimulated cells, indicating that close contacts of flotillin-1 molecules only occur in flotillin-2-containing hetero-oligomers. Comparable results were obtained for tagged flotillin-2. We further show that disruption of the actin network, depletion of intracellular calcium, and inhibition of phospholipase C all result in suppression of chemokine-induced polarization and flotillin cap formation, but do not abolish FRET between tagged flotillin-1 and -2. Our results support predominant formation of flotillin-1 and -2 hetero-oligomers in resting and chemokine-stimulated human T-cells which may importantly contribute to structuring of the uropod.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Service Sector > Institute of Pathology
04 Faculty of Medicine > School of Dental Medicine

UniBE Contributor:

Baumann, Tommy and Niggli, Verena

Subjects:

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

ISSN:

0021-9258

Publisher:

American Society for Biochemistry and Molecular Biology

Language:

English

Submitter:

Tommy Baumann

Date Deposited:

04 Oct 2013 14:33

Last Modified:

27 Nov 2015 12:22

Publisher DOI:

10.1074/jbc.M112.412742

PubMed ID:

23012365

Web of Science ID:

000311233800036

URI:

https://boris.unibe.ch/id/eprint/12980 (FactScience: 219479)

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