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
Full text not available from this repository.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, 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: |
05 Dec 2022 14:10 |
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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