title: Evidence for chemokine-mediated coalescence of preformed flotillin hetero-oligomers in human T-cells.
abstract: 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.
authors.affiliation: Institute of Pathology, University of Bern, Murtenstrasse 31, 3010 Bern, Switzerland.
authors.forename: Tommy
authors.initials: T
authors.lastname: Baumann
authors.forename: Sarah
authors.initials: S
authors.lastname: Affentranger
authors.forename: Verena
authors.initials: V
authors.lastname: Niggli
mesh_terms: D002478:Cells, Cultured; D018925:Chemokines / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization / Q000502:physiology*; D013601:T-Lymphocytes / Q000378:metabolism*
chemical_list: D018925:Chemokines; D008565:Membrane Proteins; C106354:flotillins; D049452:Green Fluorescent Proteins