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.
Highlights
Coexpression of the membrane microdomain-associated proteins flotillin-1 and -2 is required for their location in uropods of chemokine-stimulated T-cells
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 fluorescence resonance energy transfer (FRET) between tagged flotillin-1 and -2
We provide evidence for significant FRET between C-terminally tagged flotillin-2-enhanced green fluorescent protein (EGFP) and flotillin-1-mCherry in resting and chemokine-stimulated human T-cells, which is not abolished by treatments that suppress T-cell polarity and flotillin cap formation such as disruption of F-actin, depletion of cytosolic calcium, or inhibition of phospholipase C (PLC)
Summary
Coexpression of the membrane microdomain-associated proteins flotillin-1 and -2 is required for their location in uropods of chemokine-stimulated T-cells. 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. Transfection of T-cells with a dominant negative mutant of flotillin-2 impaired cell polarization and uropod capping of endogenous flotillin-1 and the adhesion receptor P-selectin glycoprotein-1, indicating a functional role of flotillins in cell polarization [7] These findings are in line with those obtained by Ludwig et al [8], who studied neutrophils from flotillin-1 knock-out mice. We provide evidence for significant FRET between C-terminally tagged flotillin-2-EGFP and flotillin-1-mCherry in resting and chemokine-stimulated human T-cells, which is not abolished by treatments that suppress T-cell polarity and flotillin cap formation such as disruption of F-actin, depletion of cytosolic calcium, or inhibition of phospholipase C (PLC).. We provide evidence for significant FRET between C-terminally tagged flotillin-2-EGFP and flotillin-1-mCherry in resting and chemokine-stimulated human T-cells, which is not abolished by treatments that suppress T-cell polarity and flotillin cap formation such as disruption of F-actin, depletion of cytosolic calcium, or inhibition of phospholipase C (PLC). We present evidence strongly suggesting the predominant presence of hetero-oligomeric flotillin complexes in resting and chemokine-stimulated human T-cells
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