Institute of Pathology, University of Bern, Murtenstrasse 31, 3010 Bern, Switzerland.
J Biol Chem. 2012 Nov 16;287(47):39664-72. doi: 10.1074/jbc.M112.412742. Epub 2012 Sep 25.
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.
我们之前已经表明，内源性的浮游蛋白-1 和 -2，与膜微区支架有关的密切相关的蛋白质，在趋化因子刺激的人类中性粒细胞和 T 细胞的尾足中迅速募集，并参与细胞极化。共表达的浮游蛋白-1 和 -2，但不是单独表达的蛋白质，也被靶向到 T 细胞和中性粒细胞的尾足。生化研究表明，在其他细胞类型中形成浮游蛋白同型和异型寡聚体，但到目前为止，缺乏白细胞中浮游蛋白原位组织的知识。我们现在使用荧光共振能量转移（FRET）分析了人类 T 细胞中的浮游蛋白组织。在没有和存在趋化因子的情况下，共表达的 C 端标记的浮游蛋白-1-mCherry 和浮游蛋白-2-增强型绿色荧光蛋白（EGFP）在完整的人类 T 细胞中显示出显著的 FRET。相比之下，在趋化因子添加前后，共表达的浮游蛋白-1-mCherry 和浮游蛋白-1-EGFP 之间很少观察到 FRET，表明主要形成异二聚体和/或寡聚体。有趣的是，未标记的浮游蛋白-2 的共表达强烈增强了静止和趋化因子刺激的细胞中不同标记的浮游蛋白-1 分子之间的 FRET，表明浮游蛋白-1 分子的紧密接触仅发生在含有浮游蛋白-2 的异源寡聚体中。对于标记的浮游蛋白-2 也得到了类似的结果。我们进一步表明，破坏肌动蛋白网络、耗尽细胞内钙和抑制磷脂酶 C 都会导致趋化因子诱导的极化和浮游蛋白帽形成的抑制，但不会消除标记的浮游蛋白-1 和 -2 之间的 FRET。我们的结果支持在静止和趋化因子刺激的人类 T 细胞中主要形成浮游蛋白-1 和 -2 异源寡聚体，这可能对尾足的结构重要贡献。
