Rossy Jérémie, Schlicht Dominique, Engelhardt Britta, Niggli Verena
Department of Pathology, University of Bern, Bern, Switzerland.
PLoS One. 2009;4(4):e5403. doi: 10.1371/journal.pone.0005403. Epub 2009 Apr 30.
Neutrophils polarize and migrate in response to chemokines. Different types of membrane microdomains (rafts) have been postulated to be present in rear and front of polarized leukocytes and disruption of rafts by cholesterol sequestration prevents leukocyte polarization. Reggie/flotillin-1 and -2 are two highly homologous proteins that are ubiquitously enriched in detergent resistant membranes and are thought to shape membrane microdomains by forming homo- and hetero-oligomers. It was the goal of this study to investigate dynamic membrane microdomain reorganization during neutrophil activation.
METHODOLOGY/PRINCIPAL FINDINGS: We show now, using immunofluorescence staining and co-immunoprecipitation, that endogenous flotillin-1 and -2 colocalize and associate in resting spherical and polarized primary neutrophils. Flotillins redistribute very early after chemoattractant stimulation, and form distinct caps in more than 90% of the neutrophils. At later time points flotillins accumulate in the uropod of polarized cells. Chemotactic peptide-induced redistribution and capping of flotillins requires integrity and dynamics of the actin cytoskeleton, but does not involve Rho-kinase dependent signaling related to formation of the uropod. Both flotillin isoforms are involved in the formation of this membrane domain, as uropod location of exogenously expressed flotillins is dramatically enhanced by co-overexpression of tagged flotillin-1 and -2 in differentiated HL-60 cells as compared to cells expressing only one tagged isoform. Flotillin-1 and -2 associate with P-selectin glycoprotein ligand 1 (PSGL-1) in resting and in stimulated neutrophils as shown by colocalization and co-immunoprecipitation. Neutrophils isolated from PSGL-1-deficient mice exhibit flotillin caps to the same extent as cells isolated from wild type animals, implying that PSGL-1 is not required for the formation of the flotillin caps. Finally we show that stimulus-dependent redistribution of other uropod-located proteins, CD43 and ezrin/radixin/moesin, occurs much slower than that of flotillins and PSGL-1.
CONCLUSIONS/SIGNIFICANCE: These results suggest that flotillin-rich actin-dependent membrane microdomains are importantly involved in neutrophil uropod formation and/or stabilization and organize uropod localization of PSGL-1.
中性粒细胞会响应趋化因子而发生极化和迁移。据推测，不同类型的膜微区（脂筏）存在于极化白细胞的后部和前部，通过胆固醇螯合破坏脂筏会阻止白细胞极化。Reggie/小窝蛋白-1和-2是两种高度同源的蛋白质，它们普遍富集于抗去污剂膜中，并被认为通过形成同型和异型寡聚体来塑造膜微区。本研究的目的是研究中性粒细胞激活过程中动态膜微区的重组。
方法/主要发现：我们现在通过免疫荧光染色和免疫共沉淀表明，内源性小窝蛋白-1和-2在静息球形和极化的原代中性粒细胞中共定位并相互关联。趋化因子刺激后，小窝蛋白很快重新分布，并在超过90%的中性粒细胞中形成明显的帽状结构。在稍后的时间点，小窝蛋白在极化细胞的尾足中积累。趋化肽诱导的小窝蛋白重新分布和帽状结构形成需要肌动蛋白细胞骨架的完整性和动态性，但不涉及与尾足形成相关的 Rho激酶依赖性信号传导。两种小窝蛋白亚型都参与了这个膜结构域的形成，因为与仅表达一种标记亚型的细胞相比，在分化的HL-60细胞中共同过表达标记的小窝蛋白-1和-2会显著增强外源性表达的小窝蛋白在尾足的定位。通过共定位和免疫共沉淀表明，小窝蛋白-1和-2在静息和受刺激的中性粒细胞中都与P-选择素糖蛋白配体1（PSGL-1）相关联。从PSGL-1缺陷小鼠分离的中性粒细胞与从野生型动物分离的细胞一样表现出小窝蛋白帽状结构，这意味着PSGL-1对于小窝蛋白帽状结构的形成不是必需的。最后我们表明，其他位于尾足的蛋白质CD43和埃兹蛋白/根蛋白/膜突蛋白的刺激依赖性重新分布比小窝蛋白和PSGL-1的要慢得多。
结论/意义：这些结果表明，富含小窝蛋白的肌动蛋白依赖性膜微区重要地参与了中性粒细胞尾足的形成和/或稳定，并组织了PSGL-1在尾足的定位。
