Frasch S Courtney, Henson Peter M, Nagaosa Kaz, Fessler Michael B, Borregaard Niels, Bratton Donna L
Department of Pediatrics, Division of Cell Biology, National Jewish Medical and Research Center, Denver, Colorado 80206, USA.
J Biol Chem. 2004 Apr 23;279(17):17625-33. doi: 10.1074/jbc.M313414200. Epub 2004 Feb 6.
Movement of phosphatidylserine (PS) to the plasma membrane outer leaflet is a nearly universal marker of apoptosis and occurs during activation of many cells. Neutrophils stimulated with the chemotactic peptide formylated Met-Leu-Phe (fMLP) demonstrated transient PS exposure. Stimulated outward movement of PS was accompanied by enhanced inward movement of several phosphorylcholine lipid probes and was associated with enhanced FM 1-43 staining indicative of phospholipid packing changes. Unlike apoptosis, inward movement of exogenously added fluorescent PS did not decline, and DNA was not cleaved during fMLP stimulation. Movement of phospholipids occurred within minutes following stimulation, was independent of endocytosis/pinocytosis, and was consistent with bidirectional, transbilayer phospholipid flip-flop. While the role of phospholipid scramblase 1 (PLSCR1) is controversial in flip-flop, we sought evidence for its role in enhanced phospholipid movements during fMLP stimulation. Using antibodies to the carboxyl-terminal domain of PLSCR1, its presence in the plasma membranes of non-permeabilized neutrophils was confirmed by flow cytometry. Additionally subcellular fractionation demonstrated that PLSCR1 was also located in secretory vesicles and tertiary and secondary granules. Activation of neutrophils with fMLP, however, did not significantly alter surface labeling suggesting that stimulated phospholipid flip-flop does not require additional mobilization of PLSCR1 to the plasma membrane. As expected for palmitoylated proteins, PLSCR1 was enriched in detergent-insoluble membranes and co-localized with raft markers at the neutrophil uropod after stimulation. Of note, PS exposure, phospholipid uptake, and FM 1-43 staining also localized to the uropod following stimulation demonstrating that both PLSCR1 and phospholipid flip-flop characterize this specialized domain of polarized neutrophils.
磷脂酰丝氨酸（PS）向质膜外小叶的移动是细胞凋亡几乎通用的标志，且在许多细胞激活过程中都会发生。用趋化肽甲酰甲硫氨酸-亮氨酸-苯丙氨酸（fMLP）刺激的中性粒细胞表现出短暂的PS暴露。PS的刺激外向移动伴随着几种磷酸胆碱脂质探针的增强内向移动，并与指示磷脂堆积变化的增强的FM 1-43染色相关。与细胞凋亡不同，外源性添加的荧光PS的内向移动没有下降，并且在fMLP刺激期间DNA未被切割。磷脂的移动在刺激后几分钟内发生，与内吞作用/胞饮作用无关，并且与双向跨膜磷脂翻转一致。虽然磷脂翻转酶1（PLSCR1）在翻转中的作用存在争议，但我们寻求其在fMLP刺激期间增强的磷脂移动中作用的证据。使用针对PLSCR1羧基末端结构域的抗体，通过流式细胞术证实其存在于未通透的中性粒细胞的质膜中。此外，亚细胞分级分离表明PLSCR1也位于分泌小泡以及三级和二级颗粒中。然而，用fMLP激活中性粒细胞并没有显著改变表面标记，这表明刺激的磷脂翻转不需要额外的PLSCR1向质膜的动员。正如对棕榈酰化蛋白的预期，PLSCR1在去污剂不溶性膜中富集，并在刺激后与中性粒细胞尾足处的脂筏标记物共定位。值得注意的是，刺激后PS暴露、磷脂摄取和FM 1-43染色也定位于尾足，这表明PLSCR1和磷脂翻转都表征了极化中性粒细胞这个特化结构域的特征。
