Sheterline P, Hopkins C R
J Cell Biol. 1981 Sep;90(3):743-54. doi: 10.1083/jcb.90.3.743.
An experimental approach is described that enables the analysis of interactions between exogenous surface ligands and components of the cytoplasm in neutrophil leukocytes. Neutrophils treated with the nonionic detergent Lubrol PX, under controlled conditions, yield intact detergent-insoluble ghosts. Morphological analysis of neutrophil ghosts shows that they retain the original dimensions of the cell and consist almost entirely of a peripheral filamentous network, representing the submembranous cortical web, concentric to nuclear remnants. All intracellular membrane-bounded organelles, plasma membrane, and background cytoplasmic electron density are absent. Biochemical analysis of the ghosts shows that less than 10% of enzyme markers for the soluble and granule fractions remain, and that greater than 90% of total cell phospholipid is removed during detergent extraction. The major proteins remaining in the ghosts comigrate, on polyacrylamide gels in the presence of SDS, with chicken gizzard actin, myosin, filamin, and a 110-kdalton protein. Patches and caps induced on neutrophils with either fluorescein isothiocyanate-concanavalin A or ferritin-concanavalin A retain their original location and morphology on ghosts after lysis, as determined by both fluorescence and electron microscopy. In similar experiments, but using 125I-labeled lectins, 37% of total cell bound concanavalin A (Con A) and 25% succinylated Con A remain attached to the ghosts. A major 125I-labeled membrane glycoprotein (80 kdaltons) is associated with ghosts prepared from intact neutrophils iodinated in the presence of exogenous lactoperoxidase. Further 125I-labeled membrane glycoproteins (217, 170, and 147 kdaltons) become associated with ghosts prepared from iodinated cells treated before lysis with Con A, but not with succinylated Con A. These data taken together suggest that linkages exist in neutrophils between proteins exposed on the outer surface of the plasma membrane and the peripheral filamentous network independent of the presence of lipid bilayer. The implications of these findings for surface motile phenomena will be discussed.
本文描述了一种实验方法，该方法能够分析中性粒细胞中外源表面配体与细胞质成分之间的相互作用。在可控条件下，用非离子去污剂Lubrol PX处理中性粒细胞，可产生完整的不溶于去污剂的幽灵细胞。对中性粒细胞幽灵细胞的形态学分析表明，它们保留了细胞的原始尺寸，几乎完全由外周丝状网络组成，该网络代表与核残余物同心的膜下皮质网络。所有细胞内膜结合细胞器、质膜和背景细胞质电子密度均不存在。对幽灵细胞的生化分析表明，可溶性和颗粒部分的酶标记物保留不到10%，并且在去污剂提取过程中超过90%的总细胞磷脂被去除。在SDS存在下，聚丙烯酰胺凝胶上，幽灵细胞中剩余的主要蛋白质与鸡胗肌动蛋白、肌球蛋白、细丝蛋白和一种110 kDa的蛋白质共同迁移。用异硫氰酸荧光素 - 伴刀豆球蛋白A或铁蛋白 - 伴刀豆球蛋白A在中性粒细胞上诱导产生的斑块和帽状物，在裂解后在幽灵细胞上保留其原始位置和形态，这通过荧光显微镜和电子显微镜均已确定。在类似实验中，但使用125I标记的凝集素，总细胞结合的伴刀豆球蛋白A（Con A）的37%和琥珀酰化Con A的25%仍附着在幽灵细胞上。一种主要的125I标记膜糖蛋白（80 kDa）与在外源乳过氧化物酶存在下碘化的完整中性粒细胞制备的幽灵细胞相关。另外的125I标记膜糖蛋白（217、170和147 kDa）与在用Con A裂解前处理的碘化细胞制备的幽灵细胞相关，但与琥珀酰化Con A无关。综合这些数据表明，在中性粒细胞中，质膜外表面暴露的蛋白质与外周丝状网络之间存在独立于脂质双层存在的连接。将讨论这些发现对表面运动现象的影响。
