Seveau S, Lopez S, Lesavre P, Guichard J, Cramer E M, Halbwachs-Mecarelli L
INSERM U90 Hôpital Necker, Paris, France.
J Cell Sci. 1997 Jul;110 ( Pt 13):1465-75. doi: 10.1242/jcs.110.13.1465.
We investigated a possible association of leukosialin (CD43), the major surface sialoglycoprotein of leukocytes, with neutrophil cytoskeleton. We first analysed the solubility of CD43 in Triton X-100 and observed that CD43 of resting neutrophils was mostly soluble. The small proportion of CD43 molecules, which 'spontaneously' precipitated in Triton, appeared associated with F-actin, as demonstrated by the fact that this insolubility did not occur when cells were incubated with cytochalasin B or when F-actin was depolymerized with DNase I in the Triton precipitate. Cell stimulation with anti-CD43 mAb (MEM59) enhanced this CD43-cytoskeleton association. By immunofluorescence as well as by electron microscopy, we observed a redistribution of CD43 on the neutrophil membrane, initially in patches followed by caps, during anti-CD43 cross-linking at 37 degrees C. This capping did not occur at 4 degrees C and was inhibited by cytochalasin B and by a myosin disrupting drug butanedione monoxime, thus providing evidence that the actomyosin contracile sytem is involved in the capping and further suggesting an association of CD43 with the cytoskeleton. Some of the capped cells exhibited a front-tail polarization with CD43 caps located in the uropod at the rear of the cell. Surprisingly, colchicine and the chemotactic factor fNLPNTL which induce neutrophil polarization associated with cell motility, also resulted in a clustering of CD43 in the uropod, independently of a cross-linking of the molecule by mAbs. An intracellular redistribution of F-actin, mainly at the leading front and of myosin in the tail, was observed during CD43 clustering induced by colchicine and in cells polarized by anti-CD43 mAbs cross-linking. We conclude that neutrophil CD43 interacts with the cytoskeleton, either directly or indirectly, to redistribute in the cell uropod under antibodies stimulation or during cell polarization by colchicine, thus highly suggesting that CD43 may be involved in cell polarization.
我们研究了白细胞主要表面唾液糖蛋白白细胞涎酸蛋白（CD43）与中性粒细胞细胞骨架之间可能存在的关联。我们首先分析了CD43在Triton X-100中的溶解性，观察到静息中性粒细胞的CD43大多是可溶的。在Triton中“自发”沉淀的一小部分CD43分子似乎与F-肌动蛋白相关，这一事实表明，当细胞与细胞松弛素B一起孵育时，或者当F-肌动蛋白在Triton沉淀物中被脱氧核糖核酸酶I解聚时，这种不溶性不会发生。用抗CD43单克隆抗体（MEM59）刺激细胞增强了这种CD43-细胞骨架的关联。通过免疫荧光以及电子显微镜，我们观察到在37℃下抗CD43交联期间，CD43在中性粒细胞膜上重新分布，最初形成斑块，随后形成帽状。这种帽状形成在4℃时不会发生，并且受到细胞松弛素B和一种破坏肌球蛋白的药物丁二酮单肟的抑制，从而提供了证据表明肌动球蛋白收缩系统参与了帽状形成，并进一步表明CD43与细胞骨架相关。一些形成帽状的细胞表现出前后极化，CD43帽位于细胞后部的尾足。令人惊讶的是，秋水仙碱和诱导与细胞运动相关的中性粒细胞极化的趋化因子fNLPNTL，也导致CD43在尾足聚集，与单克隆抗体对该分子的交联无关。在秋水仙碱诱导的CD43聚集期间以及在抗CD43单克隆抗体交联极化的细胞中，观察到F-肌动蛋白主要在前端和肌球蛋白在尾部的细胞内重新分布。我们得出结论，中性粒细胞CD43直接或间接与细胞骨架相互作用，在抗体刺激下或在秋水仙碱诱导的细胞极化过程中在细胞尾足中重新分布，因此强烈表明CD43可能参与细胞极化。
