Yürüker B, Niggli V
Department of Pathology, University of Bern, Switzerland.
J Cell Sci. 1992 Feb;101 ( Pt 2):403-14. doi: 10.1242/jcs.101.2.403.
We have studied the reorganization of vinculin and alpha-actinin during the process of adhesion in human neutrophils using immunofluorescence microscopy and interference reflection microscopy (IRM). Neutrophils in contact with uncoated glass formed black IRM areas in the cell periphery, indicative of very close contact with the substratum. Eight to twelve minutes after addition of cells to glass, vinculin was found to become concentrated in small patches at the cell periphery, partially colocalizing with the black IRM areas and with small F-actin-containing adherent protrusions. In contrast, vinculin was not significantly enriched in the less adherent F-actin-rich large pseudopods. alpha-Actinin became enriched during cell adhesion in retraction fibers and, in 40-50% of the inspected cells, also in large less adherent pseudopods where it colocalized with F-actin. The latter finding suggests a continuous dynamic reorganization of pseudopods, with incorporation of alpha-actinin at a certain stage. Disruption of the actin network with cytochalasin D revealed a differential interaction of alpha-actinin and vinculin with the actin network. alpha-Actinin was strongly influenced by cytochalasin D, comparable to F-actin, and both proteins formed colocalizing peripheral caps in 10(-5) M of the drug. Vinculin organization in contrast was not affected by up to 10(-6) M cytochalasin. At 10(-5) M of the drug, however, the patches disappeared completely, vinculin now assuming a diffuse cytoplasmic location. Our results suggest a specialized function of vinculin in adhesion sites of human neutrophils, whereas alpha-actinin may structure the actin network in retraction fibers and in less adherent pseudopods.
我们利用免疫荧光显微镜和干涉反射显微镜（IRM）研究了人中性粒细胞黏附过程中纽蛋白和α-辅肌动蛋白的重组情况。与未包被的玻璃接触的中性粒细胞在细胞周边形成黑色IRM区域，这表明与基质紧密接触。将细胞加入玻璃后8至12分钟，发现纽蛋白集中在细胞周边的小斑块中，部分与黑色IRM区域以及含有小F-肌动蛋白的黏附突起共定位。相比之下，在黏附性较差、富含F-肌动蛋白的大伪足中，纽蛋白没有显著富集。α-辅肌动蛋白在细胞黏附过程中在收缩纤维中富集，并且在40%-50%被检查的细胞中，在黏附性较差的大伪足中也有富集，在那里它与F-肌动蛋白共定位。后一发现表明伪足存在持续的动态重组，在某个阶段会掺入α-辅肌动蛋白。用细胞松弛素D破坏肌动蛋白网络揭示了α-辅肌动蛋白和纽蛋白与肌动蛋白网络的不同相互作用。α-辅肌动蛋白受细胞松弛素D的影响很大，与F-肌动蛋白相当，在10^(-5) M的该药物中，这两种蛋白质都形成共定位的周边帽。相比之下，纽蛋白的组织在高达10^(-6) M的细胞松弛素中不受影响。然而，在10^(-5) M的该药物中，斑块完全消失，纽蛋白现在呈弥漫性细胞质定位。我们的结果表明纽蛋白在人中性粒细胞的黏附位点具有特殊功能，而α-辅肌动蛋白可能在收缩纤维和黏附性较差的伪足中构建肌动蛋白网络。
