Fernández-Segura E, García J M, Santos J L, Campos A
Departamento de Biología Celular e Histología, Facultad de Medicina, Universidad de Granada, Spain.
Anat Rec. 1995 Apr;241(4):519-28. doi: 10.1002/ar.1092410410.
The exposure of human neutrophils to uniform concentrations of chemoattractants, such as N-formyl peptides, induces morphological cell polarization. In this study we report the temporal sequence of changes in cell shape, F-actin, and cell surface morphology during cellular polarization induced by N-formylmethionyl-leucyl-phenylalanine (fMLP) in human neutrophils in suspension.
Neutrophil shape changes induced by 10(-8) M fMLP were observed with DIC microscopy. Size and cellular granularity were analyzed by flow cytometry measuring their forward and side scattered light. To visualize F-actin distribution, neutrophils were labeled with the fluorescence probe FITC-phalloidin, and were examined with fluorescence and confocal laser scanning microscopy. Cell surface morphology was assessed with scanning electron microscopy (SEM).
The stimulation of round-smooth neutrophils with nanomolar concentrations (10(-8) M) of fMLP in suspension induced a temporal sequence of morphological changes during cell polarization, characterized by 1) increase in size as determined by forward angle scattered light, 2) rapid redistribution of F-actin from a diffuse cytoplasmic localization to the cell periphery, and 3) rapid reorganization of cell surface morphological features, with accumulation of plasma membrane in the front of polar cells. Four cell shapes were identified with SEM after stimulation of round-smooth neutrophils: round-ridged, round-ruffled, nonpolar ruffled, and polar cells. These cell shapes were correlated with a cortical localization, focal aggregates, and multipolar distribution of F-actin. In polar neutrophils, F-actin became concentrated in the front of the cell.
These findings show the relation between reorganization of the microfilamentous cytoskeleton and modifications in cell shape and surface features during cell polarization induced after fMLP activation in neutrophils. This approach offers a powerful tool for further analysis of receptor distribution in polarized, motile neutrophils.
人类中性粒细胞暴露于均匀浓度的趋化因子，如N-甲酰肽，会诱导细胞形态极化。在本研究中，我们报告了悬浮状态下人类中性粒细胞在N-甲酰甲硫氨酰-亮氨酰-苯丙氨酸（fMLP）诱导的细胞极化过程中细胞形状、F-肌动蛋白和细胞表面形态变化的时间顺序。
用微分干涉差显微镜观察10⁻⁸ M fMLP诱导的中性粒细胞形状变化。通过流式细胞术测量前向和侧向散射光来分析细胞大小和细胞颗粒度。为了可视化F-肌动蛋白分布，用荧光探针异硫氰酸荧光素-鬼笔环肽标记中性粒细胞，并用荧光显微镜和共聚焦激光扫描显微镜进行检查。用扫描电子显微镜（SEM）评估细胞表面形态。
在悬浮液中用纳摩尔浓度（10⁻⁸ M）的fMLP刺激圆形光滑的中性粒细胞，在细胞极化过程中诱导了一系列形态变化，其特征为：1）由前向角散射光测定的细胞大小增加；2）F-肌动蛋白从弥漫性胞质定位迅速重新分布到细胞周边；3）细胞表面形态特征迅速重组，质膜在极化细胞前端积累。用SEM观察圆形光滑中性粒细胞受刺激后的四种细胞形状：圆形脊状、圆形皱褶状、非极性皱褶状和极性细胞。这些细胞形状与F-肌动蛋白的皮质定位、局灶性聚集和多极分布相关。在极性中性粒细胞中，F-肌动蛋白集中在细胞前端。
这些发现表明了中性粒细胞在fMLP激活后诱导的细胞极化过程中，微丝细胞骨架重组与细胞形状和表面特征改变之间的关系。这种方法为进一步分析极化、运动性中性粒细胞中的受体分布提供了一个有力工具。
