Hock R S, Sanger J M, Sanger J W
Department of Anatomy, University of Pennsylvania School of Medicine, Philadelphia 19104-6058.
Cell Motil Cytoskeleton. 1989;14(2):271-87. doi: 10.1002/cm.970140213.
To investigate the role of talin in the anchoring of actin-containing stress fibers to the cell membrane of nonmuscle cells, a fluorescent analog of the adhesion plaque protein talin was developed, characterized, and microinjected into living cells. Purified chicken gizzard talin was covalently labeled with the fluorescent dye lissamine rhodamine B sulfonyl chloride. The fluorescently labeled protein was then chromatographed on Sephadex G-25 and DEAE-cellulose in order to remove free dye and denatured protein. The fluorescent talin was able to bind purified vinculin and was localized in adhesion plaques, membrane ruffles, microspikes, and polygonal networks in acetone-permeabilized nonmuscle cells. In cells that were double-stained with fluorescent talin and an affinity-purified anti-talin antibody, a one-to-one correspondence of adhesion plaque staining was seen. Living epithelial cells (PtK2) were microinjected during interphase with fluorescent talin. Computer-enhanced video microscopy was used to document adhesion plaque dynamics such as 1) changes in plaque shape, 2) alterations in plaque positions, and 3) the appearance, growth, and dissolution of plaques. In cells that were followed during mitosis, the adhesion plaques disappeared during cell rounding and then subsequently reappeared upon spreading of the two daughter cells. Treatment of microinjected cells with DMSO in order to disassemble stress fibers resulted in an altered localization of the fluorescent talin. Upon recovery of the cell from the drug, the talin was visualized in its characteristic submembraneous position. These results are the first to document the role and distribution of talin in dynamic processes occurring in living microinjected nonmuscle cells.
为了研究踝蛋白在含肌动蛋白的应力纤维与非肌肉细胞细胞膜锚定过程中的作用，我们开发、表征了一种粘着斑蛋白踝蛋白的荧光类似物，并将其显微注射到活细胞中。将纯化的鸡砂囊踝蛋白与荧光染料丽丝胺罗丹明B磺酰氯共价标记。然后将荧光标记的蛋白在葡聚糖凝胶G - 25和二乙氨基乙基纤维素上进行层析，以去除游离染料和变性蛋白。荧光踝蛋白能够结合纯化的纽蛋白，并定位于丙酮通透的非肌肉细胞中的粘着斑、膜皱褶、微刺和多边形网络中。在用荧光踝蛋白和亲和纯化的抗踝蛋白抗体进行双重染色的细胞中，可见粘着斑染色呈现一一对应关系。在间期将荧光踝蛋白显微注射到活的上皮细胞（PtK2）中。利用计算机增强视频显微镜记录粘着斑的动态变化，如1）斑块形状的变化，2）斑块位置的改变，以及3）斑块的出现、生长和溶解。在有丝分裂过程中跟踪观察的细胞中，粘着斑在细胞变圆时消失，然后在两个子细胞铺展时再次出现。用二甲基亚砜处理显微注射的细胞以拆解应力纤维，导致荧光踝蛋白的定位发生改变。细胞从药物处理中恢复后，踝蛋白在其特征性的膜下位置可见。这些结果首次记录了踝蛋白在显微注射的活非肌肉细胞中发生的动态过程中的作用和分布。
