Parast M M, Otey C A
Department of Cell Biology, University of Virginia, Charlottesville, Virginia 22908, USA.
J Cell Biol. 2000 Aug 7;150(3):643-56. doi: 10.1083/jcb.150.3.643.
Here, we describe the identification of a novel phosphoprotein named palladin, which colocalizes with alpha-actinin in the stress fibers, focal adhesions, cell-cell junctions, and embryonic Z-lines. Palladin is expressed as a 90-92-kD doublet in fibroblasts and coimmunoprecipitates in a complex with alpha-actinin in fibroblast lysates. A cDNA encoding palladin was isolated by screening a mouse embryo library with mAbs. Palladin has a proline-rich region in the NH(2)-terminal half of the molecule and three tandem Ig C2 domains in the COOH-terminal half. In Northern and Western blots of chick and mouse tissues, multiple isoforms of palladin were detected. Palladin expression is ubiquitous in embryonic tissues, and is downregulated in certain adult tissues in the mouse. To probe the function of palladin in cultured cells, the Rcho-1 trophoblast model was used. Palladin expression was observed to increase in Rcho-1 cells when they began to assemble stress fibers. Antisense constructs were used to attenuate expression of palladin in Rcho-1 cells and fibroblasts, and disruption of the cytoskeleton was observed in both cell types. At longer times after antisense treatment, fibroblasts became fully rounded. These results suggest that palladin is required for the normal organization of the actin cytoskeleton and focal adhesions.
在此,我们描述了一种名为帕拉丁的新型磷蛋白的鉴定过程,它与α - 辅肌动蛋白共定位于应力纤维、粘着斑、细胞间连接和胚胎Z线中。帕拉丁在成纤维细胞中以90 - 92 kD的双条带形式表达,并在成纤维细胞裂解物中与α - 辅肌动蛋白形成复合物进行共免疫沉淀。通过用单克隆抗体筛选小鼠胚胎文库分离出了编码帕拉丁的cDNA。帕拉丁在分子的NH(2) - 末端一半具有富含脯氨酸的区域,在COOH - 末端一半具有三个串联的Ig C2结构域。在鸡和小鼠组织的Northern和Western印迹中,检测到了帕拉丁的多种同工型。帕拉丁在胚胎组织中广泛表达,而在小鼠的某些成年组织中表达下调。为了探究帕拉丁在培养细胞中的功能,使用了Rcho - 1滋养层细胞模型。当Rcho - 1细胞开始组装应力纤维时,观察到帕拉丁表达增加。使用反义构建体来减弱Rcho - 1细胞和成纤维细胞中帕拉丁的表达,并且在两种细胞类型中都观察到了细胞骨架的破坏。在反义处理后的较长时间,成纤维细胞变得完全圆润。这些结果表明,帕拉丁是肌动蛋白细胞骨架和粘着斑正常组织所必需的。