Dystrophin isoform Dp71 is present in lamellipodia and focal complexes in human astrocytoma cells U-373 MG
Dystrophin isoform Dp71 is present in lamellipodia and focal complexes in human astrocytoma cells U-373 MG
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DOI：
10.1078/0065-1281-00654
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发表时间：
2002-01-01
影响因子：
2.5
通讯作者：
Hernández, JM
中科院分区：
文献类型：
--
作者：
García-Tovar, CG;Luna, J;Hernández, JM
Dp71是dmd基因在大脑中最丰富的产物。至少有2种异构体是由78外显子的选择性剪接而产生的（Dp71d，其中包含78外显子和Dp71f剪接异构体），但每种异构体的精确定位和功能仍然未知。在本研究中，我们通过RT-PCR证明了Dp71f异构体存在于星形细胞瘤细胞系U-373 MG中，其亚细胞定位在细胞质中，特别是在核周区域，向外周的数量较少，但在板足和局灶复合体的领导边界增加。双标记间接免疫荧光显示Dp71f与肌动蛋白样β -肌动蛋白样β -肌动蛋白样糖酐和β -1整合素共定位。我们还通过三重标记证明了Dp71f与肌动蛋白和整合素复合物的两个成员（α -肌动蛋白和血管蛋白）在局灶复合物中共定位。腹侧质膜富集，在含有局灶复合蛋白的质膜中，我们发现了Dp71f、肌动蛋白和血管蛋白的共定位。由此可见，U-373 MG细胞将Dp71f作为板足和局灶复合体蛋白的一部分表达，并可能通过二叉聚糖复合体与整合素复合体连接。
Dp71 is the most abundant product of the dmd gene in the brain. There are at least 2 isoforms derived from alternative splicing of exon 78 (Dp71d, which contains exon 78 and Dp71f, the spliced isoform) but the precise localization and function of each isoform is still unknown. In the present study, we demonstrate by RT-PCR that the Dp71f isoform is present in an astrocytoma cell line U-373 MG, and its subcellular localization was determined in the cytoplasm, particularly in perinuclear areas, with lower amounts towards the periphery but increasing in the leader borders of lamellipodia and focal complexes. Double labeling indirect immunofluorescence showed that Dp71f colocalized with actin-like beta-dystroglycan and beta-1 integrin. We also demonstrated by triple labeling that Dp71f was colocalized with actin and two members of integrin complexes, alpha-actinin and vinculin, in focal complexes. Ventral plasma membranes were enriched and in those containing focal complex proteins, we found colocalization of Dp71f, actin and vinculin. It is concluded that U-373 MG cells express Dp71f as part of lamellipodia and focal complex proteins, and possibly connected via distroglycan complexes to integrin complexes.