Gipson I K, Spurr-Michaud S, Tisdale A, Elwell J, Stepp M A
Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts 02114.
Exp Cell Res. 1993 Jul;207(1):86-98. doi: 10.1006/excr.1993.1166.
As basal cells of stratified squamous epithelia become migratory in response to wounding, they lose their cell-substrate adhesion junctions, the hemidesmosomes. We report here studies to determine the fate of the hemidesmosome components, alpha 6 beta 4 integrin and the bullous pemphigoid antigens (BPAGs), as recognized by bullous pemphigoid autoantisera (BPA), in migrating epithelium. In addition, we report studies to determine whether relative synthesis and amount of alpha 6 beta 4 is altered during migration. Mouse corneas with 1.5- to 2-mm-diameter central epithelial debridements were allowed to heal in vitro or in vivo for 1-18 h. In order to do preembedding immunoelectron microscopic localization of alpha 6 beta 4, sheets of stationary and migrating corneal epithelium were removed from their basal laminae after organ culture. BPA and antibodies to alpha 6 and beta 4 were used for immunofluorescence microscopy on frozen sections of intact corneas healing in vivo 1-18 h. Both alpha 6 and beta 4 were found to redistribute from their clustered location within hemidesmosomes to a more even distribution within the substrate-associated membrane of basal cells of the tip of the leading edge of migrating epithelium. Behind the tip of the leading edge, basal cells bound the integrin antibodies around their entire membrane. BPAGs moved from their location along the basal cell membrane of stationary epithelium to a diffuse location within the cytoplasm of migrating cells at the leading edge of migration. Quantitative immunoprecipitation and immunoblotting of alpha 6 beta 4 as well as beta 1 integrin from stationary and migrating epithelium were done to determine whether the synthesis or total amount of the integrins were altered during migration. The relative syntheses of alpha 6 beta 4 and beta 1 per milligram of protein or per cell do not appear to differ between stationary and migrating epithelium and the total amount of the beta 4 and beta 1 does not change despite increased rates of protein synthesis in migrating epithelium. Taken together, these studies suggest that as hemidesmosomes disassemble, their clustered integrin component distributes more evenly in the basal cell membrane, the components recognized by BPA and associated with intermediate filaments are released from the membrane, and these events occur in the absence of any measurable change in the synthesis or total amount of the alpha 6 beta 4 component.
随着复层鳞状上皮的基底细胞因受伤而迁移，它们失去了细胞与基质的黏附连接，即半桥粒。我们在此报告一些研究，以确定半桥粒成分α6β4整合素和大疱性类天疱疮抗原（BPAGs）（可被大疱性类天疱疮自身抗血清（BPA）识别）在迁移上皮细胞中的命运。此外，我们还报告了一些研究，以确定在迁移过程中α6β4的相对合成量和总量是否发生改变。对直径为1.5至2毫米的中央上皮清创的小鼠角膜进行体外或体内愈合1至18小时。为了对α6β4进行包埋前免疫电子显微镜定位，在器官培养后从其基膜上取下静止和迁移的角膜上皮片。BPA以及抗α6和β4的抗体用于对体内愈合1至18小时的完整角膜冰冻切片进行免疫荧光显微镜检查。发现α6和β4均从它们在半桥粒内的聚集位置重新分布到迁移上皮前缘顶端基底细胞的基质相关膜内更均匀的分布。在前沿顶端之后，基底细胞在其整个膜周围结合整合素抗体。BPAGs从它们在静止上皮基底细胞膜上的位置移动到迁移前沿迁移细胞细胞质内的弥散位置。对静止和迁移上皮中的α6β4以及β1整合素进行定量免疫沉淀和免疫印迹，以确定在迁移过程中整合素的合成或总量是否发生改变。每毫克蛋白质或每个细胞中α6β4和β1的相对合成量在静止和迁移上皮之间似乎没有差异，并且尽管迁移上皮中蛋白质合成速率增加，但β4和β1的总量并未改变。综上所述，这些研究表明，随着半桥粒解体，它们聚集的整合素成分在基底细胞膜中分布得更加均匀，被BPA识别并与中间丝相关的成分从膜上释放，并且这些事件发生时α6β4成分的合成或总量没有任何可测量的变化。
