Langhofer M, Hopkinson S B, Jones J C
Department of Cell, Molecular and Structural Biology, Northwestern University Medical School, Chicago, IL 60611.
J Cell Sci. 1993 Jul;105 ( Pt 3):753-64. doi: 10.1242/jcs.105.3.753.
Hemidesmosomes are important adhesion devices found in epithelial cells. They connect the intermediate filament cytoskeleton network with components of the basement membrane zone. 804G cells are an unusual epithelial cell line, since they form bona fide hemidesmosomes when plated on glass or plastic. In this study we tested an hypothesis: that this ability is a consequence of an extracellular component produced by the 804G cells. As probes for our study we generated a rabbit antiserum (J18) and monoclonal antibodies against components of urea-solubilized 804G matrix. Antibodies in the J18 serum recognize major lectin-binding polypeptides of 150, 140 and 135 kDa in the 804G matrix. A monoclonal antibody (5C5) that shows reactivity with the 150 and 135 kDa polypeptides in western immunoblots immunoprecipitates all three molecular mass species, indicating that these polypeptides are part of a matrix complex. Moreover, one, at least, of these matrix elements is immunologically related to laminin, since J18 antibodies selected on fusion protein fragments of a newly characterized laminin variant, laminin B2t (Kallunki et al., J. Cell Biol., 119, 679-694, 1992), react with the 140 kDa polypeptide component of the 804G cell matrix. To undertake functional analyses of 804G matrix, cells of the human epidermal carcinoma line SCC12, which do not assemble bona fide hemidesmosomes in vitro, were cultured on 804G matrix for 24 h and then analysed by confocal immunofluorescence and electron microscopy. In SCC12 cells maintained on 804G cell matrix, hemidesmosomal antigens localize in a distinctive leopard spot pattern that mirrors the distribution of 804G matrix elements. Furthermore, ultrastructural analysis reveals that the 804G cell matrix supports the formation of 'mature' hemidesmosomes by SCC12 cells. Thus 804G cell matrix is a remarkable tool for hemidesmosome studies and it will now be of great importance to determine the exact composition of the 804G matrix, especially its structural and antigenic relationship to laminins.
半桥粒是上皮细胞中重要的黏附结构。它们将中间丝细胞骨架网络与基底膜区的成分相连。804G细胞是一种特殊的上皮细胞系，因为当接种在玻璃或塑料上时，它们能形成真正的半桥粒。在本研究中，我们验证了一个假说：这种能力是804G细胞产生的一种细胞外成分的结果。作为我们研究的探针，我们制备了一种兔抗血清（J18）以及针对尿素溶解的804G基质成分的单克隆抗体。J18血清中的抗体识别804G基质中150、140和135 kDa的主要凝集素结合多肽。一种在蛋白质免疫印迹中与150和135 kDa多肽发生反应的单克隆抗体（5C5）免疫沉淀了所有这三种分子量的物质，表明这些多肽是基质复合物的一部分。此外，这些基质成分中至少有一种在免疫上与层粘连蛋白相关，因为在新鉴定的层粘连蛋白变体层粘连蛋白B2t（Kallunki等人，《细胞生物学杂志》，119，679 - 694，1992）的融合蛋白片段上筛选的J18抗体与804G细胞基质的140 kDa多肽成分发生反应。为了对804G基质进行功能分析，将在体外不能组装真正半桥粒的人表皮癌细胞系SCC12的细胞在804G基质上培养24小时，然后通过共聚焦免疫荧光和电子显微镜进行分析。在维持在804G细胞基质上的SCC12细胞中，半桥粒抗原以独特的豹斑模式定位，这与804G基质成分的分布相对应。此外，超微结构分析表明，804G细胞基质支持SCC12细胞形成“成熟”的半桥粒。因此，804G细胞基质是半桥粒研究的一个出色工具，现在确定804G基质的确切组成，尤其是其与层粘连蛋白的结构和抗原关系将非常重要。
