Thacher S M, Malone K L, Dave K, Zhao S M
Department of Medical Biochemistry and Genetics, Texas A & M College of Medicine, College Station 77843-1114.
Exp Cell Res. 1991 Jun;194(2):238-47. doi: 10.1016/0014-4827(91)90360-7.
The hemidesmosome is the major attachment structure of the epidermal basal cell visible ultrastructurally in skin. The importance of its components to cultured cell attachment to substratum is not understood, however. A component of the hemidesmosome, the 230-kDa bullous pemphigoid antigen (p230), has been shown to be present in an insoluble or particulate fraction of cultured cells. In order to more fully characterize its potential importance for cell-matrix adhesion in cultured keratinocytes, specific antibodies were raised to the C-terminal region of p230 expressed as a bacterial fusion protein. Such antibodies recognize the hemidesmosome of epidermis, binding on the cytoplasmic region of its plaque. In addition, keratinocytes cultured in a 0.15 mM Ca(2+)-defined medium contain a detergent-resistant pool of p230 which appears to lie in the same focal plane as the culture substrate and has a patchy or irregular distribution by indirect immunofluorescence. Treatment of cultured cells at 4 degrees C with trypsin or pronase sufficient to release keratinocytes from the culture dish does not affect the electrophoretic migration of p230 on SDS-gels, suggesting that p230 is not exposed to the extracellular space. In cells cultured in 0.15 mM Ca2+, 230-kDa BP antigen is localized to discrete clusters resting near the basal plasma membrane of the cell by immunogold staining following brief detergent treatment and fixation. These clusters are approximately 0.1 micron in diameter, which is similar in size to the in vivo hemidesmosome. Fully formed electron dense hemidesmosomal plaques are not observed under the same culture conditions, however. It appears that these clusters are early precursors of the hemidesmosome.
半桥粒是皮肤中在超微结构上可见的表皮基底细胞的主要附着结构。然而，其组成成分对培养细胞附着于底物的重要性尚不清楚。半桥粒的一个组成成分，即230-kDa大疱性类天疱疮抗原（p230），已被证明存在于培养细胞的不溶性或颗粒性部分中。为了更全面地描述其对培养角质形成细胞中细胞-基质黏附的潜在重要性，制备了针对以细菌融合蛋白形式表达的p230 C末端区域的特异性抗体。此类抗体可识别表皮的半桥粒，结合在其斑块的细胞质区域。此外，在0.15 mM Ca(2+)限定培养基中培养的角质形成细胞含有一个耐去污剂的p230池，通过间接免疫荧光观察，该池似乎与培养底物处于同一焦平面，且分布呈斑片状或不规则状。在4℃用足以使角质形成细胞从培养皿中释放的胰蛋白酶或链霉蛋白酶处理培养细胞，并不影响p230在SDS凝胶上的电泳迁移，这表明p230未暴露于细胞外空间。在0.15 mM Ca2+中培养的细胞中，经短暂去污剂处理和固定后，通过免疫金染色，230-kDa BP抗原定位于靠近细胞基底质膜的离散簇中。这些簇的直径约为0.1微米，大小与体内半桥粒相似。然而，在相同培养条件下未观察到完全形成的电子致密半桥粒斑块。这些簇似乎是半桥粒的早期前体。
