KINETICS OF DESMOSOME ASSEMBLY IN MADIN-DARBY CANINE KIDNEY EPITHELIAL-CELLS - TEMPORAL AND SPATIAL REGULATION OF DESMOPLAKIN ORGANIZATION AND STABILIZATION UPON CELL CELL CONTACT .2. MORPHOLOGICAL ANALYSIS
KINETICS OF DESMOSOME ASSEMBLY IN MADIN-DARBY CANINE KIDNEY EPITHELIAL-CELLS - TEMPORAL AND SPATIAL REGULATION OF DESMOPLAKIN ORGANIZATION AND STABILIZATION UPON CELL CELL CONTACT .2. MORPHOLOGICAL ANALYSIS
复制标题
DOI：
10.1083/jcb.106.3.687
复制
发表时间：
1988-03-01
影响因子：
7.8
通讯作者：
NELSON, WJ
中科院分区：
文献类型：
--
作者：
PASDAR, M;NELSON, WJ
在Madin-Darby犬肾（MDCK）上皮细胞中，桥粒的两种细胞质斑块蛋白，桥粒斑块蛋白I（250，000 Mr）和II（215，000 Mr）的组装动力学的生物化学分析表明，这些蛋白存在于可溶性和不溶性池中，如通过它们在Triton X-100高盐缓冲液（CSK缓冲液）中的提取能力所定义的。在细胞-细胞接触时，以可溶性池为代价，不溶性池的容量快速增加;随后，不溶性池稳定化，而残留在可溶性池中的蛋白质继续快速降解（Pasdar，M.，和W.纳尔逊。1988. 106：677-685）。在本文中，我们试图确定的空间分布的可溶性和不溶性池的桥粒斑蛋白I和II，和他们的组织中存在和不存在的细胞-细胞接触，通过使用差分提取程序和间接免疫荧光显微镜。在没有细胞-细胞接触的情况下，观察到桥粒斑蛋白I和II的两种形态和空间上不同的染色模式：在细胞质和核周区域中的离散斑点模式，其不溶于CSK缓冲液;和弥散核周染色模式，其可溶于CSK缓冲液，但当细胞在-20 ℃固定在100%甲醇中时保留。C.在细胞-细胞接触时，在不存在或存在蛋白质合成的情况下，桥粒斑蛋白I和II的点状染色模式在细胞-细胞接触区域（< 180分钟）中从细胞质到质膜被快速有效地清除。弥漫性核周染色模式的分布保持相对不变，并成为主要形式的桥斑蛋白I和II在细胞质中诱导细胞-细胞接触后180分钟。此后，弥漫图案的染色的相对强度逐渐减弱，并且在细胞-细胞接触诱导后2-3天完全不存在。 值得注意的是，双重免疫荧光显示，在质膜上的桥粒组装过程中，两种染色模式都与细胞角蛋白中间丝的亚群相吻合。与前面的生化分析一起，我们认为，在MDCK上皮细胞中的桥粒斑蛋白I和II的组装在桥粒形成过程中的三个离散阶段进行调节。
Biochemical analysis of the kinetics of assembly of two cytoplasmic plaques proteins of the desmosome, desmoplakins I (250,000 Mr) and II (215,000 Mr), in Madin-Darby canine kidney (MDCK) epithelial cells, demonstrated that these proteins exist in a soluble and insoluble pool, as defined by their extract ability in a Triton X-100 high salt buffer (CSK buffer). Upon cell-cell contact, there is a rapid increase in the capacity of the insoluble pool at the expense of the soluble pool; subsequently, the insoluble pool is stabilized, while proteins remaining in the soluble pool continue to be degraded rapidly (Pasdar, M., and W. J. Nelson. 1988. J. Cell Biol. 106:677-685). In this paper, we have sought to determine the spatial distribution of the soluble and insoluble pools of desmoplakins I and II, and their organization in the absence and presence of cell-cell contact by using differential extraction procedures and indirect immunofluorescence microscopy. In the absence of cell-cell contact, two morphologically and spatially distinct patterns of staining of desmoplakins I and II were observed: a pattern of discrete spots in the cytoplasm and perinuclear region, which is insoluble in CSK buffer; and a pattern of diffuse perinuclear staining, which is soluble in CSK buffer, but which is preserved when cells are fixed in 100% methanol at -20.degree. C. Upon cell-cell contact, in the absence or presence of protein synthesis, the punctate staining pattern of desmoplakins I and II is cleared rapidly and efficiently from the cytoplasm to the plasma membrane in areas of cell-cell contact (< 180 min). The distribution of the diffuse perinuclear staining pattern remains relatively unchanged and becomes the principal form of desmoplakins I and II in the cytoplasm 180 min after induction of cell-cell contact. Thereafter, the relative intensity of staining of the diffuse pattern gradually diminishes and is completely absent 2-3 d afer induction of cell-cell contact. Significantly, double immunofluorescence shows that during desmosome assembly on the plasma membrane both staining patterns coincide with a subpopulation of cytokeratin intermediate filaments. Taken together with the preceding biochemical analysis, we suggest that the assembly of desmoplakins I and II in MDCK epithelial cells is regulated at three discrete stages during the formation of desmosomes.