Wang A Z, Wang J C, Ojakian G K, Nelson W J
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, California 94305-5426.
Am J Physiol. 1994 Aug;267(2 Pt 1):C473-81. doi: 10.1152/ajpcell.1994.267.2.C473.
Madin-Darby canine kidney epithelial cells form three-dimensional cysts in spinner culture with a defined cell surface polarity. Transfer of cysts from spinner culture to a collagen gel matrix results in rapid loss of apical membrane proteins from the outside surface of the cyst, degradation of extracellular matrix (ECM) from the cyst lumen, and de novo formation of the apical membrane at the luminal surface. Degradation of endogenous ECM was inhibited with 1,10-phenanthroline, an inhibitor of metalloproteinases, resulting in cysts in which cells are surrounded by either cell-cell or cell-substratum contacts. The consequence of the lack of a free cell surface on the formation of a new apical membrane domain in these cysts was analyzed. Changes in cell surface polarity were followed with antibodies to marker proteins of the apical or basolateral membranes. In the absence of a free cell surface, the apical membrane formed de novo by accumulation and fusion of presorted vesicles containing apical membrane proteins; the coalescence of these vesicles results in the formation of a central lumen. These results provide novel insights into the generation of membrane domains and formation of a lumen in complex, three-dimensional epithelial structures in development.
Madin-Darby犬肾上皮细胞在旋转培养中形成具有明确细胞表面极性的三维囊肿。将囊肿从旋转培养转移到胶原凝胶基质中，会导致囊肿外表面的顶端膜蛋白迅速丢失，囊肿腔内的细胞外基质（ECM）降解，以及在腔表面重新形成顶端膜。用金属蛋白酶抑制剂1,10-菲咯啉抑制内源性ECM的降解，会导致囊肿中的细胞被细胞-细胞或细胞-基质接触所包围。分析了这些囊肿中缺乏自由细胞表面对新顶端膜结构域形成的影响。用针对顶端或基底外侧膜标记蛋白的抗体追踪细胞表面极性的变化。在没有自由细胞表面的情况下，顶端膜通过含有顶端膜蛋白的预分选囊泡的积累和融合而重新形成；这些囊泡的聚结导致中央腔的形成。这些结果为发育过程中复杂三维上皮结构中膜结构域的产生和腔的形成提供了新的见解。
