Colosetti Pascal, Tunwell Richard E A, Cruttwell Caroline, Arsanto Jean-Pierre, Mauger Jean-Pierre, Cassio Doris
INSERM U-442, Signalisation cellulaire et calcium, Bât 443, Université Paris-Sud, 91405 Orsay Cedex, France.
J Cell Sci. 2003 Jul 1;116(Pt 13):2791-803. doi: 10.1242/jcs.00482. Epub 2003 May 20.
The subcellular localization of inositol 1,4,5-trisphosphate (InsP3)-induced Ca2+ signals is important for the activation of many physiological functions. In epithelial cells the spatial distribution of InsP3 receptor is restricted to specific areas, but little is known about the relationship between the receptor's distribution and cell polarity. To investigate this relationship, the best known polarized cell model, MDCK, was examined. This cell line is characterized by a strong expression of the type 3 InsP3 receptor and the subcellular localization of this receptor was followed during cell polarization using immunofluorescence and confocal analysis. In non-polarized cells, including ras transformed f3 MDCK cells, the type 3 InsP3 receptor was found to co-localize with markers of the endoplasmic reticulum in the cytoplasm. In contrast, in polarized cells, this receptor was mostly distributed at the apex of the lateral plasma membrane with the markers of tight junctions, ZO-1 and occludin. The localization of the type 3 InsP3 receptor in the vicinity of tight junctions was confirmed by immunogold electron microscopy. The culture of MDCK cells in calcium-deprived medium, led to disruption of cell polarity and receptor redistribution in the cytoplasm. Addition of calcium to these deprived cells induced the restoration of polarity and the relocalization of the receptor to the plasma membrane. MDCK cells were stably transfected with a plasmid coding the full-length mouse type 1 InsP3 receptor tagged with EGFP at the C-terminus. The EGFP-tagged type 1 receptor and the endogenous type 3 co-localized in the cytoplasm of non-polarized cells and at the tight junction level of polarized cells. Thus, the localization of InsP3 receptor in MDCK depends on polarity.
肌醇1,4,5 - 三磷酸（InsP3）诱导的Ca2+信号的亚细胞定位对于激活许多生理功能很重要。在上皮细胞中，InsP3受体的空间分布局限于特定区域，但关于该受体分布与细胞极性之间的关系却知之甚少。为了研究这种关系，我们检测了最著名的极化细胞模型——MDCK细胞。该细胞系的特征是强烈表达3型InsP3受体，并在细胞极化过程中使用免疫荧光和共聚焦分析追踪该受体的亚细胞定位。在非极化细胞中，包括ras转化的βMDCK细胞，发现3型InsP3受体与内质网标记物在细胞质中共定位。相反，在极化细胞中，该受体主要分布在外侧质膜顶端，与紧密连接标记物ZO - 1和闭合蛋白在一起。免疫金电子显微镜证实了3型InsP3受体在紧密连接附近的定位。在缺钙培养基中培养MDCK细胞会导致细胞极性破坏和受体在细胞质中重新分布。向这些缺Ca2+的细胞中添加Ca2+会诱导极性恢复以及受体重新定位到质膜。用编码在C末端标记有EGFP的全长小鼠1型InsP3受体的质粒稳定转染MDCK细胞。带有EGFP标记的1型受体与内源性3型受体在非极化细胞的细胞质中以及极化细胞的紧密连接水平上共定位。因此，MDCK细胞中InsP3受体的定位取决于极性。
