Straight Samuel W, Shin Kunyoo, Fogg Vanessa C, Fan Shuling, Liu Chia-Jen, Roh Michael, Margolis Ben
University of Michigan, Department of Biological Chemistry, Ann Arbor, Michigan 48109, USA.
Mol Biol Cell. 2004 Apr;15(4):1981-90. doi: 10.1091/mbc.e03-08-0620. Epub 2004 Jan 12.
Prior work in our laboratory established a connection between the PALS1/PATJ/CRB3 and Par6/Par3/aPKC protein complexes at the tight junction of mammalian epithelial cells. Utilizing a stable small interfering RNA expression system, we have markedly reduced expression of the tight junction-associated protein PALS1 in MDCKII cells. The loss of PALS1 resulted in a corresponding loss of expression of PATJ, a known binding partner of PALS1, but had no effect on the expression of CRB3. However, the absence of PALS1 and PATJ expression did result in the decreased association of CRB3 with members of the Par6/Par3/aPKC protein complex. The consequences of the loss of PALS1 and PATJ were exhibited by a delay in the polarization of MDCKII monolayers after calcium switch, a decrease in the transepithelial electrical resistance, and by the inability of these cells to form lumenal cysts when grown in a collagen gel matrix. These defects in polarity determination may be the result of the lack of recruitment of aPKC to the tight junction in PALS1-deficient cells, as observed by confocal microscopy, and subsequent alterations in downstream signaling events.
我们实验室之前的工作在哺乳动物上皮细胞的紧密连接处建立了PALS1/PATJ/CRB3与Par6/Par3/aPKC蛋白复合物之间的联系。利用稳定的小干扰RNA表达系统，我们显著降低了MDCKII细胞中紧密连接相关蛋白PALS1的表达。PALS1的缺失导致其已知结合伴侣PATJ的表达相应缺失，但对CRB3的表达没有影响。然而，PALS1和PATJ表达的缺失确实导致CRB3与Par6/Par3/aPKC蛋白复合物成员的结合减少。PALS1和PATJ缺失的后果表现为钙转换后MDCKII单层细胞极化延迟、跨上皮电阻降低，以及这些细胞在胶原凝胶基质中生长时无法形成管腔囊肿。如共聚焦显微镜观察到的，极性确定中的这些缺陷可能是由于PALS1缺陷细胞中aPKC缺乏募集到紧密连接处，以及随后下游信号事件的改变所致。
