Loss of PALS1 Expression Leads to Tight Junction and Polarity Defects

Abstract

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.

PALS1 Regulates E-Cadherin Trafficking in Mammalian Epithelial Cells

“…Lysates were cleared by centrifugation (12,000 ϫ g for 15 min at 4°C). Western blotting was performed as described previously (Straight et al, 2004).…”

“…Prior work in our laboratory showed that when the expression of the tight junction-associated protein PALS1 is markedly reduced in MDCKII cells by SiRNA expression, the expression level of PATJ is lowered accordingly, and the formation of tight junctions is significantly delayed (Straight et al, 2004). To further investigate the role of PALS1 in junction formation, we designed two canine-specific SiRNA sequences and created PALS1 KD cell lines by stably expressing the SiRNA-encoding plasmid in MDCKII cells.…”

“…Like Sdt, the PDZ domain of PALS1 binds the C-terminal tail of mammalian CRB isoforms, and PALS1 also interacts with a multi-PDZ domain protein, PALS1-Associated Tight Junction protein (PATJ), through binding to the N-terminal L27 (L27N) domain of PALS1 (Roh et al, 2002). The PALS1-PATJ-CRB complex localizes to the tight junction of mammalian epithelial cells, and the disruption of the complex leads to defects in cell polarity (Straight et al, 2004). The C-terminal L27 (L27C) domain of PALS1 interacts with an L27 domain within Lin-7 (Kamberov et al, 2000), and prior work in our laboratory has shown that an evolutionarily conserved region in the N terminus of PALS1 mediates its interaction with Par6.…”

“…Prior work in our laboratory has demonstrated that the knockdown of PALS1 in Madin Darby canine kidney (MDCK) cells leads to tight junction and polarity defects (Straight et al, 2004). In this study, we created new PALS1 stable knockdown (KD) cell lines with more profound reduction of PALS1 expression.…”

Rôle des protéines Crumbs dans le contrôle de la morphogenèse des cellules épithéliales et des photorécepteurs

“…PATJ se lie également à Pals1 (protein associated with Lin-7, homologue de Sdt), une MAGUK localisée dans les jonctions serrées, et peut donc se lier potentiellement à d'autres protéines ayant un rôle à jouer dans l'organisation de la région apicale des jonctions [34]. La perte d'expression de Pals1 perturbe l'établis-sement des jonctions serrées, indiquant que ce complexe de protéines est important pour ce processus [35]. CRB3 est exprimé par la majorité des épithéliums et sa surexpression entraîne aussi un défaut de formation des jonctions serrées [36,37].…”