MDCKII cells were transfected using Amaxa Nucleofector (Lonza) with indicated cDNAs and cultured in Eagles minimal essential medium (MEM) with 10% FCS
MDCKII cells were transfected using Amaxa Nucleofector (Lonza) with indicated cDNAs and cultured in Eagles minimal essential medium (MEM) with 10% FCS. morphogenesis. The plasma membrane of mammalian epithelial cells is asymmetrically organized into apical and basolateral domains; the two domains serve differently to integrate epithelial function. The apical membrane, facing a lumen, is separated from the basolateral one by Alvespimycin tight junctions (TJs), which participate in epithelial barrier function (Goldstein and Macara, 2007; Bryant and Mostov, 2008; Prehoda, 2009; Knoblich, 2010; St Johnston and Rabbit Polyclonal to MC5R Ahringer, 2010). Formation of apico-basal polarity in epithelial cells likely involves atypical protein kinase C (aPKC), which is considered to serve as a master enzyme in animal cell polarization (Goldstein and Macara, 2007; Bryant and Mostov, 2008; Prehoda, 2009; Knoblich, 2010; St Johnston and Ahringer, 2010). aPKC constitutively interacts with Par6, an evolutionarily conserved adaptor protein, which interaction is mediated via N-terminal PB1 (Phox and Bem1p 1) domains of both proteins (Noda et al., 2003; Sumimoto et al., 2007). In Par6, the PB1 domain is followed by a semi-CRIB (Cdc42/Rac interactive binding) motif and a PDZ (PSD95/Dlg/ZO-1) domain (Kemphues, 2000; Noda et al., 2003; Suzuki and Ohno, 2006; Sumimoto et al., 2007). During epithelial cell polarization in the fruit fly epithelial cells, wild-type Par6 localizes to the apical membrane, but a mutant protein defective in binding to Cdc42 delocalizes to the cytoplasm, resulting in impaired formation of apico-basal polarity (Hutterer et al., 2004). Although this finding suggests that Cdc42 localizes to the apical surface for anchoring of Par6, apical localization of Cdc42 in these cells has not been well evidenced. This may be because anti-Cdc42 antibodies suitable for immunostaining have been unavailable or because fixation conditions used have been unsuitable for immunostaining. Similarly, in monolayer culture of mammalian epithelial cells such as Madin-Darby canine kidney (MDCK) cells, localization of endogenous Cdc42 has not been well studied, although it has been reported that, in 3D culture of MDCK cells, GFP-fused Cdc42 is recruited to the apical surface and appears to participate in apical localization of aPKC (Martin-Belmonte et al., 2007). The role of Cdc42 in aPKC targeting to the apical surface, however, has been Alvespimycin questioned using experiments of 3D culture of human colon carcinoma-derived Caco-2 cells (Jaffe et al., 2008). The type I transmembrane protein Crumbs, another Par6 target, is known to serve as an evolutionarily conserved apical determinant (Bulgakova and Knust, 2009; Datta et al., 2011). The C-terminal cytoplasmic region of Crumbs contains a canonical PDZ-binding motif, which directly interacts with the Par6 PDZ domain (Lemmers et al., 2004) and also with the PDZ domain of Pals1, an adaptor protein that is enriched together with Patj at TJs but not in the apical surface (Makarova et Alvespimycin al., 2003). In epithelial cells, Par6 localization to the apical surface appears to require Crumbs (Kempkens et al., 2006). Its dominant homologue in mammalian epithelial cells is Crumbs3 (Crb3; Makarova et al., 2003; Lemmers et al., 2004). Crb3 has been shown to be capable of recruiting Par6 to the membrane in unpolarized mammalian cells (Hurd et al., 2003). It has recently been reported that depletion of Crb3 results in a failure of aPKC to localize to the forming apical membrane of MDCK cells at the two-cell stage in 3D culture.