To test this hypothesis, we measured the dynamics of membrane redistribution of DE-Cad and Arm within the AJs by carrying out diffusion or exchange-based FRAP assays in which a spot of ~0.84 m diameter on an AJ segment was bleached by a 488 nm laser and its recovery recorded every 2 seconds (supplementary material Fig. in polarized epithelial cells. Consistently, biochemical assays showed that the binding of Arm to DE-Cad is weaker in polarizing cells than in polarized cells. Our data revealed that the molecular interaction between DE-Cad and Arm is modulated during apicalbasal polarization, suggesting a new mechanism that might be crucial for establishing apicalbasal polarity through regulating the AJ dynamics. Key words:Adherens junction, Apicalbasal polarity, DE-cadherin, Armadillo,Drosophila == Introduction == Establishing and maintaining apicalbasal polarity is essential for epithelial cell morphology, function and tissue integrity. One hallmark of apicalbasal polarity in epithelial cells is the demarcation of their membrane domains by polarized formation of cell junctions such as adherens junctions (AJs). The Thalidomide cell adhesion molecule E-cadherin and its cytosolic partner -catenin (Armadillo, Arm) are the major components of the AJ complex. As a transmembrane protein, E-cadherin in AJs is constantly under turnover through vesicle trafficking, and such a dynamic nature of the AJ complex is required for modulating the adhesion properties during tissue morphogenesis (Classen et al., 2005;Gumbiner, 2005;Ulrich et al., 2005). Formation of AJs is a crucial process in apicalbasal polarization in epithelial cells and it is conceivable that establishing and maintaining the polarity requires proper regulation of AJ dynamics. However, systematic and quantitative assays of in vivo AJ dynamics during apicalbasal polarization have yet to be done. It is known thatDrosophilaE-cadherin (DE-Cad) is regulated by actin-based endocytosis in polarized pupal epithelial cells (Georgiou et al., 2008;Leibfried et al., 2008) and in morphogenetically active neuroectoderm (Duncan and Peifer, 2008;Harris and Tepass, 2008), but quantitative data measuring such turnover are not available. AJ dynamics can be regulated by distinct mechanisms involving dramatically different spatial-temporal scales. The biosynthesis-based turnover of the AJ complex often is a rather slow process, for instance the membrane E-cadherin in AJs of polarized MDCK cells showed a long half-life of approximately 5 hours (Shore and Nelson, 1991). Similarly, ectopically expressed DE-Cad::mEosFP fusion protein in AJs of polarizingDrosophilaembryonic epithelia also showed very little turnover within a 1 hour period (Cavey et al., 2008). These results seem to suggest that the biosynthetic turnover of membrane E-cadherin remains slow in AJs of either polarized or polarizing cells, and that its dynamics are not subjected to stage-specific regulations during polarization. However, such interpretations are complicated by the fact that the DE-Cad::mEosFP was ectopically expressed and assayed in a heterogeneous background, which might obscure the dynamics of endogenous DE-Cad. Furthermore, MDCK cells polarize through mesenchymalepithelial transition that is based on contact-initiated cell adhesion, whereasDrosophilaembryonic epithelial cells polarize through pre-existing polarity cues Thalidomide (Cavey and Lecuit, 2009), making their results Thalidomide not directly comparable. In contrast to the slow biosynthetic turnover of E-cadherin, its local turnover at the membrane (i.e. membrane redistribution) is a much faster process, with half-times often measured in seconds (Cavey et al., 2008;de Beco et al., 2009), and has long been assumed to be based on the adhesion-dependent lateral diffusion of E-cadherins (Cavey et al., 2008). Recently, de Beco et al. showed that membrane redistribution of E-cadherin in matured AJs of polarized MDCK cells contained no lateral diffusion, but was an exchange-based process requiring dynamin-mediated endocytosis (de Beco et al., 2009). Again, little is known about how membrane Rabbit polyclonal to VCAM1 redistribution dynamics of E-cadherin and Arm are regulated during apicalbasal polarization and how such regulations might be implicated in establishing the cell polarity. For the purpose of assaying the in vivo AJ dynamics during apicalbasal polarization,Drosophilaembryonic epithelia can serve as an excellent genetically tractable system. The apicalbasal polarity inDrosophilaembryonic primary epithelia is established in a stepwise fashion that correlates well with embryogenesis (Tepass, 1997) (Fig. 1A), making it possible to precisely and reproducibly identify cells at distinct stages of polarization simply by staging the embryos. In addition, we have recently made two new and genetically.