Differential regulation of adherens junction dynamics during apical–basal polarization

Abstract

SummaryAdherens junctions (AJs) in epithelial cells are constantly turning over to modulate adhesion properties under various physiological and developmental contexts, but how such AJ dynamics are regulated during the apical-basal polarization of primary epithelia remains unclear. Here, we used new and genetically validated GFP markers of Drosophila E-cadherin (DE-cadherin, hereafter referred to as DE-Cad) and bcatenin (Armadillo, Arm) to quantitatively assay the in vivo dynamics of biosynthetic turnover and membrane redistribution by fluorescence recovery after photobleaching (FRAP) assays. Our data showed that membrane DE-Cad and Arm in AJs of polarizing epithelial cells had much faster biosynthetic turnover than in polarized cells. Fast biosynthetic turnover of membrane DE-Cad is independent of actin-and dynamin-based trafficking, but is microtubule-dependent. Furthermore, Arm in AJs of polarizing cells showed a faster and diffusion-based membrane redistribution that was both quantitatively and qualitatively different from the slower and exchange-based DE-Cad membrane distribution, indicating that the association of Arm with DE-Cad is more dynamic in polarizing cells, and only becomes stable 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 apical-basal polarization, suggesting a new mechanism that might be crucial for establishing apical-basal polarity through regulating the AJ dynamics.Key words: Adherens junction, Apical-basal polarity, DE-cadherin, Armadillo, Drosophila Introduction Establishing and maintaining apical-basal polarity is essential for epithelial cell morphology, function and tissue integrity. One hallmark of apical-basal polarity in epithelial cells is the demarcation of their membrane domains by polarized formation of cell junctions such as adherens junctions (AJs). The cell adhesion molecule E-cadherin and its cytosolic partner b-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 apical-basal 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 apical-basal polarization have yet to be done. It is known that Drosophila E-cadherin (DECad) 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 ...

Phosphorylation potential of Drosophila E-Cadherin intracellular domain is essential for development and adherens junction biosynthetic dynamics regulation

“…3A). In addition, DECadASSA has significantly reduced biosynthetic turnover in polarizing cells and increased turnover in polarized cellsessentially a reversed differential regulation pattern of AJ dynamics compared with wild-type DE-Cad during cell polarization (Huang et al, 2011). Consistent with our whole-cell FRAP results, latrunculin treatment also showed that in DECadS4A-αCat and DE-CadASSA embryos the AJ-localized DECad, and presumably AJs, are more resistant to the loss of F-actin (Fig.…”

“…Stage 9-11 embryos were selected for assaying polarizing epithelial cells and stage 15 embryos for assaying polarized cells. Whole-cell FRAP assays were performed in lateral epidermis (Huang et al, 2011). Although all DE-Cad* show reduced levels of β-Catenin in AJs in vivo, overall it appears that loss of phosphorylation potential of the SLSSLAS motif in DE-Cad does not significantly increase the biosynthetic instability of DE-Cad in AJs ( Fig.…”

“…To investigate whether phosphorylation of the S-motif could regulate AJ turnover dynamics, we carried out whole-cell fluorescence recovery after photobleaching (FRAP) assays (Huang et al, 2011) to measure specifically the biosynthetic turnover rates of selected DE-Cad* mutants in polarizing and polarized cells ( Fig. 3A; Fig.…”

“…In addition, cell culture studies showed that phosphorylation of specific serine residues within the cluster could play distinct roles in regulating AJ formation and stability (Choi et al, 2015;Lickert et al, 2000;McEwen et al, 2014). In Drosophila, we previously reported that the biosynthetic turnover of AJs is differentially regulated during apical-basal polarization in Drosophila embryonic epithelia (Huang et al, 2011). The increased AJ stability in polarized cells coincides with the stronger binding between Drosophila E-Cadherin (DE-Cad, also known as Shotgun or Shg) and β-Catenin (also known as Armadillo or Arm; Peifer and Wleschaus, 1990) (Huang et al, 2011), making DE-Cad phosphorylation an attractive mechanism for modulating the DECad/β-Catenin interaction during cell polarization.…”

Compensatory branching morphogenesis of stalk cells in the Drosophila trachea

“…The following fly strains were used: (Huang et al, 2011)], UAS-Lamp1:GFP (gift of H. Kramer, University of Texas Southwestern, Dallas, USA), SRF-GAL4 (gift of M. Metzstein, University of Utah, Salt Lake City, USA), btl-GAL4 and drm-GAL4 (Bloomington). EMS alleles of okg and cnj were generated previously (Ghabrial et al, 2011 RNAi, UAS-Tor.TED, Tor HMS01114 RNAi at 29°C.…”