β1-Integrin Orients Epithelial Polarity via Rac1 and Laminin

Abstract

Epithelial cells polarize and orient polarity in response to cell-cell and cell-matrix adhesion. Although there has been much recent progress in understanding the general polarizing machinery of epithelia, it is largely unclear how this machinery is controlled by the extracellular environment. To explore the signals from cell-matrix interactions that control orientation of cell polarity, we have used three-dimensional culture systems in which Madin-Darby canine kidney (MDCK) cells form polarized, lumen-containing structures. We show that interaction of collagen I with apical ␤1-integrins after collagen overlay of a polarized MDCK monolayer induces activation of Rac1, which is required for collagen overlay-induced tubulocyst formation. Cysts, comprised of a monolayer enclosing a central lumen, form after embedding single cells in collagen. In those cultures, addition of a ␤1-integrin function-blocking antibody to the collagen matrix gives rise to cysts that have defects in the organization of laminin into the basement membrane and have inverted polarity. Normal polarity is restored by either expression of activated Rac1, or the inclusion of excess laminin-1 (LN-1). Together, our results suggest a signaling pathway in which the activation of ␤1-integrins orients the apical pole of polarized cysts via a mechanism that requires Rac1 activation and laminin organization into the basement membrane. INTRODUCTIONPolarization of cells is a fundamental process in biology. Epithelial cells polarize into apical and basolateral poles. Much has been learned recently about the mechanisms of epithelial polarization. Three major polarization complexes, the Par3/Par6/atypical protein kinase C (aPKC), Crumbs/ PATJ/Stardust, and Scribble/Discs Large/Lethal Giant Larvae complexes are conserved from Caenorhabditis elegans and Drosophila to mammals and are essential for epithelial polarization (Roh and Margolis, 2003;Macara, 2004). Cell polarization also requires interactions of the cells with each other and the extracellular matrix (ECM), as well as polarized organization of the cytoskeleton and membrane traffic, although how these processes are connected to the three polarization complexes is largely obscure (Vega-Salas et al., 1987;Yeaman et al., 1999;O'Brien et al., 2002;Mostov et al., 2003;Nelson, 2003;Zegers et al., 2003).Formation of epithelial tissues requires that the orientation of polarity of individual epithelial cells be coordinated in space and time. This was underappreciated in studies using cells grown on filter supports, because the filter provides an overriding extrinsic cue to orient the cells with the basolateral surface facing the filter and the apical surface opposite. Building on earlier observations (Wang et al., 1990a), we found that generation of epithelial polarity can be uncoupled from the orientation of that polarity (O'Brien et al., 2001). Madin-Darby canine kidney (MDCK) epithelial cells grown in three-dimensional (3D) collagen gels form cysts, where the apical surface of a polarized monolayer of cells fa...

Different responses in transformation of MDCK cells in 2D and 3D culture by v-Src as revealed by microarray techniques, RT-PCR and functional assays

“…A tight junction (TJ) marker ZO-1 was very tightly delineating the apical part of the cells and an apoptotic marker, caspase-3 cleaved form antibody stained apoptotic cell fragments inside the lumen of untransformed MDCK cell cysts (Figure 4c). The results are consistent with previous observations shown by Rahikkala et al 17 and Yu et al 30,31 Ts-Src-transformed MDCK cells formed spherical clusters filled with cells and there was no cavity or lumen in the centre (Figure 4d). Instead, there were clearly two cell populations: the cells facing the extracellular matrix were cubic with cadherin molecules delineating the lateral membranes and basal surfaces, and the others remaining inside the spheres were rounded, void of apico-basal axis and showed very strong actin staining.…”

Rotational motion during three-dimensional morphogenesis of mammary epithelial acini relates to laminin matrix assembly

“…Properly assembled basement membrane plays a critical role during establishment of cell polarity and epithelial morphogenesis (31). Defects in ECM assembly will also affect the ability of cancer cells to invade during metastasis (32).…”

The microenvironmental determinants for kidney epithelial cyst morphogenesis

“…Multicellular, spherical, polairzed cyst formation of MDCK cells in collagen gel has been used to study epithelial polarization and morphogenesis (Hall et al, 1982;McAteer et al, 1987;Warren and Nelson, 1987;Mangoo-Karim et al, 1989;Wang et al, 1990a, b;Montesano et al, 1991a;Rahikkala et al, 2001;Yu et al, 2005). Because of the simplicity of its morphogenetic process, MDCK cyst formation is an ideal model for determining the cellular events critical for epithelial morphogenesis and the molecular mechanism(s) governing it.…”

“…Because of the simplicity of its morphogenetic process, MDCK cyst formation is an ideal model for determining the cellular events critical for epithelial morphogenesis and the molecular mechanism(s) governing it. The signaling involved in MDCK cyst formation has emerged from various studies (Mangoo-Karim et al, 1989;Orellana and Marfella-Scivittaro, 2000;O'Brien et al, 2001;Schramek et al, 2003;Yu et al, 2005). The importance of the microenvironment in cystogenesis is well documented (Lin and Bissell, 1993;Kleinman et al, 2003).…”