Cellular polarization involves the generation of asymmetry along an intracellular axis. In a multicellular tissue, the asymmetry of individual cells must conform to the overlying architecture of the tissue. However, the mechanisms that couple cellular polarization to tissue morphogenesis are poorly understood. Here, we report that orientation of apical polarity in developing Madin-Darby canine kidney (MDCK) epithelial cysts requires the small GTPase Rac1 and the basement membrane component laminin. Dominant-negative Rac1 alters the supramolecular assembly of endogenous MDCK laminin and causes a striking inversion of apical polarity. Exogenous laminin is recruited to the surface of these cysts and rescues apical polarity. These findings implicate Rac1-mediated laminin assembly in apical pole orientation. By linking apical orientation to generation of the basement membrane, epithelial cells ensure the coordination of polarity with tissue architecture.

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...

MDCK cells expressing RhoA or Rac1 mutants under control of the tetracycline repressible transactivator were used to examine short-term effects of known amounts of each mutant before, during, or after development of cell polarity. At low cell density, Rac1V12 cells had a flattened morphology and intact cell–cell contacts, whereas Rac1N17 cells were tightly compacted. Abnormal intracellular aggregates formed between Rac1N17, F-actin, and E-cadherin in these nonpolarized cells. At all subsequent stages of polarity development, Rac1N17 and Rac1V12 colocalized with E-cadherin and F-actin in an unusual beaded pattern at lateral membranes. In polarized cells, intracellular aggregates formed with Rac1V12, F-actin, and an apical membrane protein (GP135). At low cell density, RhoAV14 and RhoAN19 were localized in the cytoplasm, and cells were generally flattened and more fibroblastic than epithelial in morphology. In polarized RhoAV14 cells, F-actin was diffuse at lateral membranes and prominent in stress fibers on the basal membrane. GP135 was abnormally localized to the lateral membrane and in intracellular aggregates, but E-cadherin distribution appeared normal. In RhoAN19 cells, F-actin, E-cadherin, and GP135 distributions were similar to those in controls. Expression of either RhoAV14 or RhoAN19 in Rac1V12 cells disrupted Rac1V12 distribution and caused cells to adopt the more fibroblastic, RhoA mutant phenotype. We suggest that Rac1 and RhoA are involved in the transition of epithelial cells from a fibroblastic to a polarized structure and function by direct and indirect regulation of actin and actin-associated membrane protein organizations.

Tight junctions (TJ) govern ion and solute diffusion through the paracellular space (gate function), and restrict mixing of membrane proteins and lipids between membrane domains (fence function) of polarized epithelial cells. We examined roles of the RhoA and Rac1 GTPases in regulating TJ structure and function in MDCK cells using the tetracycline repressible transactivator to regulate RhoAV14, RhoAN19, Rac1V12, and Rac1N17 expression. Both constitutively active and dominant negative RhoA or Rac1 perturbed TJ gate function (transepithelial electrical resistance, tracer diffusion) in a dose-dependent and reversible manner. Freeze-fracture EM and immunofluoresence microscopy revealed abnormal TJ strand morphology and protein (occludin, ZO-1) localization in RhoAV14 and Rac1V12 cells. However, TJ strand morphology and protein localization appeared normal in RhoAN19 and Rac1N17 cells. All mutant GTPases disrupted the fence function of the TJ (interdomain diffusion of a fluorescent lipid), but targeting and organization of a membrane protein in the apical membrane were unaffected. Expression levels and protein complexes of occludin and ZO-1 appeared normal in all mutant cells, although ZO-1 was more readily solubilized from RhoAV14-expressing cells with Triton X-100. These results show that RhoA and Rac1 regulate gate and fence functions of the TJ, and play a role in the spatial organization of TJ proteins at the apex of the lateral membrane.

The cadherin-catenin complex is important for mediating homotypic, calcium-dependent cell-cell interactions in diverse tissue types. Although proteins of this complex have been identified, little is known about their interactions. Using a genetic assay in yeast and an in vitro protein-binding assay, we demonstrate that 13-catenin is the linker protein between E-cadherin and c-catenin and that E-cadherin does not bind directly to a-catenin. We show that a 25-amino acid sequence in the cytoplasmic domain of E-cadherin and the amino-terminal domain of a-catenin are independent binding sites for 8-catenin. In addition to 13-catenin and plakoglobin, another member of the armadillo family, p120 binds to E-cadherin. However, unlike 13-catenin, p120does not bind a-catenin in vitro, although a complex of p120 and endogenous oi-catenin could be immunoprecipitated from cell extracts. In vitro protein-binding assays using recombinant E-cadherin cytoplasmic domain and x-catenin revealed two catenin pools in cell lysates: an -1000-to '2000-kDa complex bound to E-cadherin and an -220-kDa pool that did not contain E-cadherin. Only 18-catenin in the -220-kDa pool bound exogenous E-cadherin. Delineation of these molecular linkages and the demonstration of separate pools of catenins in different cell lines provide a foundation for examining regulatory mechanisms involved in the assembly and function of the cadherin-catenin complex.The cadherin superfamily comprises glycoproteins responsible for calcium-dependent, homotypic cell interactions (1). Ecadherin is generally expressed in epithelial tissues and has been shown to regulate cell-cell adhesion (2), cell migration (3), morphogenesis (4), and the establishment of membrane polarity (5).Homotypic interactions between extracellular domains of cadherins are necessary but not sufficient for cell-cell adhesion (2). Linkage of the cadherin cytoplasmic domain to three cytosolic proteins, named a-catenin, ,B-catenin, and plakoglobin (y-catenin), is required (6-8). Although a-catenin, 3-catenin, and plakoglobin can be coimmunoprecipitated in a complex with E-cadherin (1, 7), the binding order of proteinprotein interactions has not been resolved. Insight into this problem is important for understanding functions of the cadherin-catenin complex and the regulation of cadherincatenin complex assembly. Here, we define the binding order of protein-protein interactions in the cadherin-catenin complex using genetic and biochemical approaches. MATERIALS AND METHODSStrain and Microbiological Techniques. All cloning procedures and bacterial transformation were performed by standard procedures outlined by Sambrook et at (9). Yeast strain Y190 (MATa gal4 gal80 his3-200 trpl-901 ade2-101 ura3-52

Polarized epithelial cells maintain the asymmetric composition of their apical and basolateral membrane domains by at least two different processes. These include the regulated trafficking of macromolecules from the biosynthetic and endocytic pathway to the appropriate membrane domain and the ability of the tight junction to prevent free mixing of membrane domain-specific proteins and lipids. Cdc42, a Rho family GTPase, is known to govern cellular polarity and membrane traffic in several cell types. We examined whether this protein regulated tight junction function in Madin-Darby canine kidney cells and pathways that direct proteins to the apical and basolateral surface of these cells. We used Madin-Darby canine kidney cells that expressed dominant-active or dominant-negative mutants of Cdc42 under the control of a tetracycline-repressible system. Here we report that expression of dominant-active Cdc42V12 or dominant-negative Cdc42N17 altered tight junction function. Expression of Cdc42V12 slowed endocytic and biosynthetic traffic, and expression of Cdc42N17 slowed apical endocytosis and basolateral to apical transcytosis but stimulated biosynthetic traffic. These results indicate that Cdc42 may modulate multiple cellular pathways required for the maintenance of epithelial cell polarity.

Podocalyxin was initially identified in glomerular podocytes to critically maintain the structural and functional integrity of the glomerular ultrafiltrative apparatus. Lately, it has emerged as a malignant marker in tumors arising from a variety of tissue origins. By immunohistochemistry, we identified that 9.6% of renal cell carcinoma patients overexpress this protein. This subset of patients had significantly shorter disease-specific and overall survivals, and, importantly, we established podocalyxin overexpression as an independent prognostic factor for latent distant metastasis with multivariate analysis. Podocalyxin down-regulation by small interfering RNA led to defective migration in model renal tubular cells, which was corrected by re-expression of podocalyxin. The activity of the small GTPase Rac1, a wellcharacterized modulator of cell migration, was diminished by podocalyxin knock-down. Conversely , podocalyxin overexpression in human embryonic kidney cells up-regulated Rac1 activity, which depended on a complex formed by podocalyxin, ERMbinding phosphoprotein 50, ezrin, and ARHGEF7, a Rac1 activator. Therefore, podocalyxin can serve as a biomarker to identify renal cell carcinoma patients with higher metastatic potential for more aggressive intervention at earlier clinical stages. (Am J Pathol

SummaryGroup A streptococcus (GAS) induces its own entry into eukaryotic cells in vitro and in vivo. Fibronectin (Fn) bound to protein F1, a GAS surface protein, acts as a bridge connecting the bacterium to host cell integrins. This triggers clustering of integrins, which acquire a polar pattern of distribution similar to that of protein F1 on the GAS surface. A unique and transient adhesion complex is formed at the site of GAS entry, which does not contain a-actinin. Vinculin is recruited to the site of GAS entry but is not required for uptake. The invading GAS recruits focal adhesion kinase (FAK), which is required for uptake and is tyrosine phosphorylated. The Src kinases, Src, Yes and Fyn, enhance the efficiency of GAS uptake but are not absolutely required for GAS entry. In addition, Rac and Cdc42, but not Rho, are required for the entry process. We suggest a model in which integrin engagement by Fn-occupied protein F1 triggers two independent signalling pathways. One is initiated by FAK recruitment and tyrosine phosphorylation, whereas the other is initiated by the recruitment and activation of Rac. The two pathways subsequently converge to trigger actin rearrangement leading to bacterial uptake.