Defining interactions and distributions of cadherin and catenin complexes in polarized epithelial cells

The cadherin/catenin complex plays important roles in cell adhesion, signal transduction, as well as the initiation and maintenance of structural and functional organization of cells and tissues. In the preceding study, we showed that the assembly of the cadherin/catenin complex is temporally regulated, and that novel combinations of catenin and cadherin complexes are formed in both Triton X-100-soluble and - insoluble fractions; we proposed a model in which pools of catenins are important in regulating assembly of E-cadherin/catenin and catenin complexes. Here, we sought to determine the spatial distributions of E- cadherin, alpha-catenin, beta-catenin, and plakoglobin, and whether different complexes of these proteins accumulate at steady state in polarized Madin-Darby canine kidney cells. Protein distributions were visualized by wide field, optical sectioning, and double immunofluorescence microscopy, followed by reconstruction of three- dimensional images. In cells that were extracted with Triton X-100 and then fixed (Triton X-100-insoluble fraction), more E-cadherin was concentrated at the apical junction relative to other areas of the lateral membrane. alpha-Catenin and beta-catenin colocalize with E- cadherin at the apical junctional complex. There is some overlap in the distribution of these proteins in the lateral membrane, but there are also areas where the distributions are distinct. Plakoglobin is excluded from the apical junctional complex, and its distribution in the lateral membrane is different from that of E-cadherin. Cells were also fixed and then permeabilized to reveal the total cellular pool of each protein (Triton X-100-soluble and -insoluble fractions). This analysis showed lateral membrane localization of alpha-catenin, beta- catenin, and plakoglobin, and it also revealed that they are distributed throughout the cell. Chemical cross-linking of proteins and analysis with specific antibodies confirmed the presence at steady state of E-cadherin/catenin complexes containing either beta-catenin or plakoglobin, and catenin complexes devoid of E-cadherin. Complexes containing E-cadherin/beta-catenin and E-cadherin/alpha-catenin are present in both the Triton X-100-soluble and -insoluble fractions, but E-cadherin/plakoglobin complexes are not detected in the Triton X-100- insoluble fraction. Taken together, these results show that different complexes of cadherin and catenins accumulate in fully polarized epithelial cells, and that they distribute to different sites. We suggest that cadherin/catenin and catenin complexes at different sites have specialized roles in establishing and maintaining the structural and functional organization of polarized epithelial cells.

[1]  R. Kemler,et al.  The cytoplasmic domain of the cell adhesion molecule uvomorulin associates with three independent proteins structurally related in different species. , 1989, The EMBO journal.

[2]  L. Reichardt,et al.  Distinguishing roles of the membrane-cytoskeleton and cadherin mediated cell-cell adhesion in generating different Na+,K(+)-ATPase distributions in polarized epithelia , 1993, The Journal of cell biology.

[3]  M. Takeichi,et al.  Cell binding function of E‐cadherin is regulated by the cytoplasmic domain. , 1988, The EMBO journal.

[4]  J. Madara.  Intestinal absorptive cell tight junctions are linked to cytoskeleton. , 1987, The American journal of physiology.

[5]  Frans,et al.  Loss of epithelial differentiation and gain of invasiveness correlates with tyrosine phosphorylation of the E-cadherin/beta-catenin complex in cells transformed with a temperature-sensitive v-SRC gene , 1993, The Journal of cell biology.

[6]  N. Korman,et al.  Demonstration of an adhering-junction molecule (plakoglobin) in the autoantigens of pemphigus foliaceus and pemphigus vulgaris. , 1989, The New England journal of medicine.

[7]  J. Papkoff,et al.  Wnt-1 modulates cell-cell adhesion in mammalian cells by stabilizing beta-catenin binding to the cell adhesion protein cadherin , 1994, The Journal of cell biology.

[8]  C W Turck,et al.  A homolog of the armadillo protein in Drosophila (plakoglobin) associated with E-cadherin. , 1991, Science.

[9]  M. Takeichi,et al.  Transmembrane control of cadherin-mediated cell adhesion: a 94 kDa protein functionally associated with a specific region of the cytoplasmic domain of E-cadherin. , 1989, Cell regulation.

[10]  D. Agard,et al.  Fluorescence microscopy in three dimensions. , 1989, Methods in cell biology.

[11]  S. Butz,et al.  Plakoglobin and β-Catenin: Distinct But Closely Related , 1992, Science.

[12]  E. Rodriguez-Boulan,et al.  Morphogenesis of the polarized epithelial cell phenotype. , 1989, Science.

[13]  E. Adamson,et al.  Transcytosis of epidermal growth factor. The epidermal growth factor receptor mediates uptake but not transcytosis. , 1991, The Journal of biological chemistry.

[14]  M. Takeichi,et al.  The 102 kd cadherin-associated protein: Similarity to vinculin and posttranscriptional regulation of expression , 1991, Cell.

[15]  S. Butz,et al.  Plakoglobin and beta-catenin: distinct but closely related. , 1992, Science.

[16]  H. McNeill,et al.  Novel function of the cell adhesion molecule uvomorulin as an inducer of cell surface polarity , 1990, Cell.

[17]  F. Masiarz,et al.  Association of the APC gene product with beta-catenin. , 1993, Science.

[18]  M. Itoh,et al.  Submembranous junctional plaque proteins include potential tumor suppressor molecules , 1993, The Journal of cell biology.

[19]  S. Tsukita,et al.  Molecular linkage between cadherins and actin filaments in cell-cell adherens junctions. , 1992, Current opinion in cell biology.

[20]  N. K. Wessells,et al.  Microfilaments in Cellular and Developmental Processes , 1971, Science.

[21]  M. Takeichi,et al.  Cadherin cell adhesion receptors as a morphogenetic regulator. , 1991, Science.

[22]  J. Papkoff,et al.  Dynamics of cadherin/catenin complex formation: novel protein interactions and pathways of complex assembly , 1994, The Journal of cell biology.

[23]  R. Kemler,et al.  Classical cadherins. , 1992, Seminars in cell biology.

[24]  Marc Levoy,et al.  Display of surfaces from volume data , 1988, IEEE Computer Graphics and Applications.

[25]  W. Nelson,et al.  Remodeling the cell surface distribution of membrane proteins during the development of epithelial cell polarity , 1992, The Journal of cell biology.

[26]  E. Wieschaus,et al.  Spatial expression of the Drosophila segment polarity gene armadillo is posttranscriptionally regulated by wingless , 1990, Cell.

[27]  T. Akiyama,et al.  Specific proto-oncogenic tyrosine kinases of src family are enriched in cell-to-cell adherens junctions where the level of tyrosine phosphorylation is elevated , 1991, The Journal of cell biology.

[28]  M. Ringwald,et al.  Uvomorulin-catenin complex formation is regulated by a specific domain in the cytoplasmic region of the cell adhesion molecule. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[29]  K. Kinzler,et al.  Association of the APC tumor suppressor protein with catenins. , 1993, Science.

[30]  F. Lottspeich,et al.  The uvomorulin-anchorage protein alpha catenin is a vinculin homologue. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[31]  R. Hynes,et al.  Plakoglobin: A protein common to different kinds of intercellular adhering junctions , 1986, Cell.

[32]  S. J. Smith,et al.  Spatial and temporal dissection of immediate and early events following cadherin-mediated epithelial cell adhesion , 1993, The Journal of cell biology.

[33]  A. Brown,et al.  Expression of Wnt-1 in PC12 cells results in modulation of plakoglobin and E-cadherin and increased cellular adhesion , 1993, The Journal of cell biology.

[34]  J. Swedlow,et al.  Three-dimensional multiple-wavelength fluorescence microscopy for the structural analysis of biological phenomena. , 1991, Seminars in cell biology.

[35]  W. Nelson,et al.  Identification of a membrane-cytoskeletal complex containing the cell adhesion molecule uvomorulin (E-cadherin), ankyrin, and fodrin in Madin- Darby canine kidney epithelial cells , 1990, The Journal of cell biology.

[36]  M. Takeichi,et al.  Cadherin-mediated cell-cell adhesion is perturbed by v-src tyrosine phosphorylation in metastatic fibroblasts , 1992, The Journal of cell biology.