Dynamics of beta -Catenin Interactions with APC Protein Regulate Epithelial Tubulogenesis

Anne L. Pollack,* Angela I.M. Barth,Dagger Yoram Altschuler,* W. James Nelson,Dagger and Keith E. Mostov*

* Department of Anatomy, and Department of Biochemistry and Cardiovascular Research Institute, University of California, San Francisco, California 94143-0452; and Dagger  Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305-5426

Abstract
Materials and Methods
Results
Discussion
Footnotes
Acknowledgements
Abbreviations used in this paper
References


Abstract

Epithelial tubulogenesis involves complex cell rearrangements that require control of both cell adhesion and migration, but the molecular mechanisms regulating these processes during tubule development are not well understood. Interactions of the cytoplasmic protein, beta -catenin, with several molecular partners have been shown to be important for cell signaling and cell-cell adhesion. To examine if beta -catenin has a role in tubulogenesis, we tested the effect of expressing NH2-terminal deleted beta -catenins in an MDCK epithelial cell model for tubulogenesis. After one day of treatment, hepatocyte growth factor/scatter factor (HGF/ SF)-stimulated MDCK cysts initiated tubulogenesis by forming many long cell extensions. Expression of NH2-terminal deleted beta -catenins inhibited formation of these cell extensions. Both Delta N90 beta -catenin, which binds to alpha -catenin, and Delta N131 beta -catenin, which does not bind to alpha -catenin, inhibited formation of cell extensions and tubule development, indicating that a function of beta -catenin distinct from its role in cadherin-mediated cell-cell adhesion is important for tubulogenesis. In cell extensions from parental cysts, adenomatous polyposis coli (APC) protein was localized in linear arrays and in punctate clusters at the tips of extensions. Inhibition of cell extension formation correlated with the colocalization and accumulation of NH2-terminal deleted beta -catenin in APC protein clusters and the absence of linear arrays of APC protein. Continued HGF/ SF treatment of parental cell MDCK cysts resulted in cell proliferation and reorganization of cell extensions into multicellular tubules. Similar HGF/SF treatment of cysts derived from cells expressing NH2-terminal deleted beta -catenins resulted in cells that proliferated but formed cell aggregates (polyps) within the cyst rather than tubules. Our results demonstrate an unexpected role for beta -catenin in cell migration and indicate that dynamic beta -catenin-APC protein interactions are critical for regulating cell migration during epithelial tubulogenesis.


Morphogenetic movements of cells are critical to normal development of organs and tissues (Trinkaus, 1984). During embryogenesis, folding and rearrangement of sheets of cells are required for gastrulation and neurulation (Keller and Winklbauer, 1992; Keller et al., 1992a,b). During organogenesis, cell rearrangements have a central role in branching morphogenesis and tubulogenesis, processes that are essential for formation of many organs, including lung, kidney, pancreas, and mammary gland (Spooner and Wessells, 1970; Wessells, 1970; Wessells, 1977; Hogg et al., 1983; Coleman et al., 1988; Saxen, 1987; Hisaoka et al., 1992, 1993). Cell rearrangements require coordinate regulation of cell-cell adhesion and cell migration through extracellular matrix (Gumbiner, 1992). To understand the molecular mechanisms involved in cell rearrangements during development, it is important to define molecules that regulate and coordinate cell adhesion and migration during morphogenesis.

beta -catenin/armadillo plays a critical role in morphogenesis during embryonic development (McCrea et al., 1993; Peifer et al., 1993b; Funayama et al., 1995; Haegel et al., 1995; Cox et al., 1996). Originally, beta -catenin was identified in a complex with cadherins, a family of Ca2+-dependent cell adhesion proteins (Nagafuchi and Takeichi, 1989; Ozawa et al., 1989; McCrea and Gumbiner, 1991; McCrea et al., 1991). beta -catenin binds to the cytoplasmic domain of cadherins and to another cytoplasmic protein, alpha -catenin, which in turn links the cadherin-catenin complex to the actin cytoskeleton (Aberle et al., 1994; Hulsken et al., 1994; Jou et al., 1995; Rimm et al., 1995). Disruption of beta -catenin binding to cadherin (Ozawa et al., 1990; Oyama et al., 1994), or deletion of the beta -catenin gene (Peifer et al., 1993b; Haegel et al., 1995; Cox et al., 1996) results in loss of cell-cell adhesion and disorganization of cells and tissues. beta -catenin is also part of a signal transduction pathway initiated by the extracellular ligand, wingless/Wnt (McCrea et al., 1993; Peifer et al., 1993a; Noordermeer et al., 1994; Siegfried and Perrimon, 1994; Siegfried et al., 1994). Activation of this pathway results in localization of beta -catenin to the nucleus, indicating a role for beta -catenin in regulating gene expression (Funayama et al., 1995; Gumbiner, 1995). Recently, beta -catenin has been shown to bind to LEF-1, a DNA-binding transcription factor, and affects LEF-1-induced DNA bending (Behrens et al., 1996; Huber et al., 1996; Molenaar et al., 1996). beta -catenin also binds to the product of the adenomatous polyposis coli (APC)1 tumor suppressor gene in a complex that is independent of beta -catenin binding to E-cadherin (Hulsken et al., 1994; Rubinfeld et al., 1995). Mutations in the APC gene constitute the genetic defect in the inherited colon cancer syndrome, familial adenomatous polyposis, and represent an early event in a high percentage of sporadic colon cancers (Polakis, 1995). Mutations in APC protein result in accumulation of enterocytes near the crypt-villus boundary in the colon (Polakis, 1995). Näthke et al. (1996) showed that APC protein is localized in punctate clusters near the ends of microtubules that protrude into actively migrating membrane structures, suggesting a role for APC protein in cell migration.

Binding of beta -catenin to cadherin, APC protein, and LEF-1 indicates divergent functions of beta -catenin. However, these interactions are interrelated. Participation of beta -catenin in signal transduction is dependent on a high cytoplasmic level of beta -catenin that may be regulated, in part, by cadherin and APC protein (Hulsken et al., 1994). Binding of beta -catenin to cadherin antagonizes beta -catenin signaling function by sequestering beta -catenin to the cadherin complex (Heasman et al., 1994; Fagotto et al., 1996). Binding of beta -catenin to APC protein has been suggested to increase beta -catenin turnover, thereby decreasing beta -catenin levels (Munemitsu et al., 1995). That beta -catenin forms complexes with both cadherin and APC protein also raises the possibility that beta -catenin is involved in regulating functions of both proteins during epithelial morphogenesis.

To examine the function(s) of beta -catenin in epithelial morphogenesis, we expressed mutant beta -catenins containing NH2-terminal deletions of 90 (Delta N90) or 131 (Delta N131) amino acids in MDCK epithelial cells. We have determined recently that in MDCK cells grown as monolayers, Delta N90 and Delta N131 beta -catenin are significantly more stable and strongly colocalize in complexes with APC protein at the tips of membrane protrusions. Colocalization of mutant beta -catenin and APC protein correlated with a more fibroblastic cell morphology and a delay in colony formation (Barth et al., 1997). In the present study, we examined the effects of mutant beta -catenin on tubulogenesis in an in vitro assay for epithelial tubule development. This assay provides an experimentally tractable system with which to analyze the molecular mechanisms underlying the role of beta -catenin in cell adhesion and migration during morphogenetic cell rearrangements. Our results reveal that stable interaction of mutant beta -catenin with APC protein inhibits cell migration during tubulogenesis. We provide evidence that dynamic interaction of beta -catenin with APC protein is modulated by the NH2-terminal domain of beta -catenin and may regulate directed cell movements that are critical for tubulogenesis.


Materials and Methods

Cell Culture

The clones of MDCK cells used in this study are described in detail in Barth et al. (1997). The parental (T23) MDCK cell clone was derived from a polymeric immunoglobulin receptor-expressing cell line (Mostov et al., 1987) that was cotransfected with plasmid pUHD15-1 coding for expression of the tetracycline-repressible transactivator, tTA (Gossen and Bujard, 1992), and a plasmid pSV2-puro, containing a gene for resistance to puromycin. The parental T23 cell line was cotransfected with an expression vector under the control of a tetracycline-repressible promoter for full length beta -catenin (beta -cat*), luciferase (Lu), or beta -catenin mutant proteins containing NH2-terminal deletions of either 90 (Delta N90) or 131 (Delta N131) amino acids (Barth et al., 1997) and plasmid pHMR272 carrying a gene for resistance to hygromycin (Gossen and Bujard, 1992). The following clones of MDCK cells were used in this study: parental (T23); beta -cat*-10; luciferase-C and -D; Delta N90-2 and -A; Delta N131-5, -7, and -D. Multiple clones were used to ensure that our results are not due to clonal variation. All cells were passaged at subconfluent density on plastic petri dishes in minimal essential medium (MEM) containing Earle's balanced salt solution (MEM-EBSS; Cellgro; Mediatech, Inc., Hendron, VA) supplemented with 5% FCS (Hyclone, Logan, UT), 100 U/ml penicillin, and 100 mg/ml streptomycin in 5% CO2/95% air. Cells were grown in the presence or absence of Doxycyclin (Dox; 20 ng/ml; Sigma Chemical Co., St. Louis, MO). For growth in three-dimensional collagen gels, cells were isolated with trypsin and triturated into a single-cell suspension. Cells were diluted to ~2 × 104 cells/ml in a type I collagen solution containing 66% Vitrogen 100 (3 mg/ml; Celtrix, Palo Alto, CA), 1 × MEM, 2.35 mg/ml NaHCO3, 0.02 M Hepes, pH 7.6, and 12% dH2O. The cell suspension was pipetted onto NUNC filters (10 mm, 0.02-0.2 µm pore size; 162243; Applied Scientific, So. San Francisco, CA). The culture was incubated at 37°C, 5% CO2/95% air to allow the type I collagen solution to gel and then overlayered with medium. The medium was changed every 2-3 d. Cultures were grown for 10-12 d, at which time cysts comprising a closed monolayer of cells surrounding a fluid-filled lumen had formed.

Production of MRC5 Fibroblast-Conditioned Medium Containing Hepatocyte Growth Factor/Scatter Factor

MRC5 human lung fibroblasts (CCL171; American Type Culture Collection, Rockville, MD) were grown to confluence in DME containing 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, in tissue culture flasks (T75; Corning, Acton, MA). The medium was replaced with 30 ml fresh medium, and cells were cultured for 3 d. Conditioned medium was collected, centrifuged to remove cell debris, and stored frozen at -20°C.

Stimulation of Tubulogenesis in MDCK Cysts with Hepatocyte Growth Factor/ Scatter Factor-Conditioned Medium

Medium was removed from three-dimensional collagen gels containing MDCK cysts and replaced with MRC5-conditioned medium diluted 1:1 with MEM-EBSS, 5% FBS. Treatment was continued for different periods (see Fig. 1), during which time cultures were refed daily with freshly diluted, conditioned medium. (Virtually identical results were obtained using purified recombinant hepatocyte growth factor/scatter factor [HGF/ SF], a kind gift from R. Schwall, Genentech, So. San Fransisco, CA). Nomarski interference contrast images of cysts and developing tubules were obtained using a microscope (BH2-RFCA; Olympus Corp., Park Success, NY) fitted with a 20× objective (LWD CD Plan; Olympus) and connected to an MTI-CCD video camera. Images were captured with Scion 1.59 onto a Power MacIntosh (7500; Apple Computers, Cupertino, CA) and processed using NIH Image 1.6 and Adobe Photoshop 3.0.5 (Adobe Systems Inc., Mountain View, CA). For quantifying tubule formation, phase contrast images of 24-h cultures ±HGF/SF and ±Dox were photographed at 20 to 35 random sites throughout each culture dish using a microscope (Axiovert 35; Zeiss, Inc., Thornwood, NY) with a 20× LP Acrostigmat objective; focus was adjusted to the midline of cysts in the field so that the monolayer of cells in the cyst wall was in focus. Three different cultures were analyzed from each of two independent experiments for each clone ±HGF/SF and ±Dox. Images were printed, and the number of cell extensions from each cyst in the field was counted manually, binned, and tabulated. Pooled counts from three cultures were used to determine the percentage of cysts with a certain number of extensions within each bin or the average number of extensions per cyst; statistical significance was calculated by Student's t test.


Fig. 1. Tubulogenesis is not affected by Dox. Cysts derived from the parental (T23) clone of MDCK cells were grown and treated with HGF/SF in the presence (+ Dox) or absence (- Dox) of doxycycline. Cultures grown (0 d) and treated for either 1 or 4 d with HGF/SF demonstrate that Dox had no effect on cyst formation or tubule development. Bar, 50 µm.
[View Larger Version of this Image (86K GIF file)]

Immunofluorescence Confocal Microscopy

MDCK cysts were rinsed with PBS, pH 7.4, containing 1 mM CaCl2, 0.5 mM MgCl2 (PBS+), fixed for 20 min with 4% paraformaldehyde in PBS+ and 10 min with 4% paraformaldehyde in PBS+/0.025% saponin, permeabilized for 30 min with 0.025% saponin in PBS+, rinsed with PBS+, and quenched for 10 min with 75 mM NH4Cl, 20 mM glycine in PBS+, pH 8.0. Nonspecific binding sites for antibodies were blocked by incubating collagen gels for 30 min in blocking buffer (BB) containing PBS+, 0.025% saponin, 0.7% fish skin gelatin, followed by 10 min in BB containing 0.1 mg/ ml boiled RNase A. Samples were then rocked for 1-3 d at 4°C in primary antibody diluted in BB containing 0.02% Sodium-azide. Primary antibodies included: rr1 mAb supernatant (anti-E-cadherin; Gumbiner and Simons, 1986); affinity purified KT3 mAb supernatant (KT3 epitope tag on mutant beta -catenins; Barth et al., 1997); and a mouse mAb to beta -catenin (Transduction Labs, Lexington, KY). Rabbit polyclonal antibodies to APC protein were kindly provided by P. Polakis (ONYX Pharmaceuticals, Richmond, CA) and I. Näthke (Stanford University, Stanford, CA; Näthke et al., 1996). Collagen gels were washed extensively in PBS+/saponin and BB and incubated for 18 h at 4°C in BB containing FITC- or Texas red-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA; 1:100 dilution) and/or propidium iodide (ppI; Sigma Chemical Co.; 1:1,000 dilution from a 3-4 mg/ml stock). After extensive washing with PBS+/saponin and BB, collagen gels were postfixed with 4% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4, and mounted in Vectashield (Vector Labs, Burlingame, CA). Confocal images were collected using a krypton-argon laser with K1 and K2 filter sets coupled to a confocal head (MRC600; BioRad, Richmond, CA) and a Nikon microscope (Optiphot II) with a Plan Apo 60× 1.4 NA objective. Individual sections were selected from sets of serial sections collected in the x-y plane that completely spanned through cysts. Data were analyzed using Cosmos software and NIH Image 1.6. Images were converted to TIFF format, contrast levels of the images adjusted, and composites of contrast-corrected images were prepared using Adobe Photoshop (Adobe Co.) on a Power Macintosh (7200/75; Apple Computers).


Results

MDCK cells seeded in a collagen gel matrix grow clonally to form cysts that comprise a monolayer of polarized cells that surround a fluid-filled lumen (Wang et al., 1990; Montesano et al., 1991b; Santos et al., 1993; Fig. 1, 0 d). In the presence of conditioned medium from MRC5 cells that contains HGF/SF, MDCK cysts are stimulated to form branching tubules (Montesano et al., 1991a,b; Fig. 1, 1 and 4 d). Tubulogenesis of MDCK cells in vitro can be subdivided into 4 major stages: (1) extension of cell processes from the wall of the cysts; (2) formation of cell chains that remain in contact with the cyst; (3) lengthening and conversion of chains of cells to thickened cords; and (4) formation of long, blind-ended tubules that connect with the lumen of the cyst (Pollack, A.L., K.E. Mostov, manuscript submitted for publication). These events require localized changes in cell adhesion and migration through which cells emigrate and rearrange into new structures without loss of cell-cell contact (Pollack, A.L., and K.E. Mostov, manuscript submitted for publication). To investigate the role of beta -catenin in tubule development we analyzed two time periods after addition of HGF/SF (Fig. 1): day 1 (stages 1 and 2), when cell extensions form and chains of cells are initiated; and day 4 (stages 3 and 4), when cords of cells have been established and some tubule lumens have formed.

To examine functions of beta -catenin in MDCK cell tubulogenesis, we expressed mutant beta -catenins with NH2-terminal deletions of either 90 (Delta N90) or 131 (Delta N131) amino acids. Delta N90 beta -catenin retains binding sites for alpha -catenin, E-cadherin, and APC protein, whereas Delta N131 beta -catenin binds E-cadherin and APC protein but not alpha -catenin (Barth et al., 1997). For comparison, we also examined tubulogenesis of MDCK cysts derived from parental (T23) cells, cells expressing beta -cat*, and cells expressing a control protein, luciferase. Expression of exogenous genes was under the control of the Dox repressible transactivator; in the presence of Dox, gene expression was repressed, and in the absence of Dox beta -cat*, mutant beta -catenins or luciferase was expressed (for details of beta -catenin expression see Barth et al., 1997).

We first examined whether addition of Dox affected tubulogenesis. MDCK parental cell line (T23) cysts were grown and stimulated with MRC5 cell-conditioned medium in the presence or absence of Dox. Nomarski interference contrast images show that formation of initial cell extensions (day 1), complex reorganization of cells into cords (day 4), and development of tubules (data not shown) is similar for parental (T23) cells in the presence or absence of Dox (Fig. 1). Thus, Dox does not affect the timing or extent of morphological changes in cell organization during tubulogenesis.

Expression of beta -catenin Mutants (Delta N90, Delta N131) Decreases HGF/SF-stimulated Cell Extension and Cell Chain Formation (Day 1)

The following clones of MDCK cells were seeded in collagen gel matrices in the presence or absence of Dox: parental cell line (T23), luciferase-D (Lu-D), Delta N90-A beta -catenin, and Delta N131-D beta -catenin. MDCK cysts formed from cells expressing mutant beta -catenin or luciferase appear morphologically identical to cysts derived from parental cells; phase contrast microscopy shows that all cysts comprise a closed monolayer of cells surrounding a central lumen (data not shown). To stimulate tubulogenesis, cyst medium was exchanged with conditioned medium from MRC5 cells containing HGF/SF, and cysts were examined after 1 d by Nomarski interference contrast microscopy (Fig. 2).


Fig. 2. Expression of NH2-terminal deleted beta -catenin inhibits early stages of tubulogenesis. Cysts were grown in the presence or absence of Dox from T23 (Parental) MDCK cells or from T23 cells transfected with Dox- repressible expression vectors for Lu or mutant beta -catenins that contain NH2-terminal deletions of 90 (Delta N90) or 131 (Delta N131) amino acids. Nomarski interference contrast microscopy images of cysts treated 1 d with HGF/SF are shown. Top images (+ Dox) show typical examples of cysts in which expression of transfected proteins was inhibited with Dox. Bottom images (- Dox) show cysts in which Lu, Delta N90, or Delta N131 are expressed. Note that cysts expressing Delta N90 or Delta N131 beta -catenins have fewer extensions. Representative images are shown. Experiments were repeated two to four times for each clone. Bar, 50 µm.
[View Larger Version of this Image (173K GIF file)]

To determine if expression of an exogenous neutral protein affected tubulogenesis, we examined cysts formed from cells expressing Lu. Lu-D cysts cultured in either the presence or absence of Dox formed multiple cell extensions and cell chains when stimulated for 1 d with HGF/SF (Fig. 2, Lu, compare +Dox with -Dox), similar to the parental (T23) cyst response (Fig. 2, Parental, + and -Dox). Cysts derived from cells overexpressing exogenous beta -cat* also formed multiple cell extensions that were indistinguishable from those formed by parental (T23) cells (data not shown; Fig. 3); note, however, that cells accumulate higher amounts of mutant beta -catenins than full length beta -catenin, because deletion of the NH2-terminus of beta -catenin results in increased protein stability (Barth et al., 1997). Cysts derived from Delta N90-A or Delta N131-D cells, in which mutant beta -catenin expression was repressed by Dox, also formed numerous cell extensions similar to those of parental (T23) and Lu-D cysts (Fig. 2, +Dox, compare Lu with Delta N90 and Delta N131). In contrast, cysts derived from cells expressing Delta N90 or Delta N131 beta -catenin in the absence of Dox had significantly fewer and shorter cell extensions and chains compared to the same clones grown in the presence of Dox (Fig. 2, Delta N90 and Delta N131, compare +Dox with -Dox), suggesting that extension formation was inhibited in the presence of mutant beta -catenin. Note that expression of either Delta N90 or Delta N131 beta -catenin inhibited tubulogenesis, demonstrating that the inhibitory effect was independent of the ability of mutant beta -catenins to bind alpha -catenin.



Fig. 3. Quantification of tubulogenetic response after 1 d of treatment with HGF/SF. Cysts derived from T23 (Parental) MDCK cells, clone beta -cat*-10 expressing full length beta -catenin, two different luciferase clones (Lu-C and Lu-D), two different Delta N90 clones (Delta N90-A and -2), and three different clones of Delta N131 (Delta N131-D, -5, -7) were grown and treated for 1 d in the presence or absence of Dox. The number of extensions per cyst for each clone, ±Dox were counted in the midline focal plane of each cyst. Histograms in A show binned numbers of extensions per cyst. A representative experiment for each clone is shown. n, total number of cysts counted for each clone and experimental condition. The graph in B shows the average number of extensions per cyst (mean ± SEM). Quantification was obtained from two experiments for each clone. , + Dox; black-square, - Dox.
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To quantify numbers of cell extensions in cysts derived from different MDCK clones, randomly selected cysts throughout each culture were photographed with the midline plane of cells in the cyst wall in focus, and the number of cell extensions in this focal plane was counted. Cysts were examined from parental (T23) cells, clone beta -cat*-10 expressing full length beta -catenin, Lu-C and -D clones, two independent clones of cells expressing Delta N90 beta -catenin (Delta N90-2 and -A), and three independent clones of cells expressing Delta N131 beta -catenin (Delta N131-D, -5, and -7). For each clone, data were compiled from the analysis of ~25-30 cysts/culture in three separate cultures for each of two independent experiments; the data were binned and plotted as histograms (Fig. 3 A). Detailed analysis of numbers of cell extensions in cysts derived from parental (± Dox), beta -cat*-10 (± Dox), Lu-C and -D (± Dox), and Delta N90 (+ Dox) and Delta N131 (+ Dox) clones reveals that the majority of cysts contained >20 cell extensions, and very few had <9 cell extensions (Fig. 3 A). In contrast, detailed analysis of the numbers of cell extensions in cysts derived from cells expressing Delta N90 and Delta N131 beta -catenin (i.e., in the absence of Dox) reveals that the majority of cysts had <9 cell extensions, and very few had >20 cell extensions (Fig. 3 A).

The mean number of extensions for each condition (± Dox) and clone was calculated (Fig. 3 B). Results show that control cysts from parental cells, beta -cat*-10, and Lu clones that were formed and stimulated in the presence of Dox had pooled averages of 26 ± 1, 23 ± 1, and 24 ± 2 cell extensions, respectively. In the absence of Dox, parental cells, beta -cat*-10, and Lu clones had pooled averages of 25 ± 1, 25 ± 1, and 24 ± 1 cell extensions, respectively. This confirms quantitatively that neither Dox, full length beta -catenin expression, nor Lu expression affected the formation of cell extensions during initial stages of HGF/SF-induced tubulogenesis. Similarly, cysts derived from Delta N90 (Delta N90 plus Dox) and Delta N131 (Delta N131 plus Dox) cells in which mutant beta -catenin expression was repressed with Dox had pooled averages of 27 ± 2 and 27 ± 1 extensions, respectively. However, cysts derived from cells expressing Delta N90 beta -catenin (Delta N90 minus Dox) had a pooled average of 8 ± 3 extensions, and cysts derived from cells expressing Delta N131 beta -catenin (Delta N131 minus Dox) had a pooled average of 14 ± 2 extensions. The difference in number of cell extensions from cysts expressing mutant beta -catenin compared to controls is significant (P < 0.01; Student's t test). These results demonstrate that expression of mutant beta -catenins containing NH2-terminal deletions inhibits cell extension and chain formation.

Inhibition of Tubulogenic Activity Is Affected by the Level of Mutant beta -Catenin Expression

Mutant beta -catenin expression in cysts stimulated for 1 d with HGF/SF was analyzed by confocal immunofluorescence microscopy. Mutant beta -catenin was distinguishable from endogenous beta -catenin, because both Delta N90 and Delta N131 mutants were tagged with an epitope recognized specifically by the KT3 monoclonal antibody. In the presence of Dox, expression of Delta N90 and Delta N131 beta -catenin was completely repressed, and we observed diffuse background staining with KT3 antibody (KT3; Fig. 4, A and F); as expected, these cysts formed many cell extensions, as detected by staining with propidium iodide (ppI; Fig. 4, A' and F'). In the absence of Dox, cells within cysts had heterogeneous levels of mutant beta -catenin expression; in some cells KT3 staining was strong (KT3; Fig. 4, B, C, G, and H, closed arrowheads) and in other cells, levels of KT3 staining were low (KT3; Fig. 4, B, C, G and H, open arrowheads). The reasons for heterogeneity in expression of mutant beta -catenin are unknown. We note, however, that heterogeneity of expression was observed only with polarized cells in monolayer cultures (data not shown) or cysts (Fig. 4) but not in low density cultures of the same clones (Barth et al., 1997). Thus, heterogeneity in exogenous gene expression appears to be related to the differentiation status of the cells.


Fig. 4. Expression of Delta N90 or Delta N131 beta -catenin inhibits formation of cell extensions. Cysts derived from Delta N90, Delta N131, and luciferase clones were grown and treated for 1 d with HGF/SF in the presence (A, F, and K) or absence (B-E, G-J, and L-O) of Dox and processed for double immunofluorescence labeling of KT3 and ppI (A- C' and F-H') or anti-luciferase (anti-Lu) and ppI (K-M'), KT3 alone (D, E, I, and J), or anti-Lu alone (N and O). In A, F, and K, the weak KT3 and anti-Lu staining shows that Dox inhibits expression of mutant beta -catenins and luciferase. ppI staining in A', F', and K' shows that cysts from all clones treated with HGF/SF plus Dox formed multiple extensions. Cultures in B-J and L-O were treated with HGF/ SF minus Dox. Staining with KT3 or anti-Lu detects cells with high (closed arrowheads) and low (open arrowheads) Delta N90, Delta N131, and luciferase expression. (B-J) Cells expressing high amounts of mutant beta -catenins do not form extensions. Extensions formed from cells with low mutant beta -catenin expression are detected with ppI. Subcellular clusters of mutant beta -catenins are shown at tips of cells adjacent to extending cells (D and G; closed arrows) and at tips of cell extensions (D and I; open arrows). (L-O) Cells with both high and low luciferase expression form extensions. Bars: (A, E, F, J-O) 20 µm; (B-D and G-I) 10 µm.
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The heterogeneity in exogenous gene expression proved useful in assessing the effects of mutant beta -catenin expression on formation of cell extensions. All cells expressing high levels of Delta N90 or Delta N131 beta -catenin did not form cell extensions. However, cells that expressed much lower levels of Delta N90 or Delta N131 beta -catenin formed cell extensions (compare KT3 and ppI; Fig. 4, B, B', C, C', G, G', H, and H'). An even stronger correlation between expression of mutant beta -catenins and lack of formation of cell extensions is shown in slightly lower magnification images (Fig. 4, E and J). At multiple sites along each cyst wall, extensions formed from cells that had very low Delta N90 or Delta N131 beta -catenin expression (Fig. 4, E and J, open arrowheads) but did not form from any of the adjacent cells expressing high levels of Delta N90 or Delta N131 beta -catenin.

To determine if the inverse correlation between protein expression levels and inhibition of tubulogenesis was specific for mutant beta -catenins, we examined expression levels of a control protein, Lu, versus formation of HGF/SF- induced cell extensions. Fig. 4 shows representative examples of cysts derived from a Lu-expressing clone that had been stimulated with HGF/SF for 1 d in the presence (Fig. 4, K and K') or absence (Fig. 4, L-O) of Dox. Cysts formed multiple cell extensions when grown in either the presence or absence of Dox (Figs. 2 and 3). Immunofluorescence staining revealed that Lu expression levels in individual cells from cysts cultured in the absence of Dox are quite heterogeneous; some cells exhibit high levels of staining (Fig. 4, L-O, closed arrowheads), while others exhibit low, background levels of staining (Fig. 4, L-O, open arrowheads). Note that there is no correlation between the level of Lu expression and formation of cell extensions; cell extensions formed from cells that express high, medium, or low levels of Lu. These results establish that: (a) the factor(s) causing heterogeneous gene expression in these cysts do not affect formation of cell extensions; and (b) tubulogenic activity is specifically affected by the level of expression of NH2-terminal-deleted beta -catenins. Thus, some function of the NH2-terminal region of endogenous beta -catenin is essential to support early stages of tubulogenesis.

Delta N90 and Delta N131 beta -Catenins Colocalize with APC Protein at the Tips of Cell Extensions

We have shown previously that in MDCK cell monolayers Delta N-beta -catenin accumulates in APC protein clusters at the tips of extending cell membranes (Barth et al., 1997). In addition, results from Näthke et al. (1996) suggest that these APC protein clusters have a function in the formation of stable, microtubule-containing membrane extensions. Close inspection of the subcellular distribution of Delta N90 and Delta N131 beta -catenin during early stages of tubulogenesis revealed that mutant beta -catenins are localized in punctate clusters at the tips of the short cell extensions formed from cells expressing low levels of mutant beta -catenin (Fig. 4, D and I; open arrows). Therefore, we sought to determine if mutant beta -catenin and APC protein colocalize in these punctate structures and if their codistribution correlated with observed defects in the development of cell extensions during HGF/SF-induced tubulogenesis.

Cysts from parental, Delta N90- and Delta N131-expressing clones were grown in the absence of Dox and treated for 1 d with HGF/SF. In parental cells, immunofluorescence staining of APC protein revealed diffuse, finely granular staining in cells of the cyst wall (Fig. 5 A'), intense linear staining within cell extensions (Fig. 5 A', open arrows), and localized accumulation in puncta at the tip of cell extensions (Fig. 5, B and C; closed arrowheads). Double immunofluorescence revealed little or no overlap in the staining patterns of endogenous beta -catenin and APC protein at either cell-cell contacts or along the shaft of cell extensions (Fig. 5 A''). Colocalization of APC protein and endogenous beta -catenin was observed occasionally in puncta at the tip of cell extensions (Fig. 5 A'', open arrowhead), although the majority of APC protein clusters was free of detectable endogenous beta -catenin (Fig. 5, B and C).


Fig. 5. Delta N90 and Delta N131 beta -catenins accumulate in clusters with APC protein. Parental cell cysts (A, A', A'', B, and C) and cysts expressing either Delta N90 (D, D', D'') or Delta N131 (E, E', E'') beta -catenin were treated for 1 d with HGF/SF. Cultures were double labeled for APC protein (A', D', and E') and either endogenous beta -catenin (A) or KT3 (D and E). Merged images are shown in A'', B, C, D'', and E''. Extensions from parental cysts contained bright, linear staining of APC (A', open arrows) that did not colocalize with endogenous beta -catenin (A', B, C). Punctate APC protein staining is sometimes detected at tips of parental cell extensions (B and C, closed arrowheads) and occasionally colocalizes with endogenous beta -catenin (A, open arrowheads). Punctate clusters of KT3 staining (D and E, closed arrows) are strikingly detected at tips of extensions of cells expressing Delta N90 and Delta N131 beta -catenin. Punctate clusters of APC protein detected in D' and E' (closed arrowheads) colocalize with clusters of mutant beta -catenin staining (D'' and E'', open arrowheads). Bar, 10 µm.
[View Larger Version of this Image (37K GIF file)]

APC protein staining in cysts derived from cells expressing Delta N90 and Delta N131 beta -catenin was diffuse in the cytoplasm of cells within the cyst wall and was localized in punctate clusters at the tips of short cell extensions (Fig. 5, D' and E', closed arrowheads), similar to APC protein staining in parental cells. Double immunofluorescence revealed that Delta N90 and Delta N131 beta -catenin (Fig. 5, D and E; closed arrows) colocalized with APC protein in intensely stained punctate clusters at the tips of cell extensions (Fig. 5, D'' and E'', open arrowheads). In contrast to APC protein staining in parental cell extensions, staining of linear arrays of APC protein was absent in short extensions formed from cells expressing mutant beta -catenins (Fig. 5, D' and E'). Linear staining in control extensions might be due to the association of APC with a cellular structure (e.g., cytoskeleton) or may result from confinement of diffuse APC protein within long, narrow extensions. We note, however, that in control cell extensions, linear APC protein staining can be distinguished from diffuse staining near the cell membrane (Fig. 5 A'', and data not shown), suggesting that APC protein is associated with a distinct cellular structure. The lack of linear arrays of APC protein in short extensions formed from mutant beta -catenin-expressing cells likely reflects perturbation of these arrays. In addition, these results show that accumulation of Delta N90 and Delta N131 beta -catenin in the APC protein clusters correlated with the inhibition of formation of long membrane extensions.

Formation of Cell Aggregates (Polyps) in the Absence of Formation of Long Cell Extensions in Cysts Stimulated with HGF/SF for Four Days

4 d after addition of HGF/SF to parental cells, developing tubules consist of multilayered cords of cells that have begun to rearrange into lumen-containing tubules (Fig. 6, left; Pollack, A.L., and K.E. Mostov, manuscript submitted for publication); the bottom left panels of Fig. 6 show that beta -catenin and ppI staining are not continuous in the center of the extension, indicating that a small lumen has formed between the layers of cells. Endogenous beta -catenin staining is strongly localized to cell-cell contacts within the cyst wall and between cells of the cords.


Fig. 6. Stimulation of tubulogenesis from cysts, in which cell extension is inhibited, results in the formation of cell aggregates (polyps). Cysts derived from Parental (left column) and Delta N131 beta -catenin clones (right column) were stimulated for 4 d with HGF/SF in the absence of Dox. Double immunofluorescence images of endogenous beta -catenin and ppI or KT3 and ppI show that cells accumulate in aggregates at the base of short cell extensions formed from cells expressing low levels of Delta N131 beta -catenin (arrowheads) but not at the base of tubules developing from parental cysts. Bars, 10 µm.
[View Larger Version of this Image (68K GIF file)]

In contrast to parental cells, cysts derived from cells expressing Delta N131 (Fig. 6, right), Delta N90 (data not shown), and beta -catenin formed short cell extensions that had not developed further into multilayered cords of cells after 4 d in the presence of HGF/SF. These extensions were formed from cells that expressed low levels of mutant beta -catenin, whereas cells expressing relatively high levels of mutant beta -catenin did not form extensions (Fig. 4). In cysts formed from cells expressing Delta N131 beta -catenin, we noticed the occasional accumulation of cells that were not integrated into the wall of the cyst but instead appeared to form grape-like aggregates (polyps) within the wall of the cyst (Fig. 6, right, arrowheads). These cell clusters were invariably found at the base of cells that were forming short plasma membrane extensions into the surrounding collagen gel matrix. In some cases, these cell aggregates protruded into the lumenal space of the cyst. Occasionally, cells in mitosis were detected in these cell aggregates (Fig. 6, ppI, bottom right). These cell aggregates were never found in control cysts (Fig. 6, Parental, left), nor in cysts derived from cells expressing Delta N90 beta -catenin (data not shown).


Discussion

Tubulogenesis involves complex cellular rearrangements that are important for the formation of many organs and tissues during embryogenesis. For example, after mesenchymal to epithelial conversion in kidney development, cell aggregates reorganize into long tubules. These tubules are comprised of a closed epithelial monolayer surrounding a fluid-filled lumen and develop into differentiated nephrons (Saxen and Sariola, 1987). Cellular rearrangements during tubulogenesis likely involve the coordinate regulation of cell-cell interactions and cell migration (Trinkaus, 1984; Gumbiner, 1992). In this study, we demonstrate for the first time that beta -catenin, a cytoplasmic protein involved in both cell-cell adhesion and cell signaling, has a role in regulating tubulogenesis. Although beta -catenin binds to functionally divergent proteins, including E-cadherin, alpha -catenin, APC protein, fascin, and LEF-1 (Nagafuchi and Takeichi, 1989; Ozawa et al., 1989; McCrea and Gumbiner, 1991; McCrea et al., 1991; Rubinfeld et al., 1993; Behrens et al., 1996; Tao et al., 1996), we have shown that expression of NH2-terminal deleted beta -catenin inhibits tubulogenesis coincident with the colocalization and accumulation of mutant beta -catenin with APC protein. We suggest that dynamic interaction of beta -catenin with APC protein is regulated by the NH2-terminal domain of beta -catenin and is essential for normal tubule development.

To analyze the role(s) of beta -catenin in epithelial tubulogenesis we expressed mutant beta -catenins that contain a deletion of the NH2 terminus either before (Delta N90 beta -catenin) or including (Delta N131 beta -catenin) the binding site for alpha -catenin. Binding sites for E-cadherin and APC protein were retained in these mutants (Barth et al., 1997). Mutant beta -catenin expression was controlled by the Dox-repressible transactivator (Gossen and Bujard, 1992), which allowed us to examine the same clone of cells with (- Dox) or without (+ Dox) mutant beta -catenin expression. To examine stages of tubule development we exploited an in vitro tubulogenesis assay in which three-dimensional cysts formed from kidney epithelial MDCK cells are induced with HGF/SF. This assay has been shown previously to mimic cellular rearrangements that accompany tubulogenesis in vivo (Montesano et al., 1991b; Gumbiner, 1992).

Cysts formed from cells expressing Delta N90 and Delta N131 beta -catenin were similar in size and structure to cysts derived from parental (T23) cells and "control" cysts derived from Lu or full length beta -catenin-expressing cells and Delta N90- or Delta N131-beta -catenin cells grown under Dox repression. Control cysts and cysts derived from cells expressing Delta N90 or Delta N131 beta -catenin had similar growth rates, dimensions, and comprised a closed monolayer of cells surrounding a fluid-filled lumen. Thus, expression of neither Delta N90 nor Delta N131 beta -catenin affected formation of a polarized cell monolayer during cyst development. Delta N90 beta -catenin binds to both E-cadherin and alpha -catenin and, therefore, may not interfere with E-cadherin-based adhesion. Delta N131 beta -catenin, on the other hand, binds E-cadherin and not alpha -catenin and might be expected to disrupt binding of the cadherin complex to the actin cytoskeleton. However, levels of Delta N131 beta -catenin expression may not be sufficient to compete with and disrupt the function of endogenous beta -catenin in E-cadherin-based adhesion.

Although neither Delta N90 nor Delta N131 beta -catenin had detectable effects on cell-cell adhesion in polarized cysts, both mutant beta -catenins had strong inhibitory effects on tubulogenesis. Tubulogenesis begins with directed, protrusive plasma membrane activity of individual cells of the cyst wall, such that long membrane extensions formed as cell processes migrate in the direction of subsequent tubule development (Montesano et al., 1991b; Pollack, A.L., and K.E. Mostov, manuscript submitted for publication). Cysts derived from cells expressing Delta N90 and Delta N131 beta -catenin had significantly fewer cell extensions than control cysts after 1 d, and many cysts had little or no evidence of migration of cell processes from the cyst wall. The few cell extensions that formed from cysts expressing Delta N90 and Delta N131 beta -catenin were initiated exclusively from cells that expressed low levels of mutant beta -catenin. Note that inhibition of cell extensions by these mutant beta -catenins is independent of their ability to bind alpha -catenin. Therefore, NH2-terminal deletions of beta -catenin disrupt a function of beta -catenin in tubulogenesis that is distinct from its role as a functional link between E-cadherin, alpha -catenin, and the actin cytoskeleton.

How does expression of Delta N90 and Delta N131 beta -catenin inhibit directed cell extension and migration from MDCK cysts? Recently, beta -catenin has been shown to bind to a transcription factor (Behrens et al., 1996; Huber et al., 1996; Molenaar et al., 1996). It is possible that mutant beta -catenins interfere with the cellular response to HGF/SF by altering the expression of genes that are important for signal transduction. However, our results indicate that mutant beta -catenin inhibits the motogenic but not mitogenic response to HGF/SF. Taken together with the distinctive subcellular localization of mutant beta -catenin (see below), we suggest that these mutant beta -catenins affect a structural component of cell migration that is required for tubulogenesis.

The localization of APC protein in clusters at the tip of actively protruding membranes has led to the suggestion that APC protein may have a function in cell migration (Näthke et al., 1996). Accordingly, during tubulogenesis we observed that APC protein was prominently localized in clusters at the tips of membrane extensions, in cells that were actively protruding from the cyst wall and their nearest neighbors but not in cells of the cyst wall that were not forming extensions. In our previous study (Barth et al., 1997) we showed that both Delta N90 and Delta N131 beta -catenin were significantly more stable in APC protein complexes than full length beta -catenin. This increased stability resulted in the accumulation of mutant beta -catenin within APC protein clusters at the tips of membrane processes. Endogenous beta -catenin and overexpressed full length beta -catenin were rarely found in these APC protein clusters (Näthke et al., 1996; Barth et al., 1997). During tubulogenesis, full length beta -catenin was only occasionally found colocalized in distinct clusters with APC protein in cell extensions from control cysts. Inhibition of cell extension formation during tubulogenesis, however, correlated with the accumulation of mutant beta -catenin in APC protein clusters and the absence of linear cytoplasmic arrays of APC protein in the few short extensions that did form from cells expressing mutant beta -catenin. Therefore, we suggest that beta -catenin binding to APC protein regulates a function of APC protein that is important in the development of cell extensions during tubulogenesis.

What function of APC protein is regulated by beta -catenin? It is noteworthy that in actively migrating epithelial cells, bundles of microtubules invade cell extensions and coalesce at clusters of APC protein that are localized at the leading edge of cell protrusions (Näthke et al., 1996). In vitro, APC protein binds to and bundles microtubules (Munemitsu et al., 1994), and in transfected cells, exogenous APC protein codistributes along the length of microtubules (Smith et al., 1994). Significantly, addition of nocodazole to cells results in disruption of both microtubules and APC protein localization to the tips of membrane extensions and inhibition of directed cell migration (Näthke et al., 1996). An interesting corollary to these observations is that during the formation of stable extensions in growth cone outgrowth, individual microtubules actively invade cell protrusions and are subsequently organized into bundles that stabilize the direction of migration (Sabry et al., 1991; Tanaka and Kirschner, 1991; Tanaka and Sabry, 1995). Formation of cell extensions during epithelial tubulogenesis may involve similar processes in which establishing and stabilizing the direction of migration involves the reorganization and stabilization of microtubules. As evidence of this, disruption of microtubule organization or dynamics, respectively, with colcemid or taxol, inhibits elongation and directed cell migration during HGF/SF- induced tubulogenesis (Dugina et al., 1995). We suggest that beta -catenin regulates a function of APC protein in organizing microtubules that is required for the formation and/ or stabilization of cell extensions during tubulogenesis. We propose that beta -catenin-APC protein interactions are normally transient, thereby allowing APC protein to function. Stabilization of the beta -catenin-APC complex perturbs APC protein function, resulting in inhibition of cell migration.

After prolonged treatment with HGF/SF (4 d), cysts expressing mutant beta -catenin formed aggregates (polyps) that accumulated within the cyst wall subjacent to cell extensions that were inhibited in migration. A few mitotic cells were observed in these aggregates as well as in normally developing tubules of control cysts in response to mitogenic signaling by HGF/SF. We suggest that a normal proliferative response of mutant beta -catenin-expressing cells to HGF/SF, in the absence of cell migration, results in the formation of cell aggregates rather than tubules. This phenotype has an intriguing parallel to intestinal epithelium organization after mutations in APC protein. During differentiation of mammalian enterocytes, cells migrate from the crypt to the villus as a sheet of cells and are then sloughed from the villus tip (Gordon and Hermiston, 1994). However, mutations in APC protein that delete the microtubule binding site result in the accumulation of cells in polyps at the crypt/villus boundary (Polakis et al., 1995). Our results indicate that altering the function of APC protein in cell migration, through stabilization of dynamic interactions of APC protein with beta -catenin, inhibits cell rearrangements during tubulogenesis. Furthermore, in the presence of continued proliferation, inhibition of cell migration may lead to the piling up of cells and the formation of cell aggregates (polyps). Taken together with the results of other studies, we suggest that in vivo disruption of the function of APC protein, either through abnormalities in binding to microtubules or alterations in the dynamics of beta -catenin-APC protein interactions, may modify the timing and progression of cell rearrangements that affect the final outcome of epithelial morphogenesis.


Footnotes

Received for publication 20 December 1996 and in revised form 17 March 1997.

   A.L. Pollack and A.I.M. Barth contributed equally to this paper.
   Please address all correspondence to Angela I.M. Barth, Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305-5426; Tel.: (415) 725-7596; Fax: (415) 725-8021; E-mail: angelab{at}leland.stanford.edu
   A.L. Pollack's present address is Department of Cell Biology and Anatomy, University of Arizona, 1501 North Campbell Road, Tucson, AZ 85724.

We thank Dr. Ray Runyan (University of Arizona, Tucson, AZ) for help with digital capture of Nomarski images. We are also thankful to Drs. Inke Näthke (Stanford University, Stanford, CA) and Paul Polakis, (ONYX Pharmaceuticals, Richmond, CA) for providing APC protein antisera and for helpful discussions, to Dr. Rolf Kemler (Max Planck Institute for Immunobiology, Frieburg, Germany) for providing the beta -catenin cDNA, to Dr. Hermann Bujard, (ZMDH, Heidelberg, Germany) for providing the plasmids for the tetracycline repressible expression system, and to Dr. Gernot Walter (University of California, San Diego, CA) for providing the monoclonal antibody KT3.

This work was supported by grants to W.J. Nelson from the National Institutes of Health (DK45573) and The Mathers Charitable Foundation, and by grants to K.E. Mostov from the National Institutes of Health (AI25144, AI36953, HL55980). A. Barth was supported by a North Atlantic Treaty Organization (NATO) fellowship from the Deutscher Akademischer Austauschdienst and an American Heart Association fellowship.


Abbreviations used in this paper

APC, adenomatous polyposis coli; beta -cat*, full length beta -catenin; Dox, doxycycline; HGF/SF, hepatocyte growth factor/scatter factor; Lu, luciferase; ppI, propidium iodide.


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