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First published online 23 May 2006
doi: 10.1242/jcs.02973

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Wingless signaling modulates cadherin-mediated cell adhesion in Drosophila imaginal disc cells

¹ Howard Hughes Medical Institute and Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305, USA
² Abteilung Stammzellbiologie, DFG Research Center for Molecular Physiology of the Brain (CMPB), Georg-August-Universität Göttingen, Justus-von-Liebig-Weg 11, 37077 Göttingen, Germany
³ Department of Biological Sciences and Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA

View larger version (36K): [in a new window]   Fig. 1. Expression of mouse E-cadherin affects levels and subcellular localization of Arm and -catenin in cl-8 cells. (A) Whole cell lysates from cl-8 cells and cl-8 cells stably transfected with a construct driving expression of mouse E-cadherin under control of the metallothioneine promoter (cl8mEcad), were analyzed by western blotting with antibodies against mouse E-cadherin, Arm and -catenin. cl8mEcad cells expressed a baseline level of E-cadherin due to the leakiness of the metallothioneine promoter; this level of expression increased eightfold following addition of Cu2+ (+) to the culture medium. Two forms of E-cadherin were detected; a slower migrating, 135 kDa precursor and faster migrating, mature 120 kDa protein (Shore and Nelson, 1991). In cl8mEcad cells, levels of both phosphorylated (slower migrating) and hypophosphorylated (faster migrating) Arm were strongly elevated (13-fold in the absence of Cu2+, 40-fold after induction with Cu2+). -Catenin levels were also increased in cl8mEcad cells (twofold). (B) Northern blot of total RNAs from cl-8 and cl8mEcad cells, probed for Arm. Expression of E-cadherin does not alter the steady state levels of Arm mRNA. (C) Hypotonic lysates of cl-8 and cl8mEcad cells were separated into cytosolic (S) and pellet fractions by centrifugation at 100,000 g; the pellet was re-extracted in buffer containing Triton X-100 and centrifuged again, giving rise to a Triton X-100-soluble membrane fraction (P) and the insoluble pellet (I), containing proteins bound to the cytoskeleton. Fractions were analyzed by western blot with antibodies against Arm and -catenin. In cl-8 cells, Arm was present in approximately equal amounts in the cytosol (S) and membrane fraction (P). -catenin was found predominantly in the cytosolic fraction (S). In cl8mEcad cells, both Arm and -catenin were present mostly in the membrane fraction (P). Upon induction of high E-cadherin levels with Cu2+, significant amounts of both proteins were also found in the Triton X-100-insoluble pellet fraction (I).

View larger version (82K): [in a new window]   Fig. 2. Mouse E-cadherin functions as a cell adhesion molecule in Drosophila. cl-8 cells and cl8mEcad cells induced to express high levels of E-cadherin by addition of Cu2+ to the medium, were stained for E-cadherin (A), Arm (B) and -catenin (C). In cl-8 cells, mouse E-cadherin was not expressed and Arm and -catenin were barely detectable by immunofluorescence. In cl8mEcad cells, all three proteins were highly concentrated at cell-cell contacts. Note that -catenin staining was strongly increased in cl8mEcad cells although the total amount of -catenin was increased only twofold. This finding can be explained by the diffuse, mostly cytosolic localization of -catenin in cl-8 cells (see Fig. 1C), which leads to a very weak immunofluorescence signal.

View larger version (55K): [in a new window]   Fig. 3. Wg signaling and expression of E-cadherin have opposite effects on the subcellular localization of Arm. cl8Wgts/mEcad cells and cl8HSDsh/mEcad cells, grown under the indicated conditions, were fractionated by hypotonic lysis and centrifugation to generate cytosol (S) and membrane (P) fractions. Fractions were then analyzed by western blot with antibodies against E-cadherin, Arm and Dsh. Wg signaling was activated either by incubation of cl8Wgts/mEcad cells at 16°C for 6 hours, or by a 30-minute heat shock at 37°C of cl8HSDsh/mEcad cells, followed by 5.5 hours at 25°C to allow overexpression of Dsh. Expression levels of E-cadherin were dependent on the presence or absence of Cu2+ in the culture medium. In the absence of Wg signal, the fractionation profile of Arm was very similar to that of E-cadherin (lanes 1-4, 9-12). By contrast, in cells with active Wg signaling (lanes 5-8, 13-16), a hypophosphorylated form of Arm accumulated in the cytosolic fraction (lanes 5, 13), except in the presence of high levels of E-cadherin (lanes 7, 15). Note that levels of membrane-associated Arm and E-cadherin were significantly reduced in cells with activated Wg signaling (lanes 6, 8, 14 and 16; compare with lanes 2, 4, 10 and 12).

View larger version (56K): [in a new window]   Fig. 4. Wg signaling leads to an increase of cadherin-independent pools of Arm and -catenin. NP-40 lysates of heat shocked cl8mEcad cells (A), cl8Dshmyc cells induced by Cu2+ (B) and heat shocked cl8HSDsh/mEcad cells (C) were fractionated under nondenaturing conditions by FPLC gel filtration. (A) In extracts of cl8mEcad cells, Arm was detected only in fractions containing E-cadherin, and more than 90% of -catenin was also present in these fractions. (B) In cl8Dshmyc cells induced to overexpress Dsh, Arm was present exclusively in low molecular mass fractions together with the majority of -catenin. (C) Overexpression of Dsh in cl8HSDsh/mEcad cells led to the appearance of Arm and -catenin in low molecular mass fractions devoid of E-cadherin. The distribution of E-cadherin itself remained unaltered. Note that -catenin showed an altered distribution despite the fact that the total amount is unaffected by Wg signaling (cf. Fig. 7). Compared with Fig. 3, little phosphorylated Arm was detected in this experiment, presumably because of dephosphorylation during gel filtration chromatography, which was performed under non-denaturing conditions. Fractionation peaks of molecular mass markers are indicated at the bottom. The exposure times of ECL-treated western blots were optimized for each fractionation experiment and thus do not allow any quantitative comparison of protein levels between different panels.

View larger version (131K): [in a new window]   Fig. 5. Wg signaling leads to redistribution of Arm and to loss of E-cadherin from the cell surface. cl8Wgts/mEcad cells incubated for 6 hours at the restrictive temperature (25°C; A,C) or the permissive temperature (16°C; B,D) in the presence of Cu2+ were triple stained with antibodies against Arm (green), E-cadherin (red) and Wg (blue). (A) At 25°C, when Wgts protein is nonfunctional, mutant Wg exhibited a diffuse, perinuclear distribution. Arm and E-cadherin colocalized at cell-cell contacts and in intracellular dots, presumably vesicles. (B) At 16°C, Wgts protein is active and developed a punctate staining pattern. Arm was no longer localized to cell-cell contacts but was enriched in the cytoplasm and at the tips of cellular processes that appeared to be in close contact with the substratum. E-cadherin staining was strongly reduced at the cell surface and was most prominent in the perinuclear region. Little colocalization of Arm and E-cadherin was visible. (C,D) Superimposition of Arm staining (top) with phase-contrast image (bottom) of cells. (C) At the nonpermissive temperature cells formed tight contacts with each other and did not extend membrane processes. (D) Cells at the permissive temperature had less developed cell-cell contacts and extended many processes that contained high amounts of Arm. Bar, 10 µm.

View larger version (29K): [in a new window]   Fig. 6. Overexpression of Dsh causes a reduction in the stability of newly synthesized E-cadherin. cl8mEcad cells and cl8HSDsh/mEcad cells induced to overexpress Dsh were metabolically labeled and cell lysates were immunoprecipitated for E-cadherin, separated by SDS-PAGE and processed for fluorography. (A) Both cell lines exhibited comparable amounts of newly synthesized E-cadherin after 2 hours of labeling with [35S]methionine (0 hours chase, lanes 1 and 6). Note that E-cadherin is synthesized as a 135 kDa precursor polypeptide, which is later proteolytically processed to generate the mature 120 kDa protein (Shore and Nelson, 1991). Whereas conversion of precursor to mature E-cadherin was clearly visible in cl8mEcad cells during the chase period (lanes 2-5), very little mature E-cadherin was detectable in cl8HSDsh/mEcad cells (lanes 7-10). The half-life of E-cadherin in cl8HSDsh/mEcad cells was much shorter than in cl8mEcad cells, indicating a rapid turnover of E-cadherin as a consequence of Dsh overexpression. Note that Arm and -catenin were not detectable in this experiment, presumably because the relative amount of newly synthesized E-cadherin was much higher than that of Arm and -catenin during the labeling period. (B) Relative intensities of the slower migrating 135 kDa precursor band (squares) and the faster migrating band representing 120 kDa mature E-cadherin (triangles). Intensities of both bands were measured by densitometry and plotted as the ratio between the measured intensity and the intensity of the precursor band at time 0. In cl8mEcad cells, the intensity of the mature 120 kDa band increased rapidly between 0 and 1 hour chase and had decreased only by 30% after 4 hours of chase, whereas in heat shocked cl8HSDsh/mEcad cells, very little mature protein was detected.

View larger version (60K): [in a new window]   Fig. 7. Prolonged overexpression of Dsh induces expression of DE-cadherin. (A) Untransfected cl-8 cells and cl-8 cells expressing Dsh under control of the metallothioneine promoter (cl8Dshmyc) (Yanagawa et al., 1995) were incubated for various times with Cu2+, and whole cell lysates at each time point were analyzed by western blotting with the indicated antibodies. Both the amounts of overexpressed Dsh and of hypophosphorylated Arm reached steady state levels after 4 hours of induction with Cu2+. At 12 hours of induction, DE-cadherin levels began to increase, which correlated with the appearance of a slower migrating, phosphorylated form of Arm. Levels of -catenin remained unaffected by Dsh overexpression. Untransfected cl-8 cells did not show a significant change in any of the proteins analyzed after incubation with Cu2+. (B) Northern blot analysis of total RNA of cl-8 cells and cl8Dshmyc cells incubated with Cu2+ for various times. In cl8Dshmyc cells, levels of DE-cadherin mRNA were constant during the first 12 hours of Dsh induction and increased strongly between 12 and 24 hours. Levels of Arm mRNA did not change significantly between 0 and 24 hours. Incubation with Cu2+ had no effect on DE-cadherin or Arm mRNA levels in untransfected cl-8 cells.

View larger version (103K): [in a new window]   Fig. 8. Induction of DE-cadherin expression by Dsh correlates with low levels of cytosolic Arm. cl8Dshmyc cells were incubated without Cu2+ or in the presence of Cu2+ for 24 hours and triple stained with antibodies against Arm, DE-cadherin and Dsh (Dsh image not shown). In the absence of Cu2+, Arm and DE-cadherin were barely detectable by immunofluorescence (left panel). After 24 hours of Dsh overexpression, strong Arm staining was detected in the cytoplasm and nuclei of many cells, whereas other cells showed only weak staining for Arm (right panel, top), despite expression of high levels of Dsh (not shown). Staining for DE-cadherin (right panel, middle) was strong at cell-cell contacts in cells expressing low levels of cytoplasmic Arm and low in cells expressing high levels of cytoplasmic Arm (right panel, bottom). Note that Arm staining in nuclei had approximately the same intensity as cytoplasmic staining, resulting in uniform staining of cells (see Orsulic and Peifer, 1996). Bar, 10 µm.