First published online 7 April 2009
doi: 10.1242/jcs.045021
Journal of Cell Science 122, 1410-1417 (2009)
Published by The Company of Biologists 2009
Homophilic and heterophilic polycystin 1 interactions regulate E-cadherin recruitment and junction assembly in MDCK cells
Andrew J. Streets1,
Bart E. Wagner2,
Peter C. Harris3,
Christopher J. Ward3 and
Albert C. M. Ong1,*
1 Kidney Genetics Group, Academic Nephrology Unit, Sheffield Kidney Institute, University of Sheffield Medical School, Sheffield S10 2RX, UK
2 Electron Microscopy Unit, Histopathology Department, Northern General Hospital, Herries Road, Sheffield S5 7AU, UK
3 Division of Nephrology, Mayo Clinic and Foundation, Rochester, MN 55905, USA
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Fig. 1. A PC1 N-terminus-GPI anchor fusion protein is expressed at the cell surface in transfected HEK293 and L929 cells. (A) The extracellular domain of PC1 (M1-R2961) was fused in frame with a Thy-1 GPI anchor signal sequence (NT1-GPI) and cloned into a modified PCR-3 vector. The regions to which the different antibodies used in this study (7e12, LRR, IgPKD) bind, are indicated. (B) Surface expression of the N-terminus of PC1 was shown by cell surface biotinylation in transiently transfected HEK cells. Western blotting with a specific anti-PC1 N-terminus antibody (7e12) was carried out on biotinylated protein bound to and eluted from streptavidin beads. The blots show that NT1 is present at the cell surface. Immunoblotting with antibodies directed to an ER resident protein, calnexin and a cell surface marker, Na+/K+ATPase show that only cell surface proteins have been biotinylated. (C) Immunofluoresence staining was carried out on L929 cells transiently transfected with either (i-iii) NT-1/GPI or control empty vector (iv). NT1 was detected with 7e12 at points of cell-cell contact (arrows). These areas of cell-cell contact were absent in mock-transfected control L929 cells. (D) L929 cells were stably transfected with the NT1-GPI construct. Following G418 selection, a number of resistant clones were tested for expression of the NT1-GPI construct by RT-PCR using primers specific to the GPI signal sequence as well as NT1. Clones with high expression (c13) and low expression (c2) as well as mock-transfected wild-type clones were identified. Actin was used as a loading control. Western blotting with 7e12 shows that c13 expresses the NT1-GPI fusion protein. The protein is weakly expressed in c2 and is not detectable in mock-transfected control cells. Equal loading is shown by re-probing the same blot with an antibody to calnexin. (E) FACS was carried out on non-permeabilised L929 clones. Cells were stained with 7e12. There was a significant shift in fluorescence intensity in c13 compared with the negative control. c13 had the highest expression (fivefold increase over wild type) of NT1 at the cell surface, confirming the results seen by RT-PCR and western blotting.
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Fig. 2. Expression of NT1-GPI in L929 cells causes cell aggregation, which is inhibited by an anti-PC1 antibody. (A) Cell aggregation assays were carried out on the highest expressing clone (c13) and mock-transfected control cells. Images were taken and particle numbers quantified from six random fields using ImageJ (NIH). Results were expressed as Nt/N0 (where Nt is the number of particles after 4 hours incubation and N0 is the number of particles at the start) and are representative of three separate experiments. Each bar represent the mean ± s.e.m. *A significant difference (P<0.05) between c13 and mock transfectant. Expression of NT1-GPI resulted in significant cell aggregation after 4 hour with an Nt/N0 ratio of 0.14. By contrast, mock-transfected cells showed no aggregation after the 4-hour incubation and remained as a single cell suspension with an Nt/N0 ratio of 1.04. To confirm that cell aggregation was as a direct result of the expression of NT1 at the cell surface, cells were incubated with a blocking antibody (IgPKD) raised to the IgPKD domains (residues 843-2145) of the N-terminus of PC1 or non-immune rabbit serum. The antibody significantly disrupted cell aggregation in the NT1-GPI-expressing L929 cells (Nt/N0 ratio of 0.24) compared with non-immune serum (Nt/N0 ratio 1.01). (B) The NT1-GPI construct was sub-cloned into a pEBTetD-inducible vector and transiently expressed in L929 cells. Protein expression was induced by addition of 2 µg/ml tetracycline to the culture media 16 hours prior to the cell aggregation assay. Mock-transfected cells and uninduced NT1-GPI-expressing cells remained as a single cell suspension after a 4-hour incubation. Tetracycline induced NT1-GPI-expressing cells showed significant cell aggregation (*P<0.05 versus no tetracycline). Western blotting with 7e12 confirmed expression of NT1-GPI in the tetracycline-induced cells alone. Equal loading was confirmed by probing the same blot with an anti-calnexin antibody. (C) Electron microscopy shows cell-cell junction formation in L929 c13 cells stably expressing the NT1-GPI fusion protein. Sites of cell-cell contact are visible (i, arrows). Higher magnification of an area of cell-cell contact (ii) shows the appearance of cell adhesion plaques characteristic of clustering of cell adhesion molecules (arrows).
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Fig. 3. PC1 is involved in E-cadherin recruitment during junction reassembly following a calcium switch. (A) MDCK II cells were grown to confluence and subjected to a calcium switch assay. Data is representative of four separate experiments. Cells were fixed at various time points after restoring normal Ca2+ levels to the growth media. Cells were incubated either with a blocking antibody (IgPKD) or non-immune rabbit serum for the period of the calcium switch experiment. Cells were stained for E-cadherin, PC1, desmoplakin and ZO-1 expression. (B) Z section analysis of PC1 (i) and E-cadherin (ii) showing that the two proteins colocalised (iii) during junction reassembly. The presence of the IgPKD blocking antibody resulted in delayed recruitment of PC1 and E-cadherin compared with untreated cells. This effect is transient, as 2 hour after normal Ca2+ is restored, both proteins have relocalised to the lateral membrane. (C) Colocalisation and recruitment of PC1, E-cadherin and desmoplakin following a calcium switch was confirmed using a previously described antibody raised to the C-terminal of PC1 (BD3) in paraformaldehyde-fixed MDCK cells. PC1 and E-cadherin were recruited to the cell membrane after 30 minutes. At this time point, no surface recruitment of desmoplakin was observed. (D) Recruitment of E-cadherin to the cell surface following a calcium switch was detected by cell surface biotinylation. E-cadherin is absent from the cell surface at the moment when Ca2+ is restored to the culture (time 0). 30 minutes after the calcium switch, there is reduced E-cadherin cell surface expression in cells treated with IgPKD. Total E-cadherin levels in the cell lysate remain unchanged. Immunoblotting for calnexin show that only cell surface proteins have been biotinylated.
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Fig. 4. The C terminus of PC1 associates with the C-terminus of E-cadherin to form a protein complex independent of PC2 and β-catenin. (A) Reciprocal immunoprecipitation of native PC1 and E-cadherin from MDCK cell lysates show that the two proteins form a complex. Non-immune rabbit IgG and mouse IgG2a control antibodies show that the IP is specific. Data shown is from a representative experiment of two. (B) Diagrams showing (i) E-cadherin with the serine-rich domain sequence highlighted at which HA-tagged E-cadherin truncations were made at amino acids S840X and S855X; (ii) full length Myc-tagged PC1; (iii) HA-tagged PC1-R4227X; and (iv) the Myc-tagged CT1-CD8 fusion. (C) Myc-tagged PC1 (>500 kDa) coimmunoprecipitated with E-cadherin (120 kDa) from HEK cell lysates. In addition, PC1-R4227X (>500 kDa) could still be coimmunoprecipitated with E-cadherin. Data from a representative experiment of three. (D) Wild-type E-cadherin and the S855X mutant were still able to form a complex with endogenous β-catenin but the E-cadherin S840X mutant showed almost no binding. (E) Full length HA-tagged E-cadherin as well as HA-tagged C-terminal truncations are still able to form a complex with 45 kDa Myc-tagged CT1-CD8-Myc but the E-cadherin S840X mutant showed reduced binding. (F) In vitro binding assay of purified MBP-CT1 with His-Thio-tagged E-cadherin C-terminus (CT) demonstrated the absence of a direct interaction between the two proteins. Bacterially expressed MBP (43kDa), MBP-CT1 (65 kDa) or MBP-CT1R4227X (56 kDa) protein were incubated with amylose beads and mixed with His-Thio-tagged E-cadherin CT (50 kDa) or His-Thio-tagged PC2 C-terminus (50 kDa). Bead-associated proteins were eluted by boiling and immunoblotted with antibodies to MBP or thioredoxin. As a positive control, a direct interaction between MBP-CT1 and His-Thio-CT2 (CT2) was clearly detected.
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© The Company of Biologists Ltd 2009
