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First published online 27 May 2003
doi: 10.1242/jcs.00500

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The Crumbs3-Pals1 complex participates in the establishment of polarity in mammalian epithelial cells

Michael H. Roh1,*, Shuling Fan2,*, Chia-Jen Liu3 and Ben Margolis1,2,3,{ddagger}

1 Department of Biological Chemistry
2 Howard Hughes Medical Institute
3 Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109-0650, USA



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  		Fig. 1. Overexpression of human Crumbs homologue 3 in MDCK renal epithelial cells. (A) Lysates were prepared from the parental MDCK cells, MDCK stable cell line that overexpresses Crumbs 3 (CRB3) or Myc-CRB3, and cells moderately expressing Myc-CRB3. Lysates proteins (left panels) and anti-CRB3 immunoprecipitates (right panel) were resolved via SDS-PAGE and the indicated immunoblots were performed. (B-E) The indicated cell lines were initially cultured on Transwell filters in low calcium media (5 µM Ca2+). Subsequently, the media was replaced with normal calcium media (1.8 mM Ca2+). Cells were grown in this media for 12 hours and immunostained as indicated according to Materials and Methods. Ezrin and gp135 are markers of the apical surface, whereas E-cadherin marks the lateral surface. Square and rectangular panels represent X-Y photomicrographs and X-Z series (Z-sections), respectively.

 


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  		Fig. 2. Wildtype and mutant Myc-CRB3 constructs expressed in MDCK cells. (A) Sequence alignment of the intracellular portion of Crumbs homologues from indicated organisms. There are two conserved motifs: the juxtamembrane region predicted to bind a protein of the FERM superfamily and the extreme C-terminal PDZ-binding motif. Conserved residues in these two regions are shown in bold. The three most conserved residues in the FERM-binding region were all mutated to alanines (also shown in bold) in the Myc-CRB3 FERMmut construct. The Myc-CRB3{Delta}ERLI protein lacks the PDZ-binding motif. Not shown is the Myc-CRB3N->D sequence where the intracellular portion of CRB3 is unchanged; instead, the extracellular N-glycosylation site is mutated as described previously (Makarova et al., 2002). (B) MDCK stable cell lines expressing wild-type Myc-CRB3 and the indicated Myc-CRB3 mutant proteins were grown for 24 hours in normal calcium media and then co-immunostained with anti-Myc and anti-ß-catenin antibodies. The ß-catenin is used as a marker of the lateral membrane. (C) Lysates were prepared from the stable cell lines used in (B). Anti-Myc immunoprecipitates were resolved by SDS-PAGE. The various Myc-CRB3 proteins and co-precipitated endogenous Pals1 were visualized by blotting with anti-CRB3 and anti-Pals1 antibodies, respectively. (D) Increasing amounts of GST-CRB3 and GSTCRB3{Delta}ERLI fusion proteins were resolved by SDS-PAGE and immunoblotted as indicated.

 


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  		Fig. 3. The effect of CRB3 overexpression on the biogenesis of tight junctions and adherens junctions. (A) Parental MDCK cells were initially cultured in low calcium media. At t=0, the low calcium media was replaced with normal calcium media. At the indicated timepoints, cells were fixed, permeabilized, and co-immunostained with anti-ZO-1 and anti-E-cadherin. (B) The same calcium switch experiment was performed on MDCK cells expressing the indicated Myc-CRB3 proteins. At the indicated times after re-addition of calcium, cells were stained with anti-ZO-1 (green) and anti-E-cadherin (red). ZO-1 and E-cadherin are markers of the TJ and adherens junction, respectively. (C) MDCK cells expressing either Myc-CRB3 or Myc-CRB3{Delta}ERLI were immunostained with anti-Pals1 (green) and anti-ZO-1 (red) antibodies at 6 hours post-calcium switch.

 


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  		Fig. 4. Crumbs 3 overexpression leads to a disruption of apico-basal polarity in MDCK cysts cultured in collagen matrix. (A) Wild-type MDCK cells were cultured in collagen gel according to Materials and Methods to induce three-dimensional cyst formation. Cysts were co-stained with the indicated antibodies. (B,C) MDCK cells expressing the various Myc-CRB3 proteins were cultured in collagen and subsequently immunostained using the indicated antibodies.

 


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  		Fig. 5. Biochemical analysis of the Myc-Lin-2/Pals1 PDZ dominant-negative chimeric protein. (A) Schematic diagram illustrating the domain organization of wild-type Myc-Lin-2, Myc-Lin-2 missing the PDZ domain (Myc-Lin-2{Delta}PDZ), and the Myc-Lin-2 in which the PDZ domain was replaced with the Pals1 PDZ domain (shown in black). Recognized domains are denoted above the protein schematics: CKII, calmodulin serine/threonine kinase II like domain; L27, Lin-2/Lin-7 domain; PDZ, PSD-95/Discs Large/ZO-1 domain; SH3, Src homology 3 domain; 4.1B, protein 4.1 binding domain; and GUK, guanylate kinase domain. (B) Full length Myc-Lin-2 and the Myc-Lin-2/Pals1 PDZ chimera constructs were individually expressed in HEK293 cells. Pulldown experiments with GST (negative control) and GST fused to the last 20 residues of CRB3 were performed on the lysates prepared from the transiently transfected 293 cells. (C) Lysates were prepared from MDCK stable cell lines expressing the indicated proteins. Lysate proteins and anti-Myc immunoprecipitates were resolved by SDS-PAGE and the proteins visualized via anti-Myc immunoblot. (D) Myc-Lin-2/Pals1 PDZ chimeric protein and Myc-Lin-2 missing the PDZ domain were immunoprecipitated from MDCK cells. Co-precipitated endogenous CRB3 was revealed by immunoblotting with anti-CRB3 antisera.

 


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  		Fig. 6. Effects of Myc-Lin-2/Pals1 PDZ chimera expression on junctional assembly and apico-basal polarity in MDCK monolayers. (A-B) MDCK stable cell lines expressing either Myc-Lin-2/Pals1 PDZ or Myc-Lin-2{Delta}PDZ (negative control) were subjected to a calcium switch experiment as in Fig. 3. At the indicated timepoints, cells were co-immunostained with anti-ZO-1 and anti-E-cadherin antibodies (green and red, respectively). (C) MDCK cells expressing the indicated proteins were grown in low calcium media (t=0) or in normal calcium media for 24 hours. Subsequently, they were co-stained with antibodies directed against E-cadherin (red) and either gp135 or CRB3 as indicated in green.

 


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  		Fig. 7. Effects of Myc-Lin-2/Pals1 PDZ chimera expression on apico-basal polarity in MDCK cysts cultured in collagen gels. (A) Cysts derived from MDCK cells expressing Myc-Lin-2{Delta}PDZ were grown in collagen matrix and co-stained with either anti-Myc/rhodamine-phalloidin or anti-E-cadherin/anti-gp135 antibodies. (B) In parallel, cysts derived from MDCK cells expressing the Myc-Lin-2/Pals1 PDZ chimeric protein were co-immunostained with the indicated antibodies.

 

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© The Company of Biologists Ltd 2003