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First published online June 23, 2005
doi: 10.1242/10.1242/jcs.02412

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Multiple regions of Crumbs3 are required for tight junction formation in MCF10A cells

¹ Department of Internal Medicine, University of Michigan Medical School, 210 Washtenaw Avenue, Ann Arbor, MI 48109-2216, USA
² Department of Biological Chemistry, University of Michigan Medical School, 210 Washtenaw Avenue, Ann Arbor, MI 48109-2216, USA
³ Howard Hughes Medical Institute, Howard Hughes Medical Institute, University of Michigan Medical Center, Room 6183, Life Science Institute, 210 Washtenaw Avenue, Ann Arbor, MI 48109-2216, USA

View larger version (56K): [in a new window]   Fig. 1. Expression of CRB3 in mammary epithelial cell lines. (A) Immunoprecipitation of CRB3 from various epithelial cell lines. 1 mg protein lysate from each cell line was immunoprecipitated overnight with purified CRB3 antibody. Immunoprecipitates (IPs) were washed, resolved by Nu-PAGE gel electrophoresis as described in Material and Methods, transferred to nitrocellulose membranes and blotted with the purified CRB3 antibody. 2% lysate inputs are also shown. Positions of molecular weight markers (in kDa) are indicated on the left. (B) Tight junctions are poorly formed in MCF10A cells. MCF10A and MCF-7 cells were grown until 4 days post confluent on permeable Transwell filters as described in Materials and Methods. Cells on filters were fixed with 4% paraformaldehyde and stained for the tight junction marker ZO-1.

View larger version (49K): [in a new window]   Fig. 2. Retroviral-mediated expression of myc-tagged CRB3 in MCF10A cells. (A) MCF10A cells were infected with retrovirus expressing either myc-tagged CRB3 or the png retroviral vector control, as described in Materials and Methods. Stably expressing pools were isolated. Equal amounts of cell lysates were immunoprecipitated overnight with either 9E10 myc antibody or purified CRB3 antibody. Immunoprecipitates were immunoblotted with the purified CRB3 antibody. 4% lysate inputs are also shown. Positions of molecular weight markers (in kDa) are indicated on the left.

View larger version (49K): [in a new window]   Fig. 3. Expression of exogenous CRB3 induces tight junction formation in MCF10A cells. (A) Indicated cell lines were grown until 4 days post confluent on Transwell filters. Cells were fixed and stained for the tight junction marker ZO-1. (B) Cell lines were grown until 4 days post confluent on Transwell filters, then fixed and stained for ß-catenin (green), ZO-1 (red) and cell nuclei (with DAPI, shown in blue). Images were analyzed by confocal microscopy and X-Y views are shown of basal and apical confocal slices. Nuclear staining is out of plane in the apical view. Note that ß-catenin staining circumscribes individual cells at the basal level. (C) Representative Z-stack images of samples shown in B.

View larger version (69K): [in a new window]   Fig. 4. Exogenous expression of CRB3 induces the recruitment of multiple known tight junction proteins to tight junction structures containing ZO-1. (A) Cells were grown for 4 days post confluence on Transwell filters, fixed and stained with the indicated antibodies. Samples were analyzed by confocal microscopy, and apical confocal sections are shown. (B) Western blotting of tight junction proteins in png control vector and mycCRB3-expressing cells. 30 µg whole cell lysates were resolved by Nu-PAGE gel electrophoresis and immunoblotted for the indicated proteins as described in Material and Methods. Positions of molecular weight markers (in kDa) are shown on the left. Note that PALS1 runs as a characteristic doublet.

View larger version (56K): [in a new window]   Fig. 5. CRB3-induced tight junctions are functional. (A) Transmission electron microscopy analysis of mycCRB3-expressing MCF10A cells. The tight junction is marked with a white arrow and the desmosome is indicated with an asterisk. (B) Measurements of transepithelial electrical resistance (ohms cm2) in mycCRB3-expressing and control MCF10A cells grown for the indicated number of days post confluence. Bar, 100 nm.

View larger version (57K): [in a new window]   Fig. 6. Expression of mycCRB3 in individual cells. MycCRB3-expressing MCF10A cells were grown on filters until 4 days post confluence, fixed, and co-stained for the myc epitope with the 4A6 anti-myc antibody (green) and for ZO-1 (red). Note that in contrast to the cells shown in other figures, these cells were permeabilized with 0.1% Triton X-100 instead of 1% SDS in order to visualize apical staining of mycCRB3. Triton X-100 permeabilization leads to slightly less sharp ZO-1 staining. A representative apical confocal section is shown. Arrowheads indicate tight junctions expressed in cells containing undetectable levels of mycCRB3.

View larger version (51K): [in a new window]   Fig. 7. Expression of myc-tagged CRB3 mutants in MCF10A cells. (A) Amino acid sequences of the CRB3 mutants. The intracellular amino acid sequences of the myc-tagged CRB3 mutants are shown. The putative FERM binding motif is underlined, conserved RxPPxP motif is in bold, and alanine substitutions are highlighted in red. (B) Retroviral-mediated expression of CRB3 mutants in MCF10A cells. Retroviruses were constructed as described in Materials and Methods, and MCF10A cells were infected as before. Stably expressing pools were isolated. Lysates were prepared from confluent plates. 30 µg protein lysate from each cell line were separated by Nu-PAGE electrophoresis and analyzed by western blotting for the myc epitope using the 4A6 anti-myc antibody. (C) Mutations in the FERM binding motif and RxPPxP motif do not affect binding of mycCRB3 to PALS1, but deletion of the ERLI sequence does. Lysates were prepared from confluent plates of the indicated cell lines, and 1 mg of each lysate sample was immunoprecipitated overnight with the 9E10 anti-myc antibody. Immunoprecipitates were washed, resolved by NuPAGE gel electrophoresis, transferred to nitrocellulose membranes, and blotted for PALS1. PALS1 runs as a characteristic doublet. The positions of molecular weight markers in kDa are indicated on the left.

View larger version (70K): [in a new window]   Fig. 8. Both the PDZ binding motif and the putative FERM binding motif of CRB3 are critical for the induction of tight junction formation. (A) The indicated cell lines were grown on Transwell filters for 4 days post confluence. Cells were fixed and stained for ZO-1. Images shown are representative fields photographed under 40 x magnification. Arrows indicate small number of tight junctions forming in mycCRB3ERLI cells (B) Quantification of tight junction formation by different CRB3 mutants. Cells were grown until 4 days post confluent as before, and stained for ZO-1. A tight junction structure was defined as an enclosed ring of smooth, contiguous apical ZO-1 staining. The number of such structures per field was quantified under 20 x magnification as described in Materials and Methods. Graph depicts the calculated mean number±s.e.m. of tight junction structures per field from three independent experiments. (C) Quantification of tight junction formation after 9 days growth past confluence. Tight junction structures were quantified as before. Error bars represent s.e.m. of three independent experiments.

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