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Interactions of a hemidesmosome component and actinin family members

Annette M. Gonzalez1, Carol Otey2, Magnus Edlund2 and Jonathan C. R. Jones1,*

1 Department of Cell and Molecular Biology, Northwestern University Medical School, 303 E. Chicago Avenue, Chicago, IL 60611, USA
2 Department of Cell and Molecular Physiology, University of North Carolina, Chapel Hill, NC 27599, USA



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  		Fig. 1. Amino acid comparison of actinin-4 and other EF-hand proteins. The region in actinin-4 that interacts with BP180 is conserved among actinin family members and contains two EF-hand domains. The schematic includes a comparison of the BP180-binding sequence in actinin-4 and the comparable EF-hand domains in actinin-1, calmodulin and myosin regulatory light chain (MRLC).

 


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  		Fig. 2. Immunoprecipitation studies indicate that BP180 can bind actinin-4 and actinin-1 but not MRLC. Approximately 1 µg each of either HA-tagged actinin-4 (Ai), actinin-1 (Aii) or MRLC (Aiii) were incubated with a purified S-tagged BP180 fusion polypeptide in Hepes-buffered saline with 1% Brij 97 and 0.2% SDS at 4°C for 2 hours. As a control, tagged BP180 polypeptide was omitted from the immunoprecipitation mixture (Ai-iii, lane 2). A polyclonal antiserum against the BP180 cytoplasmic domain was added to the protein mixture for an additional 2 hours, followed by the addition of protein G-agarose beads (Life Technologies/BRL). The precipitated proteins were subjected to SDS-PAGE and transferred to nitrocellulose membrane. The blot was probed either with HA.11 antibody directed against the HA tag (A) or an alkaline phosphatase-conjugated S-protein that binds S-tag peptide fusion proteins (B). In lane 1 in Ai, the HA.11 antibody recognizes a single polypeptide, indicating that actinin-4 has been precipitated with BP180. Similar results were obtained when BP180 was precipitated from the BP180/actinin-1 mix (lane 1 in Aii). By contrast, MRLC was not precipitated from the MRLC/BP180 mix as indicated by the absence of a band in lane 1 in Aiii. No protein is recognized by the HA.11 antibody in Ai-iii lane 2, indicating that actinin-4, actinin-1 or MRLC were not precipitated by the BP180 antiserum. The enzyme-linked S-protein in B recognizes the immunoprecipitated BP180 polypeptide in Bi-iii lane 1. Molecular mass markers indicated on the left of the upper panels are as follows: from top to bottom 172, 111, 79, 61, 49, 36 kDa.

 


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  		Fig. 3. BP180 can co-precipitate actinin-1 from pp126 cells. Extracts of pp126 cells were prepared from cells maintained in medium containing low calcium (lanes 1,2) or cells which had been switched into medium containing 2.0 mM calcium for 4 hours (lanes 3,4). A polyclonal antiserum against the BP180 cytoplasmic domain (J17) (lanes 1,3) was added to the cell extracts which were incubated for 2 hours. No antiserum was included in the extracts shown in lanes 2 and 4. Subsequently protein G-agarose beads were added and, after an additional 2 hours, the beads collected. Precipitated proteins were subjected to SDS-PAGE, transferred to nitrocellulose membranes and processed for immunoblotting using an actinin-1 monoclonal antibody (top). The same immunoblot was then probed with the BP180 antiserum to ensure that BP180 protein had been successfully precipitated (bottom). Note that actinin-1 at 100 kDa is precipitated with BP180 in lane 3 only. An unknown protein recognized by the actinin-1 probe is indicated by an asterisk in lanes 1 and 3 in the top panels. Molecular mass markers indicated on the left of the upper panels are as follows: from top to bottom: 172, 111, 79, 61 kDa.

 


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  		Fig. 4. Sub-cellular distribution of actinin-4, actinin-1 and BP180 in pp126 oral keratinocytes maintained in low calcium medium. Cells were processed for immunofluorescence microscopy and labeled with antibodies against actinin-4 (A), actinin-1 (B) and BP180 (C). Actinin-4 localizes to the cell-edge in membrane ruffles in A while actinin-1 is present in focal contact-like structures and along the cortical microfilament cytoskeleton in B. By contrast, BP180 localizes along the basal aspect of the cell (C). Bars, 10 µm.

 


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  		Fig. 5. Increasing the concentration of calcium ions in the medium induces co-localization of BP180, actinin-4 and actinin-1 at cell-cell contact sites in pp126 oral keratinocytes. pp126 oral keratinocytes were ‘switched’ to medium containing 2.0 mM calcium for 4 hours and then were processed for double-label indirect immunofluorescence microscopy using a combination of antibodies against actinin-4 and BP180 (A,B) or actinin-1 and BP180 (D,E). Both actinins and BP180 co-distribute at cell-cell contact sites (arrows in A-E). The overlapping staining patterns appear yellow in the overlays (C,F). Bar, 10 µm.

 


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  		Fig. 6. BP180 co-distributes with actinin-4 and actinin-1 at cell-cell contact sites in MCF-10A cells. MCF-10A cells were processed for double-labeling using antibodies against either actinin-4 (A) or actinin-1 (D) in combination with an antibody preparation against BP180 (B,E). BP180 co-localizes with actinin-4 at sites of cell-cell contact (arrows in A,B). This appears yellow in the overlay of these images (C). Actinin-1 and BP180 also co-distribute at cell-cell contact sites (arrows in D,E). The areas of co-localization appear yellow in the overlay of the images (F). There is no obvious actinin-4 or actinin-1 where BP180 is found along sites of cell-substrate interaction (B,E, long arrows). Bar, 10 µm.

 


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  		Fig. 7. The fate of the product of actinin-4 transgenes in transfected MCF-10A cells. MCF-10A cells were transfected with constructs expressing full-length actinin-4 fused with a GFP-tag (A) or an HA-tag (C). In E, cells were transfected with a truncated actinin-4 (residues 1-813). At 24 hours after transfection, cells were processed for immunofluorescence microscopy. HA.11 antibody was used to visualize HA-tagged actinin-4 in transfected cells in C,E. Full-length actinin-4 is absent from the nucleus but instead distributes to sites of cell-cell contact as well as in the cytoplasm (arrows in A,C). By contrast, truncated actinin-4 localizes exclusively to the nucleus (E). B, D and F show phase contrast images of the cells. Bar, 10 µm.

 


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  		Fig. 8. BP180 co-distributes with p120 catenin in MCF-10A cells and pp126 oral keratinocytes. The localization of p120 catenin and BP180 proteins was assessed in MCF-10A cells and pp126 oral keratinocytes maintained in 2.0 mM calcium for 4 hours. Cells were processed for double-label indirect immunofluorescence microscopy using antibodies against p120 catenin (A,D) and BP180 (B,E). p120 catenin is present at cell-cell interaction sites in both cell types (short arrows in A,D) where it co-distributes with BP180 (short arrows in B,E). This is demonstrated by the yellow color in the overlays in C and F. Although BP180 antibodies also stain along the basal aspect of MCF-10A cells (long arrow in B) and in pp126 cells (long arrow in E), p120 catenin is absent from these sites (A,D). Bar, 10 µm.

 


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  		Fig. 9. BP180 co-distributes with p120 catenin, at sites of cell-cell contact in oral tissue. Tissues were processed for double-label indirect immunofluorescence microscopy using antibodies against BP180 (in red) and p120 catenin (in green). The overlay of the images generated is shown. The area indicated by the arrows toward the left-hand corner of the micrograph is shown at higher power in the inset. BP180 distributes along the basement membrane (arrow) and along lateral surfaces of basal oral epithelial cells (arrow head) where it co-distributes with p120 catenin as demonstrated by the yellow color. Bars, 20 µm.

 


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  		Fig. 10. BP180 modulates establishment of cell-cell junctions. pp126 cells maintained in low calcium medium were treated with either control IgG or an antibody against the C-terminus of BP180 for 24 hours and then switched to medium containing 2.0 mM calcium. After 4 hours, cells were processed for confocal immunofluorescence microscopy using an antibody against p120 catenin (A,C) or E-cadherin (B,E) together with a secondary antibody to identify bound BP180 antibodies (D,F). In cells treated with control IgG, p120 catenin and E-cadherin staining is linear and is concentrated along sites of cell-cell contact (A,B). In cells treated with the BP180 antibody, p120 catenin appears in a punctate pattern in rows perpendicular to cell-cell contact sites (C), whereas E-cadherin shows a diffuse staining over the cell surface (E). D and F show that BP180 antibodies bind at cell-cell contact sites. Bar, 10 µm.

 

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