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First published online 25 January 2005
doi: 10.1242/jcs.01638

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Physical and functional association of migfilin with cell-cell adhesions

Vasiliki Gkretsi^1,*, Yongjun Zhang^1,*, Yizeng Tu¹, Ka Chen¹, Donna B. Stolz², Yanqiang Yang¹, Simon C. Watkins² and Chuanyue Wu^1,

¹ Department of Pathology, Center for Biological Imaging, University of Pittsburgh, Pittsburgh, PA 15261, USA
² Department of Cell Biology and Physiology, Center for Biological Imaging, University of Pittsburgh, Pittsburgh, PA 15261, USA

View larger version (75K): [in a new window]   Fig. 3. Detergent-resistant association of migfilin with filamentous actin at cell-cell junctions. (A-C) HaCat cells were subject to cytoskeletal extraction as described in Materials and Methods. The cells were then double stained with the mouse anti-migfilin (green, A) and rabbit anti-ß-catenin (red, C) antibodies (arrowheads). The images were merged and are shown in B. (D-F) HaCat cells were Triton X-100-extracted (D), unroofed (E), or extracted and unroofed (F) as described in Materials and Methods. The cells were then co-stained with the monoclonal anti-migfilin antibody, which was detected with an Alexa 488-conjugated anti-mouse IgG antibody (green), Rhodamine-conjugated phalloidin (red) and Hoechst dye 33258 (blue). Bars, 25 µm. (G-I) Schematic representation of the Triton X-100-extracted and/or the cationic colloidal silica unroofed cells.

View larger version (106K): [in a new window]   Fig. 1. Migfilin localizes to cell-cell junctions in epithelial and endothelial cells. (A-D) Primary newborn human embryonic keratinocytes (A and B), immortalized human keratinocytes (HaCat) (C and D) and primary human microvascular endothelial cells (E and F) were double stained with a mouse monoclonal anti-migfilin antibody (clone 43) (A, C and E) and a rabbit polyclonal anti-ß-catenin antibody (B and F) or anti-FAK antibody (D). The images were recorded using a Leica DM R fluorescence microscope equipped with a Hamamatsu ORCA-ER digital camera. The cell-cell and cell-ECM adhesions are indicated by arrows and arrowheads, respectively. Note that a fraction of migfilin was co-clustered with ß-catenin at cell-cell junctions (arrows, C) that lack FAK (D). Migfilin was also detected at FAK-rich cell-ECM adhesions (arrowheads, C and D). Bars in A, C and E, 8 µm.

View larger version (103K): [in a new window]   Fig. 2. Immunofluorescent confocal microscopic analysis of cell-cell adhesion localization of migfilin. Human immortalized keratinocytes (HaCat cells) were double stained with monoclonal anti-migfilin antibody (clone 43) (A and D) and a rabbit polyclonal anti-ß-catenin antibody (B and E) observed under an Olympus Fluoview BX61 confocal microscope. The x-z sections at the lateral (A and B) and basal (D and E) levels were shown. (C,F) Images merged from the migfilin (green) and ß-catenin (red) staining at the lateral and basal levels, respectively. The arrow in C indicates the position of the x-z cross section (G). Bar, 8 µm.

View larger version (70K): [in a new window]   Fig. 4. Immunoelectron microscopic analyses of migfilin clusters at cell-cell junctions. Immunoelectron microscopic analysis of HaCat cells was performed as described in Materials and Methods. Cell adhesions connecting the two cells are shown in the orientation between the two open arrows. Note that the anti-ß-catenin antibody (10 nm gold) and the anti-migfilin antibody (5 nm gold) were colocalized at areas of cell-cell contacts (between arrowheads). (B,C) Single pre-embedding immunotransmission microscopic images of HaCat cells at low (B) and high (C) magnification. In B, migfilin (beads) was clustered and associated with the actin bundles at cell-cell junctions (arrows). N indicates the nuclei of two neighboring cells. In C, desmosomes are indicated by arrows, and migfilin clusters are shown by arrowheads. Note that migfilin clusters were associated with the actin bundles but not desmosomes. (D) HaCat cells were prepared for pre-embedding immunotransmission microscopy and double stained with anti-migfilin and anti-ß-catenin antibodies. Note that migfilin (10 nm gold particles) and ß-catenin (5 nm gold particles) were closely clustered with the actin bundles.

View larger version (74K): [in a new window]   Fig. 5. Calcium-induced migfilin localization to adherens junction. (A-D) MCF7 mammary epithelial cells were subject to calcium-chelation assay as described in Materials and Methods. The cells were fixed at 15 minutes (A and B) and 30 minutes (C and D) after switching to calcium-containing medium, and double stained with a rabbit anti-ß-catenin antibody (A and C) and the mouse monoclonal anti-migfilin antibody (B and D). (E,F) MCF7 mammary epithelial cells were transiently transfected with an expression vector encoding GFP-migfilin. The cells were subject to calcium-chelation assay in the presence of a function-blocking mouse monoclonal anti-E-cadherin antibody (SHE78-7) (G-J) or a non-specific mouse IgG as a control (E and F) as described in Materials and Methods. The cells were fixed at 60 minutes after switching to calcium-containing medium, and stained with a rabbit anti-ß-catenin antibody (E, G and I) and a Rhodamine RedTM-conjugated anti-rabbit IgG antibody. ß-Catenin (E, G and I) and GFP-migfilin (F, H and J) at the cell-cell (E-H) or cell-ECM (I and J) adhesions were detected using a Leica DM R fluorescence microscope. Bars, 5 µm.

View larger version (27K): [in a new window]   Fig. 6. Expression and schematic representation of the wild-type and mutant forms of migfilin and their localization to cell-cell and cell-ECM adhesions. (A) Expression of GFP-tagged wild-type and mutant forms of migfilin. HyCat cells were transfected with expression vectors encoding the wild-type and mutant forms of migfilin as indicated in the figure. The cell lysates (15 µg/lane) were analyzed by western blotting with an anti-GFP antibody. N-ter, residues 1-85; PR-LIM1-3, residues 85-373; LIM1-3, residues 176-373; LIM1N, residues 205-373; C243, C291 and C306, migfilin bearing C243G, C291G or C306G point mutation. C306(s), migfilin(s) bearing the C306G point mutation. (B) Summary of the localization of the wild-type and mutant forms of migfilin to cell-cell and cell-ECM adhesions, based on the results presented in this paper.

View larger version (94K): [in a new window]   Fig. 7. The C-terminal LIM domains, but not the N-terminal filamin-binding domain or the central proline-rich domain, mediate migfilin localization to adherens junctions. (A-D) The central proline-rich domain is not required for migfilin localization to cell-cell junctions. HaCat cells were transfected with expression vectors encoding GFP-migfilin (A and B) or GFP-migfilin(s) (C and D). The cells were stained with the rabbit anti-ß-catenin primary antibody and Rhodamine RedTM-conjugated anti-rabbit IgG secondary antibody, and were observed under a Leica DM R fluorescence microscope equipped with GFP (A and C) or rhodamine (B and D) filters. (E-J) The C-terminal LIM domains, but not the N-terminal filamin-binding domain, are sufficient for mediating migfilin localization to adherens junctions. HaCat cells that were transiently transfected with expression vectors encoding GFP-PR-LIM1-3 (E and F), GFP-LIM1-3 (G and H) or GFP-N-ter (I and J) were stained with the anti-ß-catenin antibody or phalloidin as indicated. Bar, 8 µm.

View larger version (72K): [in a new window]   Fig. 9. Effects of point mutations within the LIM domains on migfilin localization to cell-ECM adhesions. (A-D) Mig-2 localizes to cell-ECM but not cell-cell adhesions. Primary newborn human embryonic keratinocytes were double stained with mouse monoclonal anti-Mig-2 antibody 3A3 (A) and phalloidin (B). HaCat cells that were transiently transfected with the GFP-migfilin(s) vector (C) were stained with the mouse monoclonal anti-Mig-2 antibody (D). (E-L) HaCat cells that were transiently transfected with expression vectors encoding GFP-tagged migfilin mutants (as indicated) were stained with the mouse monoclonal anti-Mig-2 antibody (F, H, J and L). Bar, 8 µm.

View larger version (93K): [in a new window]   Fig. 8. LIM2 is essential for migfilin localization to adherens junctions. HaCat cells were transfected with the expression vectors encoding GFP-LIM1N (A), GFP-C243 (C), GFP-C291 (E) and GFP-C306(s) (G). The cells were stained with the anti-ß-catenin antibody as indicated (B, D, F and H). Notice that a small amount of GFP-tagged C306(s) mutant was detected in b-catenin-rich adherens junctions (G and H, arrows). Bar, 8 µm.

View larger version (63K): [in a new window]   Fig. 10. siRNA-mediated depletion of migfilin and its effect on ß-catenin organization. Human HT-1080 cells that were transfected with the migfilin siRNA (lane 2) or the control small RNA (lane 1) were analyzed by western blotting with monoclonal anti-migfilin antibody (clone 43) (A), monoclonal anti-actin antibody (B) or a polyclonal anti-ß-catenin antibody (C). (D-K) HT-1080 cells transfected with the control RNA (D, F, H and J) or the migfilin siRNA (E, G, I and K) were double stained with mouse monoclonal anti-migfilin antibody (clone 43) (H-K) and rabbit-polyclonal anti-ß-catenin antibody (F and G). The differential interference contrast (DIC) (D and E) and immunofluorescent (F-K) images were recorded using a Leica DM R fluorescence microscope equipped with a Hamamatsu ORCA-ER digital camera. Bar, 8 µm.

View larger version (69K): [in a new window]   Fig. 11. Depletion of migfilin compromises the organization of adherens junctions. Human HT-1080 cells that were transfected with the migfilin siRNA (lane 2) or the control small RNA (lane 1) were analyzed by western blotting with monoclonal anti-migfilin antibody (clone 43) (A) or an anti-cadherin antibody (C). Equal loading was confirmed by re-probing the migfilin membrane (A) with a monoclonal anti-actin antibody (B). (D-I) HT-1080 cells transfected with the control RNA (D, F and H) or the migfilin siRNA (E, G and I) were double stained with the mouse monoclonal anti-migfilin antibody (clone 43) (F and G) and Rhodamine-conjugated phalloidin (H and I). The mouse anti-migfilin primary antibody was detected with a FITC-conjugated anti-mouse IgG secondary antibody. (J-O) HT-1080 cells transfected with the control RNA (J, L and N) or the migfilin siRNA (K, M and O) were double stained with a rabbit polyclonal anti-ß-catenin antibody (L and M) and a mouse monoclonal anti-cadherin antibody (clone CH-19) (N and O). The DIC (D, E, J and K) and immunofluorescent (F-I and L-O) images were recorded as described in Fig. 10. Bar, 8 µm.

View larger version (45K): [in a new window]   Fig. 12. Depletion of migfilin weakens the cell-cell association. (A,B) The parental HT-1080 cells (lane 1) and HT1080 cells that were transfected with the control small RNA (lane 2) or the migfilin siRNA (lane 3) were analyzed by western blotting with the monoclonal anti-migfilin antibody (A). Equal loading was confirmed by re-probing the membrane with an anti-actin antibody (B). (C-F) Cell dissociation. The migfilin siRNA transfectants (C), control RNA transfectants (D) and the parental HT-1080 cells (E) were detached from culture plates, passed through Pasteur pipettes ten times and observed under an Olympus IX70 inverted microscope. Bar in E, 100 µm. The degree of cell dissociation (the number of particles (Np)/the number of total cells (Nc)) was calculated by analyzing at least 500 cells from each sample (F). Bars in F represent means + s.d. from two independent experiments.