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First published online 16 October 2007
doi: 10.1242/jcs.014365

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Mammalian diaphanous-related formin Dia1 controls the organization of E-cadherin-mediated cell-cell junctions

¹ Department of Molecular Cell Biology, The Weizmann Institute of Science, PO Box 26, Rehovot 76100, Israel
² The Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, Faculty of Life Sciences, Oxford Road, Manchester M13 9PT, UK

View larger version (75K): [in this window] [in a new window]   Fig. 1. Effect of Dia1 knockdown on the integrity of adherens junctions in MCF7 cells. (A) Distribution of endogenous hDia1 in MCF7 cells, as revealed by antibody staining. Note some enrichment at cell-cell contacts. (B) -catenin and (C) actin stainings of the same field. (D) Western blot of MCF7 cells transfected with a GFP expression vector and either pSuper vector (cont; lane 1) or a shRNA sequence for human Dia1 cloned in a pSuper vector (si-hDia1; lane 2). GFP-expressing cells were collected by FACS sorting 72 hours after transfection. The blot was probed with antibody against Dia1, and an antibody against tubulin as a loading control. (E,F) Depletion of Dia1 by interfering RNA. MCF7 cells co-transfected with pGFP-C1 and pSuper si-hDia1 were stained with antibody against Dia1 72 hours following transfection. Dia1 is reduced dramatically in cells coexpressing the GFP marker (F, asterisks). (G-L) E-cadherin localization in control and si-hDia1-expressing cells. E-cadherin staining in control cells (H) and hDia1-knockdown cells (K) shows that the localization of E-cadherin at cell-cell borders is dramatically reduced in Dia1-knockdown cells (arrows). GFP labeling of transfected cells is shown in (G,J) and actin staining is shown in (I,L). White boxes in the images H and K are used for quantification (see Fig. 3C,D). Bars, 10 µm. (M) Assessment of intact E-cadherin-containing cell-cell contacts in control and hDia1-knockdown cells. The mean percentage of intact E-cadherin-positive contacts is shown by bars. Quantification was done for three independent experiments, including measurements of 30 pairs of cells for each experiment. Error bars represent standard deviations (s.d.).

View larger version (62K): [in this window] [in a new window]   Fig. 2. Ectopic expression of mouse Dia1 rescues the effect of human Dia1 knockdown on cell-cell junctions. (A) The human Dia1-siRNA sequence used to knockdown the endogenous Dia1 contains three nucleotide mismatches compared with the sequence of mouse Dia1 (mDia1; black ovals). (B) The expression of GFP-mDia1 full-length protein is not affected by the hDia1-siRNA sequence. Control MCF7 cells, stably expressing the shRNA against LacZ (cont; lane 1) and cells stably expressing the shRNA against hDia1 (lane 2) were transiently transfected with the GFP-mDia1 plasmid (lanes 3, 4). Western blots of whole-cell lysates show that exogenous mDia1 is expressed at the correct molecular mass in the hDia1-knockdown cells (lane 4). (C,D) Expression of pSuper si-hDia1 leads to disappearance of E-cadherin staining from the interface between transfected (GFP-labeled) cells. (E,F) In cells expressing the GFP-mDia1 protein together with the human Dia1-siRNA plasmid, E-cadherin staining is as prominent as in the neighboring control cells. Bar, 10 µm. (G) Assessment of E-cadherin-containing cell-cell contacts in control cells, in hDia1-knockdown cells and in cells expressing the GFP-mDia1 protein together with the human Dia1-siRNA plasmid. Bars show the mean percentage of intact E-cadherin-positive contacts between transfected cells; error bars represent standard deviations (s.d.).

View larger version (36K): [in this window] [in a new window]   Fig. 3. Assessment of junction-associated protein distribution. (A-B) E-cadherin and -catenin levels are not reduced in the Dia1-knockdown cells. (A) Western blot of cell lysates from cells stably expressing the shRNA against LacZ (cont; lane 1) and cells stably expressing the shRNA against hDia1 (lane 2) blotted for E-cadherin and -catenin. Blotting for -tubulin was used as a loading control. (B) Quantitative measurement of Dia1, E-cadherin and -catenin levels, adjusted for the difference in gel loading. Values for control cells were taken as 100%. (C,D) The fluorescence intensity of E-cadherin and actin at cell-cell contacts is reduced in the Dia1-knockdown cells. Representative plot profiles of E-cadherin fluorescence intensity at cell-cell contacts in control (C) and knockdown (D) cells. The white boxes in Fig. 1H,K mark the regions corresponding to plots C and D, respectively. (E) Quantification of the fluorescence intensity of E-cadherin and actin at cell-cell contacts measured by line-scan analysis on digital images. Bars represent the average fluorescence intensity expressed in arbitrary units. Error bars represent s.d. values. Asterisks indicate the values that differ significantly from corresponding controls (Student's t-test, P<0.0001).

View larger version (60K): [in this window] [in a new window]   Fig. 4. Effect of Dia1 knockdown on the distribution of - and -catenin. Immunofluorescence of cells transfected with a GFP-encoding vector together with either an empty pSuper vector (A,B and E,F) or pSuper si-hDia1 (C,D and G,H) stained with antibodies against -catenin (B,D) and -catenin (F,H). Arrows indicate cell-cell junctions. Note that both - and -catenins are enriched and uniformly distributed along the cell-cell junctions in control cells (B,F), whereas junctions between Dia1-knockdown cells show reduced and fragmented staining (D,H). Bar, 10 µm.

View larger version (92K): [in this window] [in a new window]   Fig. 5. Effect of Dia1 knockdown on the localization of VASP and phosphotyrosine. Immunofluorescence of cells transfected with a GFP-encoding vector together with either an empty pSuper vector (A-C,G-I) or pSuper si-hDia1 (D-F,J-L) stained with antibodies against -catenin (B,E,H,K), phospho-tyrosine (C,F) and VASP (I,L). Note that Dia1 deficiency leads to disappearance of these components from the cell-cell interface. Bars, 10 µm. Arrows indicate the contacts between cells.

View larger version (100K): [in this window] [in a new window]   Fig. 6. Dia1 localizes to and strengthens the adherens junctions in a Rho-dependent manner. (A-C) GFP-tagged full-length mDia1 (GFP-mDia1) is distributed diffusely all over the transfected cells (A). The distributions of F-actin (B) and -catenin (C) in cells expressing GFP-mDia1 are not affected. (D-F) Co-transfection of GFP-mDia1 together with the constitutively active mutant of RhoA (RhoA-V14) leads to a significant enrichment of GFP-mDia1 at cell-cell junctions (D) and formation of numerous filopodia-like projections, with GFP-mDia1 localized to their tips (D, inset). It also promotes formation of prominent phalloidin-stained actin cables (E) and a significant accumulation of -catenin at the interface between transfected cells (F). (G-I) Transfection of RhoA-V14 strongly enhances the localization of endogenous Dia1 to cell-cell junctions, as revealed by staining with antibodies against Dia1 (G), promotes formation of stress fibers (H) and induces some increase in cell-cell junctions marked by staining for -catenin (I). Arrows indicate the contacts between cells. (J) Assessment of the Rho-mediated localization of GFP-mDia1 to cell-cell contacts. The fluorescence intensity of GFP-mDia1 was measured by line-scan analysis at cell-cell interfaces. A localization index was calculated as described in Materials and Methods. Means±s.d. are shown. (K) The fluorescence intensities of actin and -catenin at cell-cell contacts were measured as explained in the legend to Fig. 3. Bars represent the average fluorescence intensity expressed in arbitrary units. Error bars represent s.d. values. Asterisks indicate the values that differ significantly from those of corresponding controls (according to both Kolmogorov-Smirnov (KS) and Student's t-test, P<0.001). Quantifications were done for three independent experiments, including measurements of 30 pair of cells for each experiment.

View larger version (65K): [in this window] [in a new window]   Fig. 7. Activation of exogenous Dia1 leads to increased recruitment of E-cadherin to cell-cell junctions. (A,B) Confocal images of cells co-transfected with both GFP-mDia1- and RhoA-V14-expressing vectors and stained for E-cadherin 20 hours following transfection. A prominent junctional localization of GFP-mDia1 (A) and E-cadherin (B) is evident. (C,D) XZ projections of the images (A) and (B), respectively, taken along the white lines. Junctions between transfected cells are indicated by arrows. Bar, 10 µm. (E) Assessment of the width of the `adhesion belt' in control cells and in cells co-transfected with vectors encoding GFP-mDia1 and RhoA-V14. The lengths of cell-cell contacts at the XZ plane are indicated. Quantification was done for 30 pairs of cells for each experiment. Error bars represent s.d. values. The asterisk indicates the value differs significantly from corresponding controls (Student's t-test, P<0.0001).

View larger version (84K): [in this window] [in a new window]   Fig. 8. Localization of Dia1 and truncated mutant Dia1 proteins. (A) Structures of full-length mDia1 and its constitutively active mutants that were used in this study. The black lines indicate the region encoded by each mutant. The numbers correspond to the amino acid positions in the mDia1 sequence. None of the mutants contains an intact C-terminus. Only the CFP-DAD mutant preserves the N-terminal domain in its entirety. (B) Diffuse distribution of the Dia1 mutant GFP-N3 and staining of actin (C) and -catenin (D) in the corresponding cells. (E) Junctional localization of the Dia1 mutant CFP-DAD. (F) Co-transfection of GFP-N3 together with a vector encoding RhoA-V14 promotes the localization of the Dia1 mutant GFP-N3 to cell-cell junctions. (G) Cells expressing the mutant CFP-DAD co-transfected together with RhoA-V14 display a prominent mDia1-DAD localization to cell-cell junctions (as well as to tips of filopodia). Arrows indicate cell-cell junctions. Bar, 10 µm.

View larger version (101K): [in this window] [in a new window]   Fig. 9. Inhibition of myosin II but not disruption of microtubules alters the Dia1-induced morphology of adherens junctions. (A-I) MCF7 cells transfected with GFP-mDia1 together with a vector encoding RhoA-V14. Cells were treated with DMSO (A-C) or 1 µM nocodazole (D-F) for 24 hours. The drugs were added 4 hours following transfection. Cells were stained for -catenin (C,F) and -tubulin (A,D). Cells with intact microtubules (A) display a localization of GFP-mDia1 at cell-cell junctions (B) and a significant recruitment of -catenin to the cell-cell junctions of transfected cells, as revealed by staining (C). Cells treated with nocodazole show disrupted microtubule networks (D) and a higher cytoplasmic level of GFP-mDia1, even though GFP-mDia1 still was preferentially localized to the adherens junctions (E). Disruption of the microtubule networks did not prevent, however, the increase of the recruitment of -catenin to the junctions between transfected cells (F), as compared with their non-transfected neighbors. (G-I) Cells were treated with 30 µM blebbistatin for 6 hours preceding fixation and staining with phalloidin (G) and an antibody against E-cadherin (I). GFP-mDia1 localizes to the filopodia in transfected cells (H). Arrows indicate cell-cell junctions. Bars, 10 µm.

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