Gene silencing of Scrib in MDCK cells
Several target sequences were selected from the partial canine Scrib
gene and used to construct pSUPER vectors for the expression of small hairpin RNAs (shRNAs). Of the three sequences tested, two efficiently knocked down Scrib expression when expressed by transient transfection in MDCK II cells ( A). Immunofluorescence microscopy revealed Scrib to be associated with the lateral membranes in mammalian epithelial cells, and staining was substantially reduced by transfection with the ScrbKD1 or 2 vectors ( B). Surprisingly, however, the tight junctions appeared to be intact in these cells, as assessed by occludin staining ( B) or ZO-1 staining (not depicted). Moreover, confocal sections of cells stained for the apical marker gp135 revealed no loss of apical/basal polarization in cells expressing reduced Scrib levels (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200506094/DC1
), and cysts grown in Matrigel appeared to be polarized normally (not depicted). These results suggest that depletion of Scrib does not disrupt tight junction assembly. However, when the transfected cells were subjected to a calcium switch and stained for ZO-1, a short delay in junction assembly was observed ( C). ZO-1 accumulated rapidly at the cell–cell contacts in both the control and Scrib knockdown (KD) cells but, in the absence of Scrib, the fusion of the ZO-1 lines into a continuous band encircling each cell was delayed. The defect was particularly noticeable at vertices where several cell boundaries meet. By 20 h after calcium switch, however, the ZO-1 staining in the cells lacking Scrib was indistinguishable from that in the control cells.
Figure 1. Suppression of Scrib expression in MDCK cells causes a delay in tight junction assembly. (A) MDCK II cells were transiently transfected with either pS-Luciferase vector (Luc) as a control or three pSUPER constructs targeting different sequences of canine (more ...)
Scrib is required for maintenance of an epithelial phenotype at low cell densities
When cells were transfected with pS-ScrbKD vectors and plated at low densities, they consistently displayed a mesenchymal phenotype. Normal MDCK cells organize into discrete, tight islands with smooth boundaries, but cells lacking Scrib appeared more fibroblastic (). They spread over a much larger surface area (approximately three to five times larger; C), and the edges of the cell clusters were disorganized as though the cells were moving apart from one another. When stained with phalloidin, the normal cortical actin rings were absent in cells lacking SCRIB and were replaced by stress fibers often oriented along the long axis of the cells ( B). These observations suggested that Scrib might regulate epithelial cell adhesion and/or migration.
Figure 2. Cells lacking Scrib lose their epithelial morphology at low density. (A) Control (Luc) and ScrbKD cells were grown on 6-well plates at low density for 3 d. Images were obtained by phase-contrast microscopy using a 10× objective. (B) Control and (more ...)
Scrib inhibits cell motility and is required for oriented migration
To determine whether Scrib regulates MDCK cell motility, transfected cells were plated onto 8-μm filters in Boyden chambers and incubated in normal medium containing 10% serum (both above and below the filter). The same number of cells was plated onto each filter. After 16–20 h, cells that had migrated through the pores to the bottom surface of the filters were stained with crystal violet. Loss of Scrib substantially increased the number of cells that had migrated through the filter (). Importantly, when we expressed a GFP fusion of a human Scrib in the cells transfected with the pS-ScrbKD1 vector, the number of cells migrating through the filter was reduced to control levels (). It was conceivable that the reversion to a wild-type phenotype was caused by reexpression of the endogenous protein. To test for this possibility, we blotted cell lysates for Scrib ( C). The endogenous Scrib protein level was reduced upon expression of the ScrbKD1 shRNA and did not respond to coexpression of the human GFP–Scrib fusion. The GFP–Scrib fusion can be distinguished by its lower mobility on SDS-PAGE and was expressed at approximately two to three times the level of the endogenous protein in control cells ( C). These data prove that the effects of Scrib RNAi on motility are indeed caused by loss of the Scrib protein rather than by off-target effects of the shRNA.
Figure 3. Suppression of Scrib expression increases cell motility and attenuates orientated migration. (A) Motility of cells transfected with Luc, ScrbKD1 shRNA, or ScrbKD1 plus GFP-tagged human Scrib was measured using a Boyden chamber assay. Images were captured (more ...)
The filter assay depends not only on cell motility but also on the rate of cell attachment to and spreading on the filter surface. Therefore, as an alternative approach to measuring cell motility, we performed wounding assays on MDCK monolayers and tracked the movement of individual cells within the population at the wound edge by time-lapse microscopy. The overall rate of wound closure was similar for both the control cells and those lacking Scrib (0.35 vs. 0.32 μm/min). However, the behavior of cells lacking Scrib was remarkably different from that of the control (Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200506094/DC1
). At the edges of the wound, control cells extruded lamellipodia and moved forward as an organized sheet, but the KD cells were less organized. Some of the KD cells lost their attachment to the sheet, pulled away from the leading edge, and moved in random directions. This difference can be seen in the Rose plots of cells tracked over the period of the assay ( D). Calculation of directionality parameters confirmed that the cells lacking Scrib move at a significantly higher speed than control cells (2.54 ± 0.19 vs. 1.73 ± 0.41 μm/min; P = 0.026) and with a lower persistence coefficient (4.3 vs. 11.6 min; P = 0.02). The reduced persistence accounts for the similarity in the overall rate of wound closure. Interestingly, cells further back from the wound exhibited a continual jiggling motion, as if they had lost adhesion to their neighbors and were trying to move away from one another, and transient gaps appeared in the monolayer (Video 2). These results support the data shown in , suggesting that loss of Scrib causes a defect in cell–cell adhesion.
Scrib effects are not mediated by βPIX binding
Cell movement is regulated by Rac, and this GTPase has also been implicated in controlling adhesion between epithelial cells (Ehrlich et al., 2002
; Van Aelst and Symons, 2002
; Chu et al., 2004
). Interestingly, Scrib has been reported to bind, via its PDZ domains, to the COOH terminus of the Rac guanine nucleotide exchange factor, βPIX (Audebert et al., 2004
). To determine whether the SCRIB–βPIX interaction is important in mediating the regulation of cell migration and adhesion, we first confirmed that in MDCK cells we could detect this interaction (unpublished data) and then assessed the role of βPIX by silencing expression of the canine protein in MDCK cells.
Of four pSUPER constructs tested, three efficiently suppressed βPIX expression ( A). In particular, the PixKD1 shRNA reduced expression of the protein by >90%. However, loss of βPIX had no detectable effect on cell migration as measured using the Boyden chamber assay ( B). Moreover, in our hands, overexpression of βPIX did not increase cell migration in the filter assay (unpublished data). We then performed double KD experiments in which the expression of both Scrib and βPIX was suppressed ( C). We reasoned that one function of Scrib might be to sequester and inactivate βPIX. In this case, loss of Scrib would release the βPIX, leading to inappropriate activation of Rac and increased migration. If this hypothesis were correct, a double KD would reverse the migration phenotype by removing the excess free βPIX from the cell. As depicted in (A and B), migration through filters was increased by Scrib KD. However, the coordinate loss of βPIX did not significantly perturb this effect ( B). Note that cotransfection of the PixKD shRNA did not interfere with gene silencing of Scrib ( C). We therefore conclude that Scrib function in cell adhesion and migration is independent of βPIX binding.
Figure 4. βPIX is not involved in Scrib function at adherens junctions. (A) MDCK II cells were transiently transfected with either pS-Luciferase control vector (Luc) or three pSUPER constructs targeting different sequences of canine βPIX mRNA (PixKD). (more ...)
Because βPIX is a guanine nucleotide exchange factor for Rac, we also asked whether loss of Scrib would alter Rac activity. Rac-GTP was detected by pull-down assays using a GST fusion of the Rac binding domain of PAK. No consistent differences in Rac-GTP were detected, however, in control cells versus those lacking Scrib ( D). When the cells were subjected to a calcium switch, Rac was activated within 2 h of calcium addition in both the control and Scrib KD cells ( E). We therefore conclude that the polarity defects associated with suppression of Scrib expression are independent of βPIX and are not mediated through the Rac GTPase. Finally, we asked whether Scrib might regulate the extracellular signal–regulated kinase (ERK) signaling pathway, which is activated by scatter factor (hepatocyte growth factor [HGF]; Tanimura et al., 1998
). HGF induces an EMT in which cells lose adhesiveness and become more migratory, a phenotype similar to that observed in cells lacking SCRIB. However, no significant differences in phospho-ERK were detected when Scrib expression was knocked down either before or after addition of HGF ( E). These data suggest that Scrib does not function to regulate the HGF signaling pathway.
Scrib is required for E-cadherin–mediated adhesion
To determine whether cell–cell adhesion is compromised in the absence of Scrib, we first used an aggregation assay. Cells were trypsinized, triturated to break up clumps into individual cells, resuspended in fresh medium in a hanging drop beneath the lid of a tissue culture plate, and incubated for 18–20 h. Cell aggregation was then assessed microscopically. A dramatic loss of aggregation was apparent in cells expressing either pS-ScrbKD1 or 2 vectors, as compared with the control cells that were transfected with pS-Luc (). This effect was not a result of differential loss of E-cadherin in the cell suspensions as assessed by immunoblotting lysates from the suspended cell cultures ( B). Importantly, coexpression of human GFP-Scrib reversed the adhesion defect caused by the loss of endogenous SCRIB, proving that the effect of the shRNAs on adhesion is specifically mediated through destruction of the Scrib mRNA rather than through off-target effects (). We also tested for a possible role for βPIX on aggregation, using an shRNA directed against the canine gene (). However, loss of βPIX from the cells had no effect on the aggregation of control cells and did not reverse the loss of aggregation observed in the absence of Scrib ( D).
Figure 5. Decreased adhesiveness of cells lacking Scrib. (A) 3 × 104 control and ScrbKD cells were seeded into hanging drop cultures and allowed to aggregate overnight. After trituration by passing the cell cluster 10 times through a 200-μl pipette (more ...)
To determine whether the aggregation defect is mediated through E-cadherin or some other cell adhesion protein, we assayed the ability of the MDCK cells to attach to a surface coated with the extracellular domain of E-cadherin. Cells were disassociated using an EGTA solution (with no trypsin), centrifuged and resuspended in fresh medium, and added to 96-well plates coated with the ectodomain of E-cadherin. After 60 min, the plates were washed and remaining attached cells were counted. Results are shown in . Almost no cells attached to the plates in the absence of the E-cadherin ectodomain, demonstrating that during the 60-min incubation period integrin-mediated attachment is negligible. Control cells attached efficiently, and attachment was proportional to the amount of E-cadherin ectodomain on the plate ( B). Importantly, loss of Scrib caused a substantial drop (approximately threefold) in cell attachment, demonstrating that E-cadherin homophilic adhesion is compromised in the absence of Scrib. Addition of an arginine–glycine–aspartic acid peptide to block integrin-mediated adhesion had no significant effect (unpublished data).
Figure 6. Suppression of Scrib expression reduces E-cadherin–mediated adhesion. (A) In vitro E-cadherin binding assay. Different concentrations of recombinant E-cadherin extracellular domain were coated on plates. Control (Luc) and ScrbKD cells were collected (more ...)
Depletion of Scrib causes a defect in cell–cell adhesive junctions
These data suggest that Scrib is required for normal E-cadherin function at cell–cell junctions. We therefore examined the distribution of E-cadherin and of the Na/K-ATPase, which is a marker for the basolateral membrane. In control cells, these proteins colocalize along the lateral cell boundaries. Scrib KD caused a distinctive phenotype in which the lateral membranes of the cells became disorganized. The membranes appeared less vertical and had convoluted edges ( A). A similar phenotype was observed for β-catenin distribution ( B). However, the total amounts of E-cadherin, β-catenin, and α-catenin expressed in cells depleted of Scrib were the same as the amounts in control cells ( C). Scrib does not, therefore, regulate the expression of these junctional proteins. Moreover, when surface proteins were biotinylated, captured on streptavidin beads, and blotted for E-cadherin, no reproducible difference was observed between the control and Scrib KD cells ( D). These data demonstrate that there is no change in the amount of E-cadherin on the cell surface, and we conclude that Scrib is not involved in controlling the exocytosis or endocytosis of E-cadherin.
Figure 7. Loss of Scrib causes a defect in adherens junction structure. (A) Control and Scrib-depleted cells were plated at subconfluence and allowed to form islands of cells. They were then fixed and stained for E-cadherin (red) and Na/K-ATPase (green). Image (more ...)
When adherens junctions form, a fraction of the α- and β-catenin becomes stabilized at the cell cortex, either through clustering of the E-cadherin or perhaps through attachment to actin, and is detergent insoluble. We measured the detergent-insoluble fraction in control and Scrib KD cells and found that in the absence of Scrib the amounts of both α- and β-catenin in this fraction were substantially reduced (). Together, these results suggest that Scrib is required for the normal stabilization of α- and β-catenin at the cell cortex.
Depletion of E-cadherin phenocopies the effects of Scrib KD
If both the adhesion defect and the increased motility observed in response to Scrib silencing are caused by decreased E-cadherin activity, one would predict that depletion of E-cadherin would produce the same phenotype. We therefore expressed a shRNA targeted against the canine E-cadherin in MDCK cells and achieved a >50% reduction in E-cadherin expression ( A). Interestingly, cells depleted of E-cadherin migrated through filters significantly faster than the control ( B). Moreover, these cells were larger and more fibroblastic in appearance than control cells when plated at low densities ( C), just as observed for the Scrib KD cells. Based on these data, we conclude that both the morphological changes and increased motility in cells depleted of Scrib can be ascribed to a failure of the E-cadherin to form normal trans-adhesive interactions.
Figure 8. Effects of E-cadherin depletion and expression of an E-cadherin–α-catenin fusion protein. (A) Cells were transfected with an shRNA targeted against canine E-cadherin. Lysates were immunoblotted for E-cadherin and Scrib. Equal protein concentrations (more ...)
An E-cadherin–α-catenin fusion protein can reverse the effects of silencing Scrib expression
To determine the locus of action of Scrib, we attempted to reverse the effects of Scrib depletion by the ectopic expression either of a cadherin–GFP fusion (Ecad–GFP) or of a cadherin–α-catenin fusion protein (Ecad–αcat). This latter construct lacks β-catenin binding sites but can connect to the actin cytoskeleton through the COOH-terminal domain of the α-catenin and can promote homophilic adhesion (Nagafuchi et al., 1994
; Gottardi et al., 2001
). The Ecad–GFP has been shown previously to be fully functional (Adams et al., 1998
Both constructs were expressed only at very low levels compared with the level of endogenous E-cadherin ( D). Nonetheless, the Ecad–αcat fusion was partially able to reverse the increase in migratory behavior of the cells depleted of Scrib ( E). Importantly, however, a similar level of Ecad–GFP was unable to reduce migration of these cells. Next, using an aggregation assay, we asked whether the fusion proteins could also reverse the adhesion defect in the Scrib KD cells. Again, the Ecad–αcat provided a partial restoration of cell–cell adhesion, whereas the Ecad–GFP fusion did not (). The Ecad–αcat fusion did not appear to increase aggregation of control cells, although a small effect would not have been detectable in this assay. Therefore, the forced, constitutive linkage of E-cadherin to α-catenin can restore normal adhesive and migratory behavior on cells in which Scrib expression has been reduced, suggesting that Scrib acts to modulate this linkage.