Filopodia formation mediated by receptor tyrosine kinase Ror2 is required for Wnt5a-induced cell migration

Abstract

The receptor tyrosine kinase Ror2 plays important roles in developmental morphogenesis. It has recently been shown that Ror2 mediates Wnt5a-induced noncanonical Wnt signaling by activating the Wnt-JNK pathway and inhibiting the beta-catenin-TCF pathway. However, the function of Ror2 in noncanonical Wnt signaling leading to cell migration is largely unknown. We show, using genetically different or manipulated cultured cells, that Ror2 is critical for Wnt5a-induced, but not Wnt3a-induced, cell migration. Ror2-mediated cell migration requires the extracellular cysteine-rich domain (CRD), which is the binding site for Wnt5a, and the cytoplasmic proline-rich domain (PRD) of Ror2. Furthermore, Ror2 can mediate filopodia formation via actin reorganization, irrespective of Wnt5a, and this Ror2-mediated filopodia formation requires the actin-binding protein filamin A, which associates with the PRD of Ror2. Intriguingly, disruption of filopodia formation by suppressing the expression of either Ror2 or filamin A inhibits Wnt5a-induced cell migration, indicating that Ror2-mediated filopodia formation is essential for Wnt5a-induced cell migration.

Figure 1.

Both the CRD and PRD of Ror2 are required for Wnt5a-induced cell migration. (A) Migratory responses of Ror2^+/+ and Ror2 ^−/− MEFs in the absence or presence of control (neo CM), Wnt5a CM, or Wnt3a CM were determined in the Transwell chamber. The data are expressed as the mean ± the SD of three independent experiments. (B) Schematic representations of the wild-type (WT) Ror2 and its derived mutants. (C) L cells stably transfected with expression plasmid for Ror2-Flag (WT, DK, ΔCRD, ΔC, or Tc) or the control vector (pcDNA3), respectively, were analyzed in a Transwell migration assay in the presence of neo CM or Wnt5a CM. The data are expressed as the mean ± the SD of three independent experiments. Expression of the respective Ror2 proteins was analyzed by anti-Flag immunoblotting (inset).

Figure 2.

The PRD of Ror2 is required for Ror2-mediated filopodia formation. (A) GFP or Ror2-GFP fusion proteins (green) were expressed in HEK293T cells, and F-actin (red) was stained with rhodamine-phalloidin. Serial optical confocal z sections were obtained and stacked. (right) Merged images. (B) Ror2^+/+ and Ror2 ^−/− MEFs were trypsinized, resuspended in neo CM or Wnt5a CM containing 0.1% BSA, and incubated for 2 h at 37°C in suspension. The cells were then plated on coverslips and allowed to attach for 5 min at 37°C. Attached cells were fixed and stained with rhodamine-phalloidin. Serial optical confocal z sections were obtained and stacked. The percentages of cells with the indicated number of filopodia per cell were measured (right). The data are expressed as the mean of three independent experiments (n > 100). Bars: (A) 20 μm; (B) 10 μm.

Figure 3.

FLNa is associated with the PRD of Ror2 and is colocalized with Ror2. (A) Whole-cell lysates from HeLa and MCF7 cells were immunoprecipitated with anti-Ror2 or preimmune serum, followed by immunoblotting with anti-FLNa or anti-Ror2 antibodies. (B) MCF7 cells transfected with Ror2-GFP or Ror2ΔC-GFP (green) were fixed and stained with anti-FLNa antibody (red). (right) Merged images. Bar, 20 μm. (C) Whole-cell lysates from HEK293T cells expressing Ror2-Flag (WT or ΔC) were incubated with GST or GST-FLNa/20-24 bound to glutathione–Sepharose. Whole-cell lysates (input) and coprecipitated Ror2 were analyzed by anti-Flag immunoblotting. Precipitated GST and GST-FLNa proteins were detected by Amido black staining. (D, top) Schematic representation of FLNa dimer and C-terminal fragments containing different FLN repeats 20–24. (bottom) Whole-cell lysates from HEK293T cells expressing Ror2-Flag were incubated with GST or the indicated GST-FLNa fusion proteins bound to glutathione–Sepharose. Whole-cell lysates (input) and coprecipitated samples were analyzed as in C. (E) Whole-cell lysates form HEK293T cells expressing Myc-FLNa or -FLNaΔ20 were incubated with GST or GST-Ror2 (aa 726∼945) bound to glutathione– Sepharose. Whole-cell lysates (input) and coprecipitated FLNa were analyzed by anti-Myc immunoblotting. GST and GST-Ror2 proteins were detected by Amido black staining.

Figure 4.

Association of FLNa with Ror2 is indispensable for Ror2-mediated filopodia formation. (A) Ror2-GFP (green; top) or Ror2ΔC-GFP (green; bottom) was expressed in FLNa-deficient M2 or FLNa-expressing A7 cells, and F-actin (red) was stained with rhodamine-phalloidin. (right) Merged images. Magnified images of boxed regions are shown (magnified). (B) M2 cells were transfected with Ror2-CFP (red) along with YFP-FLNa (green; top) or YFP-FLNaΔ20 (green; bottom). (right) Merged images. Magnified images of boxed regions are shown (magnified). (C) Quantitative analysis of Ror2-induced filopodia formation. M2 and A7 cells expressing Ror2-GFP or M2 cells expressing Ror2-CFP together with YFP-FLNa or -FLNaΔ20 were stained with rhodamine-phalloidin for F-actin. The percentages of cells with the indicated number of filopodia per cell, identified by F-actin staining, on a GFP- or CFP/YFP-positive cell, were measured (n > 60). (D) M2 and A7 cells were assayed in a Transwell migration assay in the presence of neo CM or Wnt5a CM. The data are expressed as the mean ± the SD of four independent experiments. *, P < 0.005, t test. Expression levels of FLNa, Ror2, and α-tubulin were analyzed by immunoblotting (inset). Bars, 10 μm.

Figure 5.

Dvl proteins are required for Wnt5a-induced cell migration, but not Ror2-mediated, filopodia formation. (A) L cells were transfected with the respective siRNA plasmids as indicated. After 60 h in culture, whole-cell lysates from the respective transfectants were analyzed by immunoblotting with antibodies against Dvl2, Dvl3, and α-tubulin, respectively. Transfection efficiency was >90%, as assessed by the transfection of pEGFP plasmid. (B) L cells stably expressing Ror2-Flag were transfected with the respective siRNA plasmids, as indicated. After 60 h, cells were assayed in a Transwell migration assay in the presence of neo CM or Wnt5a CM. The data are expressed as the mean ± the SD of three independent experiments. (C) L and MCF7 cells were transfected with Dvl2 and Dvl3 siRNA plasmids or control siRNA plasmid. After 48 h, cells were either transfected with Ror2-GFP or left untransfected and further cultured for 16 h. Cells expressing Ror2-GFP were visualized by confocal microscopy. Bar, 20 μm. Transfection efficiency was >90%, as assessed by the transfection of pEGFP plasmid. The percentages of cells with the indicated number of filopodia per cell were measured as described in Fig. 4 C (n = 50; right).