Identification of the pro-PrP binding domains on FLNA and the motif in the GPI-PSS that binds FLNA. A, immunoblots of in vitro pulldown assays show that recombinant pro-PrP^23–253 (r-pro-PrP) binds to domains 10, 15, 16, 17, 18, 20, 21, and 23 but not domains 11, 19, 22, or 24 of FLNA. This result was confirmed at least three times. IP, immunoprecipitation; IM, immunoblot. B, immunoblots of in vitro pulldown assays show that pro-PrP lacking the last five residues is unable to bind FLNA. Furthermore, replacing two of the three polar residues in the last eight residues completely eliminated FLNA binding. This result was confirmed at least twice. C, sequence alignment shows that the FLNA binding site on the GPI-PSS of PrP from different mammalian species is highly conserved. D, shown is an in silico model of the binding interface between the GPI-PSS of PrP and domain 23 of FLNA. Amino acids at C and D strands of domain 23 of FLNA (gray carbon atoms, black letters and numbers) and PrP GPI-PSS (green carbon atoms, magenta letters and numbers) are shown as ball-and-stick. Multiple hydrogen bonds exist in the binding interface (small yellow dots).

Co-purification of FLNA and integrin β1 and the effects of down-regulation of PrP on integrin β1 and FLNA association. A, left panels, immunoblots (IM) show that PrP co-purifies (IP) with FLNA, and integrin β1 co-purifies with FLNA, but PrP does not co-purify with integrin β1 in A7 cells. This result was confirmed twice. Right panels, immunoblots show that down-regulation of PrP does not alter the expression levels of integrin β1 (top panel) but reduced the amounts of integrin β1 co-purified with FLNA (bottom panel) in the inducible shRNA model (lanes 1–6) and in the constitutively active model. This result was confirmed twice. B, microscopic images show co-localization of FLNA and integrin β1 in control A7 cells. But in PrP down-regulated A7 cells, integrin β1 is separated from FLNA. This result was independently confirmed twice. C, immunoblots show that down-regulation of PrP in A7 cells reduces the level of phosphorylated focal adhesion kinase (p-FAK) but not p-Src. This result was confirmed at least twice. D, a drawing shows that in A7 cells binding of FLNA to pro-PrP pulls FLNA closer to the inner membrane leaflet, which then promotes the binding to FLNA to integrin β1. When the level of PrP is reduced, FLNA retracts from the inner membrane leaflet, which disrupts the binding of FLNA to integrin β1.

Detection pro-PrP in melanoma in situ and invasive melanoma but not in melanocyte. A, immunostaining with anti-PrP mAb shows that PrP is undetectable in melanocytes (m). The brown spot identified by a dashed arrow is nonspecific staining. OM, ×400. B, melanocytes also do not react with the anti-GPI-PSS antibody. OM ×400; C, intra-dermal benign nevus does not react with anti-PrP mAb. OM × 400. D, Anti-PrP mAb reacts with melanoma in situ as well as invasive melanoma. OM ×200. E, anti-PrP mAb reacts with invasive melanoma cells (M) but not with melanocytes (m) in the same field ×400. F, invasive melanoma cells show strong cell surface staining. OM ×400. G, the anti-GPI-PSS antibody reacts with melanoma in situ and invasive melanoma. Invasive melanoma cells in the dermal component have the strongest staining. OM ×200. H, the anti-GPI-PSS antibody reacts with melanoma in situ. OM ×400. I, the anti-GPI-PSS antibody reacts strongly with invasive melanoma cells; OM ×400. The number of samples is listed in Table 1.

Expression of FLNA and pro-PrP in M2 and A7 cells. A, immunoblots show that only A7 cells express FLNA. Both M2 and A7 cells express PrP. PrP from M2 and A7 cells has a molecular mass of about 26 kDa. A recombinant mature PrP^23–231 (r-PrP) and a recombinant pro-PrP^23–253 (r-pro-PrP) were included as molecular mass markers. Pro-PrP from A2 and M7 cells migrates slower than recombinant pro-PrP^23–253. This likely reflects the conformational difference between recombinant pro-PrP^23–253 and native pro-PrP. Recombinant pro-PrP^23–253 has to be solubilized and refolded in urea, which might cause the protein to be more compacted. This experiment was repeated at least three times with comparable results. Histograms show that PrP on the cell surface of M2 and A7 cells reacts with multiple anti-PrP mAbs. The epitopes of the mAbs are shown in supplemental Fig. S1A. BG, background staining with irrelevant mAb D7C7. This experiment was repeated at least three times with comparable results. B, microscopic images show the expression of FLNA in A7 cells but not in M2 cells. The distributions of PrP and actin in M2 and A7 cells also differ. For organelle-specific markers, a rabbit anti-calnexin antibody was used to mark the ER, and a Bodipy™F-C5 ceramide-BSA was used to locate the Golgi. This experiment was repeated at least three times with comparable results. C, immunoblots show that in A7 cells PrP co-purifies with FLNA and vice versa. Loading controls show the levels of PrP or FLNA in cell lysates before co-immunoprecipitation (IP) and an immunoblot (IM). This experiment was repeated at least three times with comparable results. Immunoblots show that co-purification of FLNA with PrP can be competed with a PrP GPI-PSS synthetic peptide but not a control peptide. This result was confirmed at least twice. D, microscopic images show the co-localization of pro-PrP and FLNA in A7 cells. This result was confirmed at least three times. E, histograms show that cell surface PrP on A7 cells has a longer half-life. A7 and M2 cells were cultured with BFA for various lengths of time to prevent the transit of newly synthesized PrP to the cell surface. At different times after culture, cells were prepared and stained with an anti-PrP mAb, 8H4. The levels of cell surface PrP were then quantified by flow cytometry. Mean fluorescent intensities of control cells or cells treated with BFA at time 0 were arbitrarily defined as 1. This result was confirmed at least twice.

Effects of reducing PrP expression in M2 and A7 cells. A, immunoblots show that down-regulation of PrP in M2 and A7 cells reduces the levels of PrP in both cell lines but does not reduce the expression level of FLNA in A7 cells. Top panels show shRNA under the control of an inducible promoter. (shRNA-Cont, control; shRNA-PrP-10, prp knocking down sequence 10; NI, non-induced; In, induced). Bottom panels show constitutively active shRNA. This result was independently confirmed at least twice. B, microscopic photos show that down-regulation of PrP alters the spatial distribution of FLNA and organization of actin filaments in A7 cells. This result was confirmed twice. C, down-regulation of PrP reduces the expression levels of p-cofilin and LIMK1 in A7 cells but not in M2 cells. Top panels show shRNA under the control of an inducible promoter (Cont, knocking down control; PrP-12, PrP knocking down sequence 12). Bottom panels show constitutively active shRNAs. This result was confirmed twice. D, top panels, microscopic photos show that PrP-down-regulated A7-shRNA-PrP-12 cells are much less adhesive compared with control A7-shRNA-Cont cells. Solid arrows identify adherent cells, and dashed arrows identify non-adherent, floating cells. Original magnification (OM), 10×10. The graph shows quantification of the spreading results of another experiment comparing PrP down-regulated A7-shRNA-PrP-10 with control A7-shRNA-Cont cells. Results presented were the average of the two experiments ± S.D. Middle panels, microscopic photos show that PrP down-regulated A7 cells have reduced cellular migration in a wound-healing assay. Photos were taken at 15 h post-wound initiation. Two representative wells from each condition were shown. Areas between two white lines mark the original wound area. OM, 10×10. This result was confirmed twice. Bottom panels, bar graph, quantification of the wound-healing assay was taken at 15 h post-wound. PrP down-regulated A7 cells have a reduced would-healing capacity. Results presented were the average ± S.E. of two different experiments. Line graph, quantification of the would-healing assay at different time points after wound healing is shown. Results presented were the average of two different experiments ± S.E. p < 0.05 was considered to be statistically significant. The migration of shRNA-PrP-10-induced A7 cells is significantly slower (p = 0.0014) compared with shRNA-PrP-10 non-induced A7 cells.

A PrP-GPI-PSS synthetic peptide inhibits A7 cell spreading and migration. A, microscopic images show that incubation with a KKRPK-PrP-GPI-PSS synthetic peptide reduces A7 cell spreading. Arrows identify adherent cells. OM, 10×10. B, quantification of the cell spreading results. Results presented were the average ± S.E. of two experiments. OM, 10×10. C, quantification of a wound-healing assay at 15 h after the initiation of wound which shows that significant inhibition of cell migration could be achieved with as little as 0.5 μm concentrations of the peptide. Five images of every concentration were used to calculate the p values by analysis of variance one-way test. D, representative photographs of microscopic images taken at 15 h after initiation of wound, which show that incubation with a KKRPK-PrP-GPI-PSS synthetic peptide reduces A7 cell migration.