Figure 1.
Wound healing assays on cell migration and localization of focal adhesions in growth-arrested inv+/+ and inv−/− MEFs.
Graphs of time-lapse data represent mean values of four independent experiments ± S.E.M. (p<0.001, one-way ANOVA). Cells were serum starved for 24 h and allowed to recover 1 h post wounding, hence t = 0 h in the data sets relates to the initiation of monitoring. (A) Light microscopy of inv+/+ and inv−/− MEFs in wound healing assay at t = 0 h (left) and t = 6 h (right). (B) Trajectories of growth-arrested inv+/+ (N = 27) and inv−/− (N = 28) MEFs in wound healing assay. Each line represents the migration of one cell within a 6 h period. The red circles illustrate the mean translocation of the cells. (C) Mean velocity and translocation of cells into the wound. (D) IFM analysis of localization of focal adhesions by anti-vinculin (red) in cells in wound healing assays and the actin cytoskeleton was stained with phalloidin (F-Actin, blue). Open arrows mark direction of migration into the wound and green dotted lines mark the front of cells facing the wound. (E,F) SDS-PAGE and WB analysis of expression of vinculin in growth-arrested inv+/+ and inv−/− MEFs.
Figure 2.
Formation and orientation of primary cilia and localization of Inversin in growth-arrested inv+/+ and inv−/− MEFs.
(A) IFM analysis of primary cilia (closed arrows) with anti-acetylated alpha tubulin (Ac-tub, red) and their basal body region with anti-Centrin-2 that marks the two centrioles (asterisks) of the centrosome (Ctn-2, green). (B) IFM analysis of primary cilia (closed arrows) co-labelled with Ac-tub (red) and anti-glutamylated alpha-tubulin (Glu-tub, green). (C) Ciliation frequencies of inv+/+ and inv−/− MEFs upon 24 and 48 h of serum-free incubation, represented as mean ± S.E.M. (n = 3). (D) WB analysis of inv+/+ and inv−/− MEFs in the presence (+) and absence (−) of serum with anti-phospho-Retinoblastoma protein, which is downregulated in growth arrested cells. E: IFM analysis of primary cilia (Ac-tub, red, and closed arrows) formation in growth arrested cells. Nuclei (DAPI, blue) of cycling cells shows localization of anti-Ki67 (green). (F) IFM analysis of Inversin (green) localization to primary cilia (Glu-tub, red, closed arrows) of inv+/+ and inv−/− MEFs. Asterisks (*) indicate ciliary base, nuclei are stained with DAPI (blue). (G) IFM analysis of Inv-GFP (green) and Inversin (red) localization to primary cilia (Ac-tub, blue, closed arrows) in mock and Inv::GFP transfected cells. The base of the cilium is identified by Differential interference constrast microscopy, DIC (asterisks). (H) IFM analysis of inv+/+ and inv−/− in wound healing assays after 30 min (top panel) and 4 h (lower panel) migration. Open arrows indicate direction of migration. Primary cilia are stained with Ac-tub (upper panel, red, closed arrows) or Glu-Tub (lower panel, green) and nuclei are stained with DAPI (blue). In lower panel, the actin cytoskeleton is stained with phalloidin (F-actin, red). Green dotted lines mark the edge of cells facing the wound.
Table 1.
Transcriptomic analysis of up- and down-regulated genes in growth arrested inv−/− relative to inv+/+ MEFs with listed p values (n = 3) and common protein names.
Figure 3.
Wnt signaling is dysregulated in inv−/− MEFs.
(A) WB analysis of growth arrested inv+/+ and inv−/− MEFs with antibodies against Dishevelled (Dvl) -1, -2 and -3, Adenomatous polyposis coli (APC), Glycogen synthethase kinase 3 β (GSK3β), β-catenin (β-cat) and S45-phosphorylated β-catenin (p-β-cat). (B) Quantifications of WB analysis from (A). Histograms represent mean ± S.E.M. (n≥3). (C,G) IFM analysis of growth arrested inv+/+ and inv−/− MEFs. Primary cilia (Ac-tub or Glu-tub, red) are marked with closed arrows, nuclei are stained with DAPI (blue). Green protein localizations are Dvl-1 (C), Dvl-2 (D), Dvl-3 (E), p-β-cat (F) and Frizzled-3 (Fzd-3) (G). (H,I) IFM analysis on the localization of S33/37-T41-phosphorylated β-catenin (p-β-cat) (blue, open arrows) to the ciliary base (asterisks) in mock (H) and Inv::GFP (green) (I) transfected inv+/+ and inv−/− MEFs. Primary cilia (Glu-tub, red) are marked with closed arrows.
Figure 4.
Inversin affects the activity of the Rho GTPases.
Activity of Rac-1, RhoA and Cdc42 was determined by pull-down in migrating inv+/+ and inv−/− MEFs 30 min (A) or 4 h (B) after scratch. Histograms represent the mean ± S.E.M. of three independent experiments.
Figure 5.
Inversin affects the localization of the Rho GTPases and gene expression in migration-related pathways.
(A,B) IFM analysis of Rac-1 (A, green) or RhoA (B, green) localization after 4 h migration of inv+/+ and inv−/− MEFs. Arrows indicate direction of migration and arrowheads indicate Rac1/RhoA localization at the leading edge. The actin cytoskeleton is stained with phalloidin (F-actin, red) and nuclei are stained with DAPI (blue). (C,D) Quantification of leading edge staining of Rac1 (C) and RhoA (D) in inv+/+ and inv−/− MEFs represented as mean ± S.E.M. (n = 3). (E,F) Transcriptomics of growth arrested MEFs with listed p values (n = 3). Migration-related pathways with number and percentage of differentially expressed genes (DEGs) in inv−/− compared to inv+/+ MEFs (E). Downregulation of specific genes controlling actin polymerization in inv−/− relative to inv+/+ MEFs (F).
Figure 6.
Inversin affects expression, regulation and localization of ERM proteins.
(A) WB analysis of growth arrested inv+/+ and inv−/− MEFs with antibodies against total ezrin/radixin/moesin (ERM), phosphorylated ERM (T567 of ezrin, T564 of Radixin, T558 of moesin, p-ERM), ezrin, radixin and moesin, and α-tubulin as control, with indications of the 80 (1) and 75 (2) kDa bands. (B) Quantification of WB from (A); Histograms represent mean ± S.E.M. (n≥3). (C,E) IFM analysis of growth arrested inv+/+ and inv−/− MEFs in wound healing assays, with phalloidin staining of the actin cytoskeleton (F-actin, red) and nuclei with DAPI (blue). Open arrows indicate direction of migration, and arrowhead indicate leading edge staining of ezrin (C, green), Radixin (D; upper panel, green), moesin (D; lower panel, green), and p-ERM (E, green). (F,G) DIC and IFM analysis on lamellipodium formation and localization of ERM (F, red) and p-ERM (G, red) to the leading edge of migrating cells in wound healing assays in Inv-GFP (green) transfected inv−/− MEFs. The actin cytoskeleton is stained with phalloidin (F-actin, blue). Open arrows indicate direction of migration.
Figure 7.
Inversin affects the localization and expression of NHE1 and ezrin.
(A) IFM analysis of growth arrested inv+/+ and inv−/− MEFs in wound healing assays. Open arrows indicate direction of movement and arrow heads show leading edge co-localization of the Na+/H+ Exchanger 1 (NHE1, green) and ezrin (red). Nuclei are stained with DAPI (blue). (B) WB analysis of inv+/+ and inv−/− MEFs grown in the presence (+) or absence (−) of serum, with antibodies against NHE1, ezrin, and α-tubulin as control. (C) Quantification of WB from (B) represented as mean ± S.E.M. (n≥3). White dotted line marks the front of inv−/− MEFs facing the wound.