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Fig. 5. F-actin foci colocalize with Src and Tks5, and require active Src and lipid rafts for their formation in growth cones. (A) Reverse transcription PCR amplification of Tks4 and Tks5 from stage-22 and stage-25 Xenopus spinal cord. (B) Western blot of Tks5 from stage-24 Xenopus spinal cord. (C,D) Confocal images of growth cones cultured on laminin and immunolabeled for Tks5 (C) and pY418-Src (D). (C′,D′) Immunolabeling of Cttn. (C′,D′) Phalloidin labeling of F-actin. (C‴-D‴) Merged images of Tks5 or Src (blue), Cttn (green) and F-actin (red). Note colocalization of Tks5 and active Src with Cttn at growth cone invadosomes (arrows). (E) TIRF image of a live growth cone on laminin expressing both Tks5-GFP (green) and mCh-UtrCH (magenta). Note that Tks5 targets to mCh-UtrCH-labeled F-actin foci (arrows). (F) Invadosome number is significantly reduced by treatment with 5 µM SU6656, 1 µM PP2 or 20 µM LY294002 for 30 min. ***P<0.0001, Kruskal–Wallis test with Dunn's post-hoc analysis, n≥75. (G) Invadosome lifetime in GFP-β-actin expressing growth cones is significantly reduced by inhibition of Src with 1 µM PP2. ***P<0.001, unpaired t-test, n≥99. (H) TIRF images of a live growth cone on laminin expressing GFP-β-actin before (left) and after (right) treatment with 2.5 µM MβCD. (I) The number of total (left) and stable (right) invadosomes in GFP-β-actin expressing growth cones is reduced after treatment with 1 µM MβCD. *P<0.05, paired t-test, n=7. (J) Invadosome lifetime in GFP-β-actin expressing growth cones is significantly reduced by cholesterol depletion with 1 µM MβCD. ***P<0.001, paired t-test, n≥57. Scale bars: 5 µm (C-E,H). ns, not significant. |
