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Fig. 7. Spinal neurons express MMPs, and growth cones exhibit invadosome-targeted protease activity. (A) Reverse transcription PCR amplification of MMP2, MMP9 and MMP14 from stage-24 Xenopus spinal cord. (B) Western blot of MMP14 from stage-24 Xenopus spinal cord. (C) Gelatin zymogram of MMP9 (∼90 kDa) and MMP2 (70 kDa) along with their pro-forms (∼110 kDa and ∼75 kDa, respectively) from stage-24 Xenopus spinal cord. (D-F) Confocal images of a spinal neuron grown on laminin and immunolabeled for MMP14 (MT1-MMP) (D), with F-actin-labeled with phalloidin (E) and an image merge (F) of MMP14 (green) and F-actin (magenta). Note colocalization of MMP14 puncta at F-actin foci (arrows). (G,H) 3D rendering view of SIM images of a human forebrain neuron growth cone (from Fig. 6E) triple-labeled for F-actin (red), βIII tubulin (blue) and ADAM17 (green). A view of the apical surface (G) shows an F-actin foci in the central domain (arrowhead), which is revealed to be an apically directed protrusion in a lateral view (H) with ADAM17 at the protrusion tip (arrowhead). (I-L) Gelatin degradation by a spinal neuron growth cone occurs near F-actin foci. (I) Oregon Green gelatin used as the substratum. (J) Growth cone labeled with Alexa-546 phalloidin. (K) Merged image of the gelatin (green) and F-actin (magenta) labels. Note colocalization of F-actin foci with areas of gelatin degradation (arrowheads). (L) Quantification of gelatin degradation for the total growth cone (Tot. GC) and at growth cone invadosomes (GCI) compared to background fluorescence. Gelatin degradation at invadosomes was inhibited by treatment with 10 µM GM6001, 2 µM PP2, 5 µM SU6656, 2.5 µM MβCD and in growth cones expressing δPX-Tks5-GFP. ***P<0.0001, Kruskal–Wallis test with Dunn's post-hoc analysis, n≥22. Scale bars: 5 µm (D-F); 3 µm (G,H); 10 µm (I-K). |
