Structure–function studies revealed the properties of LIM and LD domains in invadosomes. (A) Schematic representation of the pax-Flag-WT and pax-Flag mutants missing the LIM domains and pax-Flag mutants deleted for the LD1, LD2, LD3, LD4, or LD5 motifs used for structure–function studies. (B) MEF-SrcY527F pax^−/− shHic-5 cells expressing pax-Flag-WT induced very compacted invadosomes, whereas pax-ΔLIM-Flag expression resulted in large rings. (C) Quantification of the percentage of cells forming invadosomes revealed that pax-ΔLIM-Flag mutants rescued invadosome formation as well as pax-Flag-WT (n = 3; 300 cells counted per condition). (D) Quantification of the normalized degraded area of fluorescent gelatin per cell revealed that expression of the pax-ΔLIM-Flag mutant poorly restored ECM degradation in comparison to pax-Flag-WT (n = 3; 200 cells counted per condition). au, arbitrary unit. (E) Immunofluorescence experiments showing the absence of invadosome rings caused by the absence of LD1, LD2, LD3, LD4, or LD5 motifs, whereas some isolated invadosomes were still visible. The expression of either pax-LD3_ΔLD3-Flag or pax-LD5_ΔLD5-Flag completely abolished the formation of isolated invadosome rings. (F) Quantification of the percentage of cells forming invadosomes showed that deletion of each LD poorly restored invadosome ring formation in comparison to pax-WT-Flag (n = 3; 300 cells counted per condition). (G) Quantification of the normalized degraded area of fluorescent gelatin per cell shows that the decrease of invadosome formation induced by each mutant was associated with low ECM degradation. ***, P < 0.001. All graphs presented as means ± SD. Differences with a probability level P < 0.05 were considered significant in one-way ANOVA. Bars: (B) 8 µm; (E) 20 µm.