Leupaxin-binding mutant kindlin-3 fails to recruit leupaxin to podosomes and to reduce paxillin phosphorylation. (A, C, and D) IF staining and confocal imaging of K3^−/− preosteoclasts retrovirally transduced with EGFP, WT EGFP-K3, or the EGFP-K3 M3 mutant (green) for paxillin (red, A), leupaxin (red, C), and phospho-paxillin Y31 (red, D) and actin (white/blue in merge). Scale bars, 5 µm (A) and 10 µm (C and D). (B) Actin core diameter of K3^−/− preosteoclasts expressing EGFP, EGPF-K3, or the EGFP-K3 M3 mutant. 10 actin cores in two regions of three to five cells were measured per experiment. n = 5. (E) Fluorescence intensity profile through three actin-cores (indicated by the black lines in A) of K3^−/− preosteoclasts expressing EGFP, WT EGPF-K3, or the EGFP-K3 M3 mutant. (F) Percentage of cells with podosome clusters, which reveal discrete localization of EGFP-K3 and EGFP-K3 M3 in the podosomal ring. 50 cells were evaluated per condition in each of three independent experiments. Dotted white lines mark cell borders.