Macrophages are built for rapidly responsive migration. (a) A primary fibroblast, grown on a fibronectin coated coverslip in α+MEM, 10% FCS, and 120 ng/ml recombinant CSF-1, was fixed and stained for phosphoY118 paxillin (green) and F-actin (red). Phospho-Y118 paxillin staining eliminates background cytoplasmic staining of unphosphorylated paxillin [1, 16]. The arrow indicates focal complexes at the leading edge and the arrowhead indicates a focal adhesion giving rise to a stress fiber. (b) MacCsf1r−/−. WT macrophage, grown on a fibronectin coated coverslip in α+MEM, 10% FCS, and 120 ng/ml recombinant CSF-1, was fixed and stained for phosphoY118 paxillin (red) and F-actin (green) and examined by TIRF microscopy. The arrow indicates a focal complex giving rise to an F-actin cable. (c) Inverted image of phosphoY118 paxillin IF staining at the leading edge of the primary fibroblast, yellow circles indicate several nascent adhesions, green ovals highlight focal complexes and red ovals outline some focal adhesions. (d) Inverted image of phospho-Y118 paxillin IF staining in the leading lamellipodium of the macrophage, yellow circles indicate point contacts with strong phosphopaxillin staining, red circles indicate point contacts with moderate phosphopaxillin staining and green ovals outline the linear focal complexes. (e) A larger view of the fibroblast stained for pY118 paxillin (left) and shown by phase contrast (right) to demonstrate the co-cultured primary macrophage migrating underneath the fibroblast (arrow) and disrupting its focal adhesions. Note the lack of macrophage focal adhesions. Scale bars = 10 μM.
