Podosome-associated MT1-MMP shows reduced lateral mobility compared with podosomal F-actin. (A and B) TIRF micrographs of macrophage expressing Lifeact-RFP to detect F-actin–rich podosome cores (A) and MT1-MMP-pHluorin (B). Images show color-coded merges of time-lapse videos with successive coloration of individual frames along the spectrum, as indicated. White boxes indicate areas of detail images shown below each panel (A′ and B′). (A″ and B″) Shown are tracks of the center of mass in yellow. Note high variability of track orientation and length of podosomes cores, whereas tracks of MT1-MMP-pHluorin are more uniform and short. Dotted line indicates cell circumference. Bar, 10 µm. Drift of the microscope stage was corrected by use of fluorescent beads, indicated by arrows in B. (C) Statistical analysis of lateral displacement of podosomal F-actin and podosomal MT1-MMP-pHluorin. Each dot represents lateral displacement of the respective center of mass of a single podosome-associated signal. Collectively, 1,411 podosomes were evaluated. Red bar indicates mean ± SEM. ****, P < 0.0001.