Podosomes are sites of matrix degradation and are contacted by MT1-MMP–mCherry vesicles. (A-F) Confocal micrographs of a macrophage seeded on rhodamine-labeled gelatin matrix, F-actin labeling podosome cores stained with Cy5-labeled phalloidin (A,Ai), gelatin matrix in red (B,Bi), merged images (C,Ci). White box in (C) indicates region enlarged in (Ai,Bi,Ci). (D) Confocal micrograph of primary human macrophage expressing MT1-MMP–mCherry and GFP-coupled Lifeact, staining F-actin-rich podosome cores. White box in (D) indicates region shown enlarged in (Di). Note close proximity between MT1-MMP–mCherry vesicles and podosome cores. (E) Still images from time-lapse video (supplemental Video 7). Macrophage expressing MT1-MMP–mCherry and a C-terminal talin1-GFP construct labeling podosome ring structures. White box in larger image indicates region shown in detail images. A podosome (labeled by dotted yellow ring) is contacted by a MT1-MMP-mCherry vesicle (white arrowhead) for approximately 10 seconds. Time in seconds is indicated in lower right corners. White scale bars indicate 10 μm. (F) Velocity of MT1-MMP–mCherry vesicles in the vicinity of podosomes. Bar diagram shows velocities in micrometers per second for vesicles moving along podosome rings in macrophages treated with control siRNA or 2 siRNAs specific for KIF5B and KIF3B (n = 17, respectively). **P < .01 compared with control. For specific values, see supplemental Table 1. Bars indicate mean values ± SD.