MT1-MMP accumulations persist beyond disruption of podosomes. TIRF still images of a cell expressing MT1-MMP-pHluorin (A and D, white) and Lifeact-RFP (B and E, red), with merges (C and F), before (A–C) and 30 min after (D–F) addition of Arp2/3 inhibitor CK-666. White boxes in D–F indicate detail regions shown as insets. Note that regions devoid of podosomes (E) still contain dot-like accumulations of MT1-MMP-pHluorin. Bar, 10 µm. (G–I) Kymographs of the cell shown in A–F. Time since start of the experiments is indicated in minutes. CK-666 was added at time point 0. Note that the localized MT1-MMP-pHluorin signal persists also in the absence of podosomes. Bars, 1 µm. (J) FRAP analysis of MT1-MMP-pHluorin turnover at CK-666 induced islets. Gallery shows TIRF micrographs taken from time-lapse movie of MT1-MMP-pHluorin expressing macrophage. Dashed line indicates single bleached islet. Time before and after bleaching is given above each panel. (K) Quantification of FRAP showing MT1-MMP-pHluorin-based, normalized fluorescence intensity over time, with fitted curve in black. Values are given as mean ± SD. For each value, 10 islets from three cells from three different donors were evaluated.