MT1-MMP islets are sites of podosome reemergence. (A–C) MT1-MMP islets are sites of de novo actin nucleation. TIRF still images of a macrophage expressing MT1-MMP-pHluorin (A, green) and Lifeact-RFP (B, red), with merge (C), treated with CK-666, with subsequent washout of the drug to allow reformation of podosomes. Cell circumference indicated by dashed line. Bars: 10 µm; (insets) 1 µm. White boxes indicate areas shown enlarged in A′–C′. Note that in A′ and B′, respective fluorescence signals have been changed to inverted grayscale. Red arrows in A′ and B′ indicate MT1-MMP islets that are also positive for Lifeact-RFP. Note absence of podosomal Lifeact-RFP signals at 7- and 15-min time points, with successive accumulation of Lifeact-RFP signals at MT1-MMP islets. Time after washout of CK-666 is indicated below (C′). (D–I) MT-MMP islets can recruit material generated by podosome fission. TIRF micrograph of macrophage expressing MT1-MMP-pHluorin (D, green) and Lifeact-RFP (E, red), with merge (F). Still images taken from Video 5. White boxes indicate detail regions shown in D′–F′. Note MT1-MMP islet (arrow) recruiting F-actin accumulation generated by fission of a podosome (white arrowheads). Time since start of the experiment is indicated in minutes and second. Bars: 10 µm; (insets) 1 µm. (G–I) Fluorescence intensity diagrams of MT1-MMP-pHluorin and Lifeact-RFP, taken along the dotted line indicated in detail images of G′–I′ at the indicated time points. Note rise of RFP-fluorescence intensity, indicative of increased actin polymerization, in the core before podosome fission, with subsequent redistribution of the daughter podosome to an MT1-MMP islet. Measurements are representative for the respective set-up, and similar curves have been reproduced multiple times.