Clathrin localizes to the phagolysosome. (A and B) Macrophages expressing clathrin-GFP were imaged live after uptake of mCherry-Lp (A) or mRFP1–E. coli (B) for >1 h. For A, deconvolved, collapsed z-stacks are shown, while insets/lower panels are deconvolved single z-planes. Boxes indicate areas enlarged in the insets/lower panels. Arrows show clathrin in close association with Lp. Scale bars: 2 µm (main panels), 0.5 µm (insets), 0.1 µm (lower panels). For B, arrows show fission associated with clathrin puncta, and arrowheads show fragments after fission. See corresponding Video 9. Scale bars: 5 µm (main panel), 2 µm (insets). (C) Quantification of fission events associated with clathrin. (D) Isolated and fixed phagosomes (asterisks) were immunolabeled for LAMP1 (red) and clathrin (green). Top row: A z-stack of a single phagosome projected as the MIP. Middle row: Single-plane image of isolated phagosome showing clathrin patches. Bottom row: Cluster of phagosomes coisolated. Images are representative of 24 phagosomes analyzed. Scale bars: 2 µm.