FIG 5 .

FM4-64 endocytosis is actin dependent in arp2 mutants. (A, top) Schematic representation of the experimental setup. Cells were treated with 10 µg/ml cytochalasin A (CA) or DMSO for 3 min, at which time LIFEACT-GFP signals were assessed. Only arp2 cells completely lost LIFEACT-GFP signals (left). In a second experiment, arp2 cells were treated with 10 µg/ml CA for 3 min, FM4-64 was added at 20 µg/ml for 5 min, unbound dye was washed away and cells were chased for another 20 min to allow dye internalization. During this 30-min procedure, either DMSO or CA was present in the corresponding solutions. Endocytosis was blocked after 30 min by adding sodium azide and sodium fluoride, and arp2 cells were assessed for endocytic capacity (right, n > 150). (B) Two control experiments demonstrating that treatment of arp2 cells for 30 min with CA does not significantly reduce viability (left), and blocking actin dynamics with CA is reversible with cells recovering almost completely after 24 h of washing CA away (right). The bottom represents the schematic setup of the experiment in panel B. All experiments were done with arp2 mutants, expect the left figure in part A.