Figure 4

Functional relevance of ubiquitin on the recruitment of autophagy effectors to phagosomes. Non-professional phagocytes were fed with RBC or IgG-opsonized particles for 30 min, fixed, and co-stained for F-actin and ubiquitin. Representative image of a nascent phagosome (A) and a phagocytic cup (B) positive for actin (first panels) and ubiquitin (second panels). The third panels show internalized RBC labelled with CFSE and non-internalized beads stained with an anti-human IgG antibody conjugated with FITC. The fourth panels are composites of the 1^st, 2^nd and 3^rd panels. Arrows and arrowheads indicate actin- and ubiquitin-positive phagosome, respectively. Bars, 10 µm. (C) Effect of PYR-41 in the ubiquitination of both RBC- and IgG-opsonized particles-containing phagosomes. Phagocytes were cultured and treated as described in Material and Methods section. (D) Quantification of PYR-41 effect on the acquisition of p62 by RBC-containing phagosomes. (E,F) Quantification of PYR-41 effect on the acquisition of the autophagy adaptor proteins, NBR1 and NDP52, respectively, by RBC- and IgG-opsonized particles-containing phagosomes. The values are means ± SEM of, at least, three independent experiments. At each time point, at least, 50 phagosomes were analyzed. *p < 0.05; **p < 0.01 comparing differences between adaptor-positive phagosomes containing RBC or IgG-opsonized particles in absence and in presence of the inhibitor PYR-41.