Figure 1
Sortilin is recruited early into mycobacterial phagosomes via GGAs/AP-1 and retromer interactions.
(A) RAW 267.4 macrophages were infected with M. bovis BCG-DsRed (2 h uptake and 1 h chase) and stained for sortilin. Panel b: 3D-colocalization plot from inset in panel a. Panel c: profile of fluorescence intensity of a phagosome (white bar in inset in panel a). (B) Fluorescence intensity of endogenous sortilin associated with BCG phagosomes from 3 independent experiments. (C) BMM were infected with M. tuberculosis-EGFP (2 h uptake and 1 h chase) and stained for endogenous sortilin. Panel b: 3D-colocalization plot obtained from inset in panel a. Panel c: profile of fluorescence intensity of a representative phagosome (white bar in inset in panel a). (D) Fluorescence intensity of endogenous sortilin associated with M. tuberculosis phagosomes from two independent experiments. (E) THP-1 cells were infected with M. bovis BCG-DsRed (2 h uptake and 1 h chase) and stained for endogenous sortilin. Panel b: 3D-colocalization plot from inset in panel a. Panel c: profile of fluorescence intensity along the phagosome (white bar in inset in panel a). (F) Fluorescence intensity of endogenous sortilin associated with BCG phagosomes from two independent experiments. (G) Analysis by live cell imaging of association of Sort/WT-EYFP with phagosomes in RAW 267.4 macrophages. Panel a shows the profile of fluorescence intensity along the phagosome (white bar panel G). Panel b: 3D-colocalization plot from inset of panel G. (H) Quantitative analysis of the association of sortilin with BCG phagosomes (n = 5). The blue curve is the mean of the phagosomes analyzed and the light dashed blue curves represent the S.E.M. (I) Schematic representation of the sortilin trafficking affected by the mutations in the cytosolic tail. (J) RAW 267.4 macrophages expressing the indicated mutants were infected with BCG-DsRed (2 h uptake and 1 h chase), fixed and stained for sortilin. Scale bars: 10 μm. Data represents the Mean ± S.E.M of three independent experiments, at least 100 cells were analysed. (**)p ≤ 0.01 from one-way ANOVA with Tukey’s post hoc test.
