Mav temporarily resides in the phagolysosomal compartment.

<p>Human MDMs were infected with Mav-CFP (red) for 10 min followed by a chase for 5 minutes to 3 days, stained for EEA1 ((A), early endosomes, green) or LAMP1 ((D, E) late endosomes/lysosomes, green) using antibodies and for nuclei using Hoechst (red), and analyzed by confocal microscopy at the indicated time points. Single labeling (left and middle images) and merged images (right images) are shown. Bottom-to-top projections of 3D-stacks from boxed areas are shown in lower panels (left to right) and represent Mav, EEA1/LAMP1⁺ membranes and merged images. Quantification of Mav localization in EEA1⁺ or LAMP1⁺ compartments was performed on 3D stacks using fluorescence intensity profiles along the indicated line (<i>FAL</i>, (B) and (F), Mav: red line; EEA1/LAMP1: green line). For each time point, at least 70 cells were recorded per donor. Quantification graphs represent mean value +/- SEM of Mav localization in EEA1⁺ (C) or LAMP1⁺ (G) compartments for 3 donors. <i>P</i> values were calculated using Fisher Exact Test (* <i>P</i> <0.05, ** <i>P</i> <0.01 and *** <i>P</i> <0.005). a.u.: arbitrary unit. Scale bar represents 10 μm.</p

TLR7/8/MyD88 regulate intracellular growth of Mav in human primary macrophages.

<p>Human MDMs were treated with siRNA against MyD88, TLR7, TLR8 or a non-targeting control before infection with Mav-CFP (10 min uptake followed by 4h to 3d chase). (A) Representative images where infected cells treated with siNTC (left) or siTLR8 (right) are outlined for analysis (Mav-CFP: red, nuclei: blue (Hoechst)). (B, C) <i>In situ</i> quantification of Mav-CFP 4 hours (B) and 3 days (C) post infection using the Corrected Total Cell Fluorescence method on infected cells. Quantification scatter plots show individual cell measures with mean +/- 95% confident intervals from 3 donors ((B, C) left graphs, n>250 cells per time point and per donor). The bar chart in (B) shows the percentage of infected cells 4 hours post infection to compare uptake. (D) Working model. Mav (dashed line, blue) is processed in LAMP1⁺ phagolysosomes. Release of mycobacterial nucleic acids engages TLR7/8, recruitment of MyD88 to the compartment, and inflammatory signaling culminating in cytokine release and growth restriction. A fraction of live Mav actively remodels the phagolysosome and/or is sorted into a new compartment (MavC) where late endosomal/lysosomal markers are excluded, TLR7/8 are not engaged and MyD88 is not recruited, thus avoiding inflammatory signaling and destruction. The program leading to Mav destruction (red line) remains to be elucidated.</p

TNF production coincides with phagolysosomal localization of Mav.

<p>Human MDMs were infected with live Mav-CFP (blue) for 10 min, chased for 4h in the presence of a protein secretion inhibitor to accumulate cytokines, fixed and stained with antibodies to LAMP1 (red) and TNF (green). (A) Single and merged images of one cell; bottom-to-top projections of 3D-stack from boxed area in lower panels show that Mav is in a LAMP1⁺ compartment. (B) Fluorescence intensity profiles along the indicated lines of the Mav phagosome (Mav: blue trace; LAMP1: red trace). (C) Quantification of the percentage of infected cells secreting TNF (black bar), and of these, the fraction with Mav in LAMP1⁺ compartments (red bar) or LAMP1^- compartments (blue bar). Quantification graphs represent the mean value +/- SEM from two different experiments. (D) Human MDMs were treated with siMyD88, siTLR8 or non-targeted control before infection with Mav for 10 min and chase for 4h. Cells were then stained with antibody to MyD88 and analyzed using confocal microscopy for recruitment of MyD88 to Mav phagosomes. Quantification graph represents MyD88-Mav association from two independent experiments. Scale bar represents 10 μm.</p

Activation of IRF-1 coincides with phagolysosomal localization of Mav.

<p>Human MDMs were infected with live or PFA-killed Mav-CFP (red) for 10 min, chased for 4h to 3d and stained with antibodies to IRF-1 (blue) and LAMP1 (green) before analysis of MDMs containing only single Mav using confocal microscopy. Nuclei were revealed by staining with Hoechst (red). Single and merged images are shown; bottom-to-top projections of 3D-stacks from boxed areas in lower panels represent Mav, LAMP1 and merged images (A, C). (A) Cell with live Mav in LAMP1⁺ compartment inducing nuclear translocation of IRF-1. (C) Cell with live Mav in LAMP1^- compartment and no nuclear translocation of IRF-1. (B, D) Quantification of live Mav localization in LAMP1⁺ compartments and nuclear localization of IRF-1 was performed on 3D stacks using fluorescence intensity profiles along the indicated lines of Mav phagosomes ((B) and (D), left (Mav: red trace; LAMP1: green trace) and right (Hoechst: red trace; IRF-1: green trace) graphs respectively). (E) The fraction of live Mav localized to LAMP1⁺ phagolysosomes in IRF-1-activated cells (mean percentage +/- SEM from 3 donors). For each time point, at least 70 cells were recorded for each donor. (F) IRF-1 nuclear translocation at indicated time points after challenge with live or PFA-killed Mav. Quantification graphs represent the mean value +/- SEM for each time point for live Mav (black bars) and PFA-killed Mav (red bars) (n>600 cells per time point and per donor, 3 donors). <i>P</i> values between live Mav and PFA-killed Mav were calculated using two-tailed t-test (* <i>P</i> <0.05, ** <i>P</i> <0.01 and *** <i>P</i> <0.005.). a.u.: arbitrary unit. Scale bar represents 10 μm.</p

MavCs, but not MavPLs, support Mav replication.

<p>Human MDMs were infected with Mav-CFP for 10 min, chased for 5 min to 3d, fixed with 4% PFA and analyzed by confocal microscopy. <i>In situ</i> quantification of the actual number of Mav inside (left panel) or outside (right panel) LAMP1⁺ compartments at different time points, including the mean value +/- 95% confidence intervals.</p

Lysosomal-Associated Transmembrane Protein 5 (LAPTM5) Is a Molecular Partner of CD1e

<div><p>The CD1e protein participates in the presentation of lipid antigens in dendritic cells. Its transmembrane precursor is transported to lysosomes where it is cleaved into an active soluble form. In the presence of bafilomycin, which inhibits vacuolar ATPase and consequently the acidification of endosomal compartments, CD1e associates with a 27 kD protein. In this work, we identified this molecular partner as LAPTM5. The latter protein and CD1e colocalize in trans-Golgi and late endosomal compartments. The quantity of LAPTM5/CD1e complexes increases when the cells are treated with bafilomycin, probably due to the protection of LAPTM5 from lysosomal proteases. Moreover, we could demonstrate that LAPTM5/CD1e association occurs under physiological conditions. Although LAPTM5 was previously shown to act as a platform recruiting ubiquitin ligases and facilitating the transport of receptors to lysosomes, we found no evidence that LATPM5 controls either CD1e ubiquitination or the generation of soluble lysosomal CD1e proteins. Notwithstanding these last observations, the interaction of LAPTM5 with CD1e and their colocalization in antigen processing compartments both suggest that LAPTM5 might influence the role of CD1e in the presentation of lipid antigens.</p> </div

Melanoma cells express LAPTM5 transcripts.

<p>RNA samples from HeLa and HEK293 cells and from the melanoma cell lines M10 and FO-1 were analyzed by RT-PCR for the expression of LAPTM5; (−) control reaction without reverse transcriptase, (+) RT-PCR assays. Beta-actin- RT-PCR assays were performed for control experiments.</p

LAPTM5 co-immunoprecipitates with CD1e.

<p>Lysates of untransfected (NT) or transfected M10 cells expressing CD1e alone (CD1e), EYFP- or V5-tagged LAPTM5 alone (YFP-LA or LA-V5), or co-expressing CD1e and EYFP- or V5-tagged LAPTM5 (CD1e YFP-LA or CD1e LA-V5), untreated (−Baf) or treated (+Baf) with bafilomycin, were used in the following two experiments. A) Proteins were immunoprecipitated from cell lysates with the anti-CD1e mAb 20.6 and analyzed by western blotting using a rabbit anti-GFP Ab or an anti-V5 mAb. B) Cell lysates were immunoprecipitated with anti-GFP or anti-V5 Abs and analyzed by western blotting using the anti-CD1e mAb VIIC7. C) V5-tagged LAPTM5 competes with endogenous LAPTM5 for CD1e binding. Transfected M10 cells expressing CD1e alone (CD1e), or CD1e and LAPTM5-V5 (CD1e LA-V5), were metabolically labeled and chased in the absence or presence of bafilomycin. CD1e molecules were immunoprecipitated with the mAb 20.6, treated with Endo F and analyzed by SDS-PAGE. The asterisk in the left upper panel indicates a non specifically immunoadsorbed protein; mCD1e and sCD1e represent membrane-associated and soluble CD1e molecules, respectively. D) Bafilomycin protects EYFP-LAPTM5 from proteolysis. Transfected M10 cells expressing CD1e and the EYFP-LAPTM5 fusion protein were pulse chase labeled in the absence (−Baf) or presence (+Baf) of bafilomycin. Fusion proteins were immunoprecipitated with anti-GFP Abs.</p