Chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion

Abstract

Macroautophagy/autophagy is a conserved transport pathway where targeted structures are sequestered by phagophores, which mature into autophagosomes, and then delivered into lysosomes for degradation. Autophagy is involved in the pathophysiology of numerous diseases and its modulation is beneficial for the outcome of numerous specific diseases. Several lysosomal inhibitors such as bafilomycin A1 (BafA1), protease inhibitors and chloroquine (CQ), have been used interchangeably to block autophagy in in vitro experiments assuming that they all primarily block lysosomal degradation. Among them, only CQ and its derivate hydroxychloroquine (HCQ) are FDA-approved drugs and are thus currently the principal compounds used in clinical trials aimed to treat tumors through autophagy inhibition. However, the precise mechanism of how CQ blocks autophagy remains to be firmly demonstrated. In this study, we focus on how CQ inhibits autophagy and directly compare its effects to those of BafA1. We show that CQ mainly inhibits autophagy by impairing autophagosome fusion with lysosomes rather than by affecting the acidity and/or degradative activity of this organelle. Furthermore, CQ induces an autophagy-independent severe disorganization of the Golgi and endo-lysosomal systems, which might contribute to the fusion impairment. Strikingly, HCQ-treated mice also show a Golgi disorganization in kidney and intestinal tissues. Altogether, our data reveal that CQ and HCQ are not bona fide surrogates for other types of late stage lysosomal inhibitors for in vivo experiments. Moreover, the multiple cellular alterations caused by CQ and HCQ call for caution when interpreting results obtained by blocking autophagy with this drug.

Keywords: Autophagy; Golgi; bafilomycin A1; degradative compartments; fusion; lysosomal degradation; lysosomal inhibitors.

Figures

Figure 1.

Quantitative automated fluorescence microscopy analysis revealed significant major differences between BafA1 and CQ treatments on DGCs. U2OS cells were treated with the vector (ctrl/0 h), 100 µM CQ or 100 nM BafA1 for 5 h, or in a time course manner between 0 and 5 h, before processing for immunofluorescence microscopy. Images were acquired and analyzed automatically using the Cellomics Arrayscan. (A) Staining of the preparations with anti-LAMP1 antibodies. (B) Quantification of the LAMP1 puncta area per cell (arbitrary units) from the immunofluorescence images such as for the examples shown in panel A. (C) Cells treated for the indicated times, were incubated with LysoTracker Red for 1 h before being processed for fluorescence microscopy. (D) Quantification of the LAMP1 puncta area per cell (arbitrary units) from images such as the examples depicted in panel C. All data are presented relative to the control at 0 h (fold). Error bars represent standard deviations (SD) of 3 independent experiments. * or ** symbols indicate significant differences of p < 0.05 and p < 0.01, respectively. Scale bars: 20 µm.

Figure 2.

Quantitative EM analysis highlights the morphological differences in the DGCs induced by CQ and BafA1. U2OS cells were treated with the vector (water; ctrl), 100 µM CQ or 100 nM BafA1 for 5 h, before processing for EM as described in Materials and Methods. (A) Representative images of the observed autophagosomes (AP). Scale bars: 250 nm. (B) Quantification of the number of AP per cell section. (C) Representative image of DGCs detected in the preparations. Enlargements of the insets highlighted with a white square in the image on left row, are shown in the right row. Arrow highlights DGCs. Scale bar: 1 µm. (D) Statistical evaluation of the number of DGCs per cell section, which were subdivided in regular (DGCs, i.e. lysosomes, amphisomes and autolysosomes) and large vacuolar DGCs (vDGCs). EM preparations were quantified as described in Materials and Methods. M, mitochondria; ER, endoplasmic reticulum; N, nucleus; PM, plasma membrane; DGCs, regular degradative compartments; vDGCs, vacuolar degradative compartments.

Figure 3.

CQ but not BafA1, has a significant impact on the endolysosomal system and the Golgi complex. U2OS cells were exposed to the vector (ctrl/0 h), 100 µM CQ or 100 nM BafA1 for 5 h or in a time-course manner between 0 and 5 h, before processing for immunofluorescence microcopy, and being automatically imaged and analyzed. Subcellular distribution of EEA1 (A), TGOLN2 (B), GOLGA2 (C) and M6PR (D), which was quantified by determining either the puncta area per cell (arbitrary units) (A) or the number of puncta per cell (B-D). All data are presented relative to the control at 0 h (fold). Error bars represent SD of 3 (A and D), 5 (B) or 4 (C) independent experiments. Images of panels A, B and D were acquired using the Cellomics Arrayscan and those of panel C using the TissueFAXS. Symbols *, ** and *** indicate significant differences of p < 0.05, p < 0.01 and p < 0.001. Scale bars: 20 µm.

Figure 4.

HCQ alters the Golgi organization in kidney and intestinal cells of treated mice. C57BL/6JOlaHsd mice were injected daily with 60 mg/kg HCQ or with saline solution (ctrl) and sacrificed 24 h after the first injection (24 h) or 24 h after the second injection (48 h). Representative images of kidney cells (A) and intestinal cells (B) stained for GOLGA2 and LC3 from one mouse are shown (images from the second and third mouse of the same groups are displayed in Figure S6). Scale bars: 10 µm.

Figure 5.

CQ impairs endocytosis-mediated degradation of EGFR. (A-B) Hela cells were exposed to 100 µM CQ or 100 nM BafA1 for 2 h, or left untreated, before being incubated with 50 ng/ml of Alexa Fluor 555-conjugated EGF from 0 to 60 min. Cells were finally processed for immunofluorescence microscopy and stained with anti-LAMP2 antibodies (A). (B) Quantification of the colocalization between EGF-labeled EGFR and LAMP2 puncta in the experiment shown in panel A. (C-D) Hela cells were exposed to 100 µM CQ or 100 nM BafA1 for 2 h, or left untreated, before being incubated with 50 ng/ml EGF from 0 to 120 min. Cells were finally lysed and protein resolved by western blot and membranes were probed with anti- EGFR (C) or anti-phospho EGFR (Y1068) (D) and anti-tubulin or anti-actin antibodies. Signals were quantified and normalized to TUBA4A/tubulin or actin (arbitrary units). Samples in C and D were probed on the same gel, and therefore the actin bands in C and D are identical. Data in panel C are presented relative to the control at 0 min (fold) and data in panel D are presented relative to the control at 5 min (fold). Error bars represent the SD of 3 independent experiments. Symbols *, ** and *** indicate significant differences of p < 0.05, p < 0.01 and p < 0.001, with control cells at the same time point. Scale bars: 10 µm.

Figure 6.

CQ does not impair endocytosis and endo-lysosomal trafficking of BSA but causes vacuolization of lysosomes. (A) U2OS cells were exposed to 100 µM CQ or 100 nM BafA1 for 2 h or left untreated, before being incubated with 375 nM BSA-TRITC (BSA-T) for 30 min. The cells were then washed and further incubated in the same medium with 100 µM CQ or 100 nM BafA1 and without BSA-TRITC for 90 min. Finally, cells were processed for immunofluorescence microscopy and stained with anti-LAMP2 antibodies. The white arrow indicates large BSA-TRITC-positive LAMP2 puncta. (B) Quantification of the colocalization between BSA-TRITC (BSA-T) and LAMP2 puncta in the experiment shown in panel A. (C) Determination of the average size of the BSA-TRITC (BSA-T)-, LAMP2- and BSA-TRITC/LAMP2 (coloc)-positive puncta (arbitrary units) in the experiment shown in panel A. (D) U2OS cells were incubated with 375 nM BSA-TRITC (BSA-T) for 30 min, washed and further incubated in medium without BSA-TRITC for 90 min before being exposed to 100 µM CQ or 100 nM BafA1 for 5 h, or left untreated. Cells were subsequently prepared for immunofluorescence microscopy and labeled with anti-LAMP2 antibodies. The white arrow indicates large BSA-TRITC-positive LAMP2 puncta. (E) Quantification of the colocalization between BSA-TRITC (BSA-T) and LAMP2 puncta in the experiment shown in panel D. (F) Determination of the average size of the BSA-TRITC (BSA-T)-, LAMP2- and BSA-TRITC/LAMP2 (coloc)-positive puncta (arbitrary units) in the experiment shown in panel D. All images were acquired using the DeltaVision microscope. Data in panels C and F are presented relative to the control (folds). Error bars represent SD of 3 independent experiments. Symbols * and ** indicate significant differences of p < 0.05 and p < 0.01, respectively. Scale bars: 10 µm.

Figure 7.

CQ treatment inhibits the autophagic flux. U2OS cells were treated with 100 µM CQ or 100 nM BafA1 for 5 h or with 50 µM CQ and 100 nM BafA1, for 24 h (A), or with 100 µM CQ or 100 nM BafA1 for 2 h (B), individually or in combination; controls were untreated cells. Cells were finally lysed and protein resolved by western blot and membranes were probed with anti-LC3 and anti-TUBA4A/tubulin antibodies. Signals were quantified and normalized to TUBA4A/tubulin (a.u., arbitrary units). (C) U2OS cells were exposed to 100 nM BafA1, 100 µM CQ, 100 nM torin1 or 10 µM SAR-405 (SAR) for 2 h or 5 h, or to 20 nM BafA1, 50 µM CQ, 4 µM SAR-405 for 24 h, as indicated, before the LDH sequestration assay was performed as described in Materials and Methods. (D) The long-lived protein turnover assay was carried out in U2OS cells treated with 100 nM BafA1 and/or 100 µM CQ for 2 h and 5 h, or to 20 nM BafA1 and/or 50 µM CQ for 24 h, as indicated, and following the protocol described in Materials and Methods. Error bars represent SD of 3 (A, C and D) or 5 (B) independent experiments. When not otherwise indicated, the statistical significances were calculated to the controls for each time point in panel C and D. The symbols *, ** and *** indicate significant differences of p < 0.05, p < 0.01 and p < 0.001, respectively.

Figure 8.

CQ treatment inhibits autophagosome-lysosome fusion without losing lysosomal acidity. RFP-GFP-LC3 HeLa cells were treated with 100 µM CQ, 100 nM BafA1, or a combination of BafA1 and CQ (B+ CQ) for 5 h or left untreated (ctrl). Cells were subsequently prepared for immunofluorescence microscopy and stained with an anti-LAMP2 antibody. (A) Representative images are shown. Image stacks acquired with the DeltaVision microscope were analyzed using the Icy software and the number of all RFP-positive puncta (the LC3 populations in the cytoplasm and in the lysosomes) (B) and the percentage of LAMP2-positive RFP-LC3 puncta (i.e., the population of LC3 in the lysosomes) (C) was determined. Data in panels B and C are presented relative to the control (folds). Error bars represent SD of 3 independent experiments. The symbols *, ** and *** indicate significant differences of p < 0.05, p < 0.01 and p < 0.001, respectively. Scale bars: 10 µm.

Figure 9.

CQ treatment blocks fusion of SQSTM1-positive autophagosomes with lysosomes and leads to the accumulation of STX17 puncta. (A-C) U2OS cells were treated with 100 µM CQ and 100 nM BafA1 for 5 h, or with 50 µM CQ and 100 nM BafA1 for 24 h, individually or in combination, or left untreated (ctrl/0 h). Cells were finally processed for fluorescence microscopy and simultaneously labeled with antibodies against SQSTM1 and LAMP2, and images were acquired using the DeltaVision microscope. (A) Representative images of the 24-h time point are shown. Green, red and yellow arrows highlight SQSTM1-negative LAMP2 puncta, LAMP2-negative SQSTM1 puncta and SQSTM1-positive LAMP2 puncta, respectively. (B) The percentage of SQSTM1 puncta that colocalize with LAMP2 puncta was determined. (C) The percentage of SQSTM1 puncta that do not colocalize with LAMP2 puncta was determined. (D-F) GFP-STX17 MEFs were treated with 100 µM CQ or 100 nM BafA1 for 5 h, or left untreated (ctrl). Cells were finally processed for fluorescence microscopy and labeled with antibodies against LC3, before acquiring images using the DeltaVision microscope. (D) Representative images and insets with a magnified area are shown. (E) The percentage of STX17 puncta that colocalize with LC3 puncta was determined and expressed relative to the control (fold). Error bars represent SD of 3 independent experiments. The symbols *, ** and *** indicate significant differences of p < 0.05, p < 0.01 and p < 0.001, respectively. Scale bars: 10 µm.