© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd doi:10.1111/tra.12143

Rab7 Is Functionally Required for Selective Cargo Sorting at the Early Endosome Emmanuelle Girard1,2,3,4 , Daniela Chmiest3,4 , Natalie Fournier1,2 , Ludger Johannes3,4 , Jean-Louis Paul1,2 , Benoˆıt Vedie1,2,† and Christophe Lamaze3,4∗,† 1

´ Georges Pompidou, Service de Biochimie, 75015 Paris, France AP-HP (Assistance Publique – Hôpitaux de Paris), Hôpital Europeen Universite´ Paris-Sud, EA 4529, UFR de Pharmacie, 92296 Châtenay-Malabry, France 3 CNRS UMR144, 75248 Paris cedex 05, France 4 Institut Curie, Centre de Recherche, Laboratoire Trafic, Signalisation et Ciblage Intracellulaires, 75248 Paris Cedex 05, France 2

∗ Corresponding

author: Christophe Lamaze, [email protected]

Abstract The small GTPases of the Rab family act as a molecular switch regulating

receptor, the interferon alpha-receptor and the Shiga toxin B-subunit.

various aspects of membrane trafficking through the selective recruit-

In contrast, epidermal growth factor (EGF) sorting at the EE or the

ment of effector proteins. Whereas Rab7 has been classically involved

recycling of transferrin and LDL-R were not affected by Rab7 depletion.

in the regulation of transport within the endolysosomal network, per-

Our findings demonstrate that in addition to regulating late endosomes

sistent controversy remains as to whether Rab7 also plays a role in

(LE) to lysosomes transport, Rab7 plays a functional role in the selective

earlier steps of endosomal trafficking. In this study, we show that Rab7

sorting of distinct cargos at the EE and that the Rab5 to Rab7 exchange

depletion or inactivation results in enlargement of both early and late

occurs early in the endosomal maturation process.

endosomes. Rab7 depletion led to the retention of a significant fraction of internalized low-density lipoproteins (LDL) mainly in enlarged early

Keywords cholesterol, endocytosis, endosome, Rab7, trafficking

endosomes (EE). As a result, LDL processing and the transcriptional

Received 17 September 2013, revised and accepted for publication 4

regulation of sterol-sensitive genes were impaired. We found that Rab7

December 2013, uncorrected manuscript published online 10 December

activity was also required for the sorting of the mannose-6-phosphate

2013, published online 13 January 2014

The endocytic pathway is a complex and highly regulated system that supports trafficking of nutrients and signaling cargos within cells (1). Early endosomes (EE) represent a major sorting station controlling and coordinating distinct trafficking steps in a temporal and spatial manner (2,3). Recent progress has been made in the identification of new machineries regulating endosome biogenesis and dynamics (4,5). Among the variety of proteins that regulate endosomal transport, the small molecular weight Rab family GTPases play a key role as molecular switches between a GTP-bound active form and a GDP-bound inactive cytoplasmic state (6). More than 60 human Rabs have been identified that control various aspects of membrane

trafficking including vesicle budding, mobility, tethering and fusion along the exocytic and endocytic pathways. Rab7 stands as a critical player in endocytic trafficking as it controls several aspects of endosomal biogenesis and dynamics. Rab7 has been classically associated with the control of homotypic fusion between lysosomes and late endosomes (LE), LE/lysosomes mobility, and autophagic maturation (7–9). On the basis of these functions, Rab7 is classically used as a bona fide marker of LE. It was recently shown that the replacement of Rab5 by Rab7 is key during the maturation process that transforms EE into LE (10,11). Yet, to date the function of Rab7 in the endocytic pathway remains controversial. If most studies established that Rab7 activity is required at late steps when cargo is transported from LE to lysosomes (12,13), some studies have suggested

†

These authors contributed equally to this work.

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an earlier role of Rab7 in the control of cargo trafficking from EE to LE (14–16). Here we analyzed the role of Rab7 in the trafficking of distinct cargos that are sorted along the endocytic pathway. By studying low-density lipoproteins (LDL) intracellular transport and processing, we could functionally link the role of Rab7 in endosomal sorting to the transcriptional control of cholesterol homeostasis. Under Rab7 inactivation or depletion, we observed an enlargement of both EE and LE. The enlargement of LE was increased by the simultaneous depletion of Rab9 in agreement with the established role of Rab7 and Rab9 in late endosomal trafficking. More importantly, we found that Rab7 depletion led to the retention, mostly in EE, of a significant fraction of internalized LDL. As a result, the processing of LDL into free cholesterol (FC) and its delivery to the endoplasmic reticulum (ER) were impaired and the regulation of sterol-sensitive gene transcription was inefficient. Furthermore, we could show that Rab7 was also required for the selective sorting of several other cargos at the EE. Altogether our results demonstrate that Rab7 plays a functional role in early endosomal sorting in a cargo-specific manner and that the Rab5 to Rab7 exchange occurs at an early stage in the endosomal maturation process.

Results Loss of Rab7 activity impairs the intracellular distribution of FC and internalized LDL LDL uptake occurs through clathrin-dependent endocytosis of the LDL-R (17) and delivers cholesterol esters (CE) first to EE and then to endolysosomal compartments, where they are hydrolyzed by acid lipase into FC. To better characterize the role of the small GTPases Rab7 and Rab9 in LDL endosomal trafficking, we depleted endogenous Rab7 and Rab9 using specific siRNAs. Transfection of siRNAs directed against Rab7 and Rab9 led to an almost complete depletion of mRNAs as assessed by quantitative real-time polymerase chain reaction (PCR) (Figure 1A). Rab7 siRNA treatment also led to a 40% decrease of Rab9 mRNA levels. These results were confirmed at the level of protein expression by western blot analysis (Figure 1B). We first analyzed the intracellular distribution of internalized LDL and FC in HeLa cells in control and knock-down conditions. After 3 h of continuous uptake of 310

fluorescently labeled LDL (DiI-LDL), Rab7 silencing led to the presence of LDL in enlarged endosomes positive for Lamp1 (Figure 1C, upper right, and Figure S1A, Supporting Information). Rab9 silencing had no visible effect whereas simultaneous silencing of Rab7 and Rab9 accentuated the Rab7 depletion phenotype leading to the presence of LDL in fewer but larger, more rounded endosomes (Figure 1C, lower right). Next we stained cells with filipin, a naturally fluorescent antibiotic that selectively binds to FC. We also observed abnormally enlarged endosomal structures loaded with FC upon Rab7 silencing (Figure 1D, upper right). Again Rab9 depletion alone had no visible effect while silencing of both Rab7 and Rab9 reduced the number and increased the size of endosomes loaded with FC. Comparable results were obtained after 24 h of LDL uptake (unpublished data). To further confirm the role of Rab7 activity in this process, we expressed constitutively activated or inactivated mutants of GFP-tagged Rab7. As observed for the knock-down of Rab7, internalized LDL and FC were also found in enlarged endosomal structures (Figure 1E,F, lower panel) in cells expressing the inactive GDP-bound Rab7 mutant (GFP-Rab7-T22N). In contrast, in cells expressing the activated GTP-bound Rab7 mutant (GFP-Rab7-Q67L), LDL and FC were distributed in endosomes that were similar to control cells. We further characterized the enlarged endosomal compartments with bona fide markers of EE and LE. We performed these experiments in the absence of added LDL to rule out possible alterations of endosomes by cholesterol loading (18). Lamp1 staining revealed that LE/lysosomes were enlarged (Figure 2A, arrows) and more dispersed in Rab7 silenced cells in agreement with published data (16). EEA1 staining also indicated the presence of enlarged EE as well (Figure 2A, arrowheads) suggesting that Rab7 depletion also affects early steps of endosomal trafficking. This is in agreement with former studies in Caenorhabditis elegans showing the enlargement of both early and late endosomes when Rab7 was silenced (19). These results were further confirmed by the expression of the Rab7 constitutively inactive mutant. Indeed, in cells expressing GFP-Rab7-T22N, both EE (arrowheads) and LE (arrows) were enlarged (Figure 2B). Importantly, we could rule out an overall unspecific perturbation of the endosomal network since we observed no colocalization between EEA1 and Lamp1, an indication that EE and LE remain molecularly distinct. Accordingly, we found Traffic 2014; 15: 309–326

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that the enlarged LE were also positive for Lysotracker (Figure 2C, arrowheads), indicating that these endosomes were indeed still able to be acidified. Thus, Rab7 depletion and inactivation impair the intracellular distribution of FC and internalized LDL, and results in the formation of abnormally enlarged EE and LE. Rab7 down-expression results in LDL retention in the early endosome Rab7 is classically involved in the regulation of intracellular trafficking between LE and lysosomes (7–9). However, in a few studies, Rab7 has also been localized on EE suggesting a role at this level (10,14,16). We therefore quantified the respective distribution of internalized LDL between early and late endosomes. After 3 h of LDL uptake, we found that internalized LDL was mostly distributed (73 ± 6%) in the late endosomal network positive for Lamp1 (Figure 2D, left panel, arrowheads). In cells silenced for Rab7, internalized LDL was distributed to the same extent (70 ± 3%) in enlarged Lamp1-positive endosomes (Figure 2D, middle panel, arrows). However, a significant fraction of internalized LDL (42 ± 5%) still remained in enlarged EEA1-positive endosomes (Figure 2D, middle panel, arrowheads). Fewer internalized LDL was present in EE at this stage in control cells (14 ± 2%), ***p < 0.0001 (Student’s t-test). Although early and late endosomal markers were mostly separated, a small portion (≈10%) of internalized LDL was localized to both EEA1 and Lamp1 endosomes in Rab7-depleted cells. These characteristics were more easily observable in Rab7/Rab9 silenced cells because of the larger endosome size induced by this condition (Figure 2D, right panel, arrows and arrowheads). Taken together these results confirm that Rab7 depletion or inactivation impairs late endosomal trafficking, as shown

by the enlargement of LE, an effect that has been previously reported by several investigators. More importantly, our data indicates that Rab7 can also serve to control trafficking events at the early endosomal level. Rab7 is required for selective cargo sorting at the early endosome Our results indicate that Rab7 may play a role in LDL trafficking from the EE. Therefore, we examined the effect of Rab7 depletion at earlier steps of endocytosis by analyzing a single wave of LDL uptake. After 10 min of internalization in control cells, LDL was detected in small and disperse endosomes that were typical of EE as confirmed by EEA1 positive staining (Figure 3A, arrows). After 40 min of uptake, LDL was transported to late endosomal structures characterized by a perinuclear localization and a positive staining for Lamp1 (Figure 3D, arrows). Sixty minutes after endocytosis, LDL localized exclusively to LE/lysosomes and was no longer detected in EEA1 positive endosomes (Figure 3E,F). Under Rab7 silencing, LDL initially followed similar kinetics of uptake and displayed the same intracellular distribution. However, after 10 min, we could already detect the presence of LDL in EEA1 positive endosomes of larger size than in control cells (Figure 3A, arrowheads). The fraction of LDL present in enlarged EE increased after 40 min of endocytosis (Figure 3C, arrowheads). At 60 min, an important fraction (53 ± 7%) of internalized LDL was still abnormally present in EE (Figure 3E, arrowheads), in contrast to normal cells (13 ± 4%), ***p < 0.0001 (Student’s t-test). We also detected internalized LDL (83 ± 4%) in Lamp1 positive endosomes after 60 min of endocytosis (Figure 3 F, arrowheads) indicating that endosomal trafficking was not totally blocked in Rab7

Figure 1: Loss of Rab7 activity impairs the intracellular distribution of FC and internalized LDL. A and B) HeLa cells were silenced for Rab7 and/or Rab9 during 48 h as indicated. A) Cells were lysed and analyzed by quantitative real-time PCR for Rab7 and Rab9 mRNAs. Values of siCtl were arbitrarily set as 1, against which experimental data were normalized. The results are expressed as means ± SD (error bars). n = 3. ***p < 0.0001 (Student’s t -test). B) Efficiency of knock-down was analyzed by immunoblotting with Rab7, Rab9 and actin (loading control) antibodies. The blot shows a representative experiment. n = 3. C) HeLa cells were transfected with the indicated siRNAs for 48 h, incubated for 3 h with 200 μg/mL DiI-LDL, and fixed. D) HeLa cells transfected with the indicated siRNAs for 48 h were loaded with 200 μg/mL LDL for 3 h, and stained with filipin. Images are representative of at least three independent experiments. Scale bar, 10 μm. E and F) HeLa cells were transfected for 48 h with plasmids encoding GFP-tagged wild type, active or inactive mutated forms of Rab7 as indicated. E) Cells were loaded for 3 h with 200 μg/mL DiI-LDL before fixation. F) Cells were incubated for 3 h with 200 μg/mL LDL and stained with filipin. Scale bar, 10 μm. n = 2. 312

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Figure 2: Rab7 down-expression leads to LDL retention in the early endosome. A) HeLa cells were transfected with Rab7 or control siRNA for 48 h, and processed for double-labeling with antibodies to EEA1 and Lamp1. Images show enlarged EE (arrowheads) and LE (arrows) in Rab7-silenced cells. Scale bar, 10 μm. n = 4. B) HeLa cells were transfected with plasmids encoding GFP-Rab7-WT or GFP-Rab7-T22N. After 48 h, cells were fixed, permeabilized and immunostained for EEA1 and Lamp1. Images show enlarged EE (arrowheads) and LE (arrows) in cells expressing Rab7-T22N. Scale bar, 10 μm. n = 2. C) After 48 h of transfection with Rab7 or control siRNA, HeLa cells were incubated for 30 min with 200 nM Lysotracker DND-99 before fixation. Cells were stained with anti-Lamp1 antibody. Arrows show enlarged acidified LE after staining with Lysotracker. Data is representative of two independent experiments. Scale bar, 10 μm. D) HeLa cells were transfected with the indicated siRNAs for 48 h, incubated for 3 h with 200 μg/mL DiI-LDL, and fixed. Arrowheads show colocalization between DiI-LDL and the EE marker EEA1. Examples of colocalization between DiI-LDL and Lamp1 are indicated by arrows. Representative images are shown. The mean values ± SD of the Pearson’s correlation coefficient (PCC) of 15–20 cells from three independent experiments are shown. Scale bar, 10 μm. silenced cells. It is important to note that the percentage of LDL present in LE did not significantly differ between control (81 ± 4%) and Rab7 silenced cells (83 ± 4%), ns (Student’s t-test). These results further confirm the early requirement of Rab7 in LDL trafficking at the EE. Traffic 2014; 15: 309–326

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Figure 3: Rab7 knock-down impairs early endosomal LDL trafficking. HeLa cells were transfected with Rab7 or control siRNA for 48 h and incubated with 200 μg/mL DiI-LDL for 40 min at 4◦ C. Cells were washed, incubated at 37◦ C for the indicated times and fixed. Cells were processed for immunofluorescent labeling with anti-EEA1 or anti-Lamp1 antibodies. Images show LDL endocytosis after 10 min (A and B), 40 min (C and D) and 60 min (E and F). Arrowheads indicate enlarged endosomes containing DiI-LDL. The mean values ± SD of the Pearson’s correlation coefficient (PCC) of 15–20 cells from three independent experiments are shown. Scale bar, 10 μm. in lysosomes. We next asked whether the early requirement of Rab7 was specific to LDL sorting or whether it could be generalized to other cargos. We therefore examined the endosomal distribution of five different transmembrane receptors that classically traffic through and are sorted at the EE, namely the cation-independent mannose-6phosphate (CI-MPR), the LDL, the transferrin (Tfn-R), 314

the interferon-α chain 1 (IFNAR1) and the epidermal growth factor (EGF-R) receptors. Endosomal trafficking of the CI-MPR allows the retrieval of newly synthesized enzymes that have been posttranslationally modified by a mannose-6-phosphate tag in the trans-Golgi network (TGN) and are destined Traffic 2014; 15: 309–326

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to the lysosome (20). Lysosomal enzymes bound to the CI-MPR are first delivered to endosomes, where they are further transported along the endocytic pathway to lysosomes. The unoccupied CI-MPR is recycled back from endosomes to the TGN trough retrograde trafficking (21). Whether CI-MPR trafficking to the TGN occurs directly from EE or LE is still not clear. We first analyzed the intracellular distribution of the CI-MPR in HeLa cells stably expressing a GFP-tagged CI-MPR (22). In control cells, the CI-MPR was strictly localized at the TGN at steady state (Figure S2A). Upon Rab7 silencing, we observed that in addition to its TGN localization, CIMPR was also present in peripheral endosomes labeled for EEA1 (Figure 4A, arrowheads). These data were further confirmed by immunostaining of the endogenous CI-MPR (Figure S2B). These results indicate that Rab7 depletion blocks at least partially the endosome-to-Golgi retrieval of the CI-MPR, in agreement with recent studies investigating the sorting machinery at the EE (16,23). After LDL-R delivery to the EE, LDL is sorted to the LE network where LDL particles are degraded and CE de-esterified by acid lipase whereas the LDL-R recycles back to the plasma membrane. Thus, we monitored by cytofluorimetry the kinetics of LDL-R recycling to the plasma membrane. As expected, LDL-R recycling occurred quickly and reached a plateau after 20 min probably due to the re-internalization of the recycled LDL-R (Figure 4B). In Rab7 silenced cells, we observed a slight delay in receptor recycling with a minor impact on the overall extent of LDL-R recycling. Unlike LDL, Tfn (transferrin) is rapidly recycled with its receptor directly from the EE or more slowly after transfer to the recycling endosome (24). We found that the amount of Tfn-R present at the cell surface as measured by antibody staining or Tfn binding at 4◦ C was slightly reduced at steady state in Rab7 silenced cells (Figure 4C). These results indicate that Rab7 depletion has little effect on receptor recycling to the plasma membrane. In contrast to the LDL-R and Tfn-R that are endocytosed and recycled in a constitutive manner, most signaling receptors including receptor tyrosine kinases such as the EGF-R are sorted to late endosomal compartments for lysosomal degradation after their endocytosis (25). To further evaluate the respective importance of Rab7 in early versus late endosomal sorting, we analyzed its role in EGF Traffic 2014; 15: 309–326

(epidermal growth factor) endosomal sorting. In this case, we switched to RPE-1 cells as they are more responsive to growth factors than HeLa cells. Cells were pulsed with fluorescent EGF for 15 min at 37◦ C and the intracellular distribution of EGF was analyzed after different times of chase at 37◦ C. As reported in many studies, EGF was found in EE after 20 min of uptake in control cells (Figure S3C, arrows). At 40 min, EGF was no longer detected in EEA1 positive endosomes in agreement with its transfer to the late endosomal compartment for degradation (Figure 4D). Indeed, after 60 min of uptake, EGF was degraded and was barely detected intracellularly (Figure S3A,D,E). Under Rab7 silencing, EGF was found as expected after 20 min of uptake in EEA1 positive endosomes that were enlarged (Figure S3C). However quantification showed no difference between control and Rab7 silenced cells (74 ± 7% vs. 78 ± 5%), ns (Student’s t-test). At 40 min, colocalization with EEA1 was no longer detected. However, after 60 min of endocytosis, in contrast to control cells, EGF was not degraded and instead was blocked in endosomal structures. These endosomes were not positive for EEA1 or Lamp1 (Figure S3D,E). Similar results were obtained in HeLa cells (unpublished data). These results are in full agreement with previous studies showing that EGF-R is sequestered in the intraluminal vesicles of enlarged LE after Rab7 inactivation or depletion (12,13). Accordingly, we further demonstrate that EGF-R degradation is strongly delayed in Rab7 silenced cells (Figure S3B). There is growing evidence that distinct endosomal populations or subdomains mediate the sorting of different cargoes, particularly in the case of signaling receptors (26). Therefore, we examined the sorting characteristics of the interferon-α receptor IFNAR, a complex made of two transmembrane chains IFNAR1 and IFNAR2. IFNAR1, like the EGF-R, undergoes ubiquitin-dependent lysosomal degradation after clathrin-dependent endocytosis (27,28). We followed IFNAR1 uptake in RPE1 cells which express higher numbers of endogenous IFNAR than HeLa cells. After stimulation with IFN-β, IFNAR1 was blocked into enlarged early endosomes (76 ± 5%) after 40 min of endocytosis in Rab7 depleted cells (Figure 4E, arrowheads). In control cells, IFNAR1 was no longer associated with EE at this time (26 ± 4%), ***p < 0.0001 (Student’s t-test). We also observed a higher intensity of EEA1 staining, certainly reflecting the higher amount of activated Rab5 present 315

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Figure 4: Rab7 is required for selective cargo sorting at the early endosome. A) HeLa cells stably expressing GFP-tagged CI-MPR were transfected for 48 h with Rab7 or control siRNA. Cells were fixed and stained for EEA1. Colocalization of CI-MPR and EEA1 is indicated by arrowheads. Scale bar, 10 μm. n = 2. B) HeLa cells transfected with Rab7 or control siRNA for 48 h, were incubated with anti-LDL-R antibodies for 45 min at 37◦ C. LDL-R recycling to the plasma membrane was analyzed by flow cytofluorimetry after labeling with secondary antibodies. Results are expressed as the percentage of the amount of recycled LDL-R to the amount of internalized LDL-R. The results are expressed as means ± SD (error bars). n = 4. *p < 0.05 (Student’s t -test). C) After 48 h transfection with control or Rab7 siRNAs, HeLa cells were incubated at 4◦ C with 5 μg/mL Alexa Fluor 633-conjugated Tfn or antibodies directed against Tfn-R and specific secondary antibodies. The levels of Tfn and Tfn-R expression at the plasma membrane were measured by flow cytofluorimetry. The results are expressed as means ± SD (error bars). n = 3, *p < 0.05 (Student’s t -test). D) Control or Rab7 silenced RPE1 cells were pulsed for 15 min with 100 ng/mL Alexa Fluor 488-conjugated EGF. Cells were washed and chased for 40 min at 37◦ C before fixation. Cells were then permeabilized and stained with antibody against endogenous EEA1. EGF was present in enlarged EE (arrowheads). The mean values ± SD of the Pearson’s correlation coefficient (PCC) of 15–20 cells from two independent experiments are shown. Scale bar, 10 μm. E) Control or Rab7 silenced RPE1 cells were incubated for 40 min at 4◦ C with IFNAR1 antibodies. Cells were then incubated 40 min at 37◦ C in medium containing 1000 U/mL IFN β. Cells were fixed and stained with anti-IgG secondary antibodies and antibody against endogenous EEA1. Arrowheads indicate the presence of IFNAR1 blocked in enlarged EE. The mean values ± SD of the Pearson’s correlation coefficient (PCC) of 15–20 cells from two independent experiments are shown. Scale bar, 10 μm. 316

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on these endosomes (see Discussion). Altogether, these results indicate that Rab7 plays a role in EE sorting in a cargo-specific manner. Rab7-dependent endosomal sorting of LDL is required for the regulation of sterol-sensitive genes The study of LDL intracellular trafficking and processing provides the opportunity to functionally link the role of Rab7 on LDL sorting to the transcriptional control of cholesterol homeostasis. Indeed, the amount of FC that is delivered to the ER tightly regulates sterol-sensing genes that control cholesterol uptake and neosynthesis (8). FC, which results from the hydrolysis of LDL-derived CE in the LE/lysosomes, can reach several intracellular membranes, including recycling endosomes, mitochondria, ER and the plasma membrane. FC can be re-esterified into CE by the ER-resident acyl cholesterol acyl transferase (ACAT). Then, the measure of intracellular CE represents an indirect but valid estimation of the amount of FC transported from the endolysosomal network to the ER (29). However, the pool of CE that is initially provided by internalized LDL also contributes to the overall amount of intracellular CE. This pool can be selectively measured by pharmacological inhibition of ACAT activity. Cells were first grown in lipoprotein-deficient serum (LPDS) for 48 h to eliminate exogenous cholesterol and then loaded with LDL for 24 h. In control cells, the total amount of CE measured by HPLC represented about 40% of the total cholesterol mass of the cell (Figure 5A). Pharmacological inhibition of ACAT (black bars) showed that the majority of CE is produced by FC re-esterification at the ER in control cells. Rab7 silencing led to a significant reduction (60%) of CE production by ACAT (Figure 5A, grey bar) indicating that less FC was delivered to the ER for re-esterification. In addition, we noticed a significant increase of the ACATindependent pool of CE (black bar). This increase probably reflects the amount of CE present in LDL that was retained in EE (Figure 2D) and as a result could not be hydrolyzed in LE/lysosomes. In contrast to the synergistic effect of Rab7 and Rab9 on endosomal enlargement, we did not observe a greater decrease in CE production when both Rab proteins were down-modulated. This indicates that Rab9 does not control the delivery of FC to the ER, in agreement with the published role of Rab9 in regulating transport from the LE to the Golgi network (30,31). Rab9 depletion alone was not significantly different from the control condition. Traffic 2014; 15: 309–326

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Figure 5: Rab7-dependent LDL sorting is required for the regulation of sterol-sensitive genes. A) HeLa cells were transfected with the indicated siRNAs for 48 h, and incubated with 200 μg/mL LDL for 24 h. The amount of CE was quantified and expressed as the percent of the total amount of cellassociated cholesterol. The amount of ACAT-independent esters was measured in the presence of 10 μg/mL ACAT inhibitor (black bars). The results are expressed as means ± SD (error bars). n = 3, ***p < 0.0001, **p < 0.01 (Student’s t -test). B) HeLa cells were transfected with the indicated siRNAs for 48 h. When indicated, cells were loaded with 200 μg/mL LDL for 24 h. Relative HMGCoAR, LDL-R and SREBF-2 mRNA levels were measured by real-time PCR. The results are expressed as means ± SD (error bars). n = 4, ***p < 0.0001, **p < 0.01 (Student’s t -test). The intracellular amount of cholesterol is constantly monitored and adapted at the ER through transcriptional regulation of cholesterol uptake and neosynthesis. The amount of FC present at the ER fine tunes the activation of ER-resident sterol-regulatory element binding proteins (SREBPs) (32). Low levels of FC activate SREBP-2 (sterol-regulatory element binding protein 2), which in turn increases the expression of many genes involved in cholesterol homeostasis including hydroxymethylglutarylcoenzyme A reductase (HMGCoAR), HMGCoA synthase, squalene synthase and the LDL-R. Conversely, high levels of FC will exert a negative feedback on this regulation (33). We therefore examined if the loss of Rab7 317

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could affect the regulation of sterol-sensitive genes in response to variations in cholesterol concentration. As expected, the addition of exogenous LDL led to a consequent down-expression of LDL-R, HMGCoAR and SREBF-2, the three major sterol-sensitive genes (Figure 5B). In Rab7-silenced cells, we observed that HMGCoAR and LDL-R genes were two times less down regulated in response to the addition of LDL. We found that SREBF-2 expression was totally insensitive to the addition of LDL, in agreement with its central role in cholesterol transcriptional regulation. Silencing both Rab7 and Rab9 did not further impair sterol-sensitive genes downregulation and Rab9 silencing alone had no impact. These results are in agreement with the significant inhibition of CE production in Rab7 silenced cells and further confirm that Rab7 endosomal sorting activity is functionally required for the delivery of FC to the ER and the subsequent transcriptional control of cholesterol homeostasis. Down-expression of late endosomal Rab7 effectors has a minor effect on LDL sorting and FC delivery to the ER While there is consensus that Rab7 plays a role in late endosomal sorting, there has been persistent controversy as to whether Rab7 could also be involved in sorting at the EE. Our results indeed support a role for Rab7 in early endosomal sorting albeit in a cargo-specific manner. Nevertheless, we sought to strengthen these data by measuring the functional impact of LDL sorting at the EE on cholesterol homeostasis. For this, we took advantage of the possibility to measure the processing of internalized LDL (i.e. CE hydrolysis in LE and FC re-esterification at the ER) to evaluate the functional contribution of Rab7 in early versus late endosomal sorting. Thus, we analyzed the role of different Rab7 effectors that are known to be present on EE or LE. We studied vacuolar associated-membrane protein 7 (VAMP7), a SNARE protein that interacts with Rab7 and mediates heterotypic fusion between LE and lysosomes (34). We also chose vacuolar protein sorting 26 (Vps26), a member of the retromer complex that interacts with Rab7 at the EE through the Vps35 subunit, and is involved in the control of retrograde transport of transmembrane proteins from EE to the TGN (35). As a control, we used syntaxin 16 (STX16), a TGN-localized t-SNARE that regulates retrograde transport from the EE to the TGN but does not interact with Rab7 (36). We could efficiently down-modulate the three effectors by RNAi treatment as shown by western 318

blotting (Figure 6E). To further confirm the selectivity of Vps26 and STX16 silencing, we followed the intracellular distribution of the Shiga toxin B-subunit (STxB), a bona fide cargo of retrograde transport between EE and the TGN (37). In agreement with published data, we found that after 45 min of endocytosis, the arrival of STxB to the TGN was strongly impaired in cells silenced for Vps26 and STX16 (Figure 6A)(16,22). The intracellular distribution of the CI-MPR, which also traffics via retrograde transport to the TGN, was similarly affected (Figure 6B) (16,38). Consistent with the findings that Rab7 controls the sorting of CI-MPR at the EE (Figure 4A), STxB sorting to the TGN was also impaired by Rab7 depletion, further supporting a role in EE sorting (unpublished data). In agreement with the established role of VAMP7 in late endosomal transport (39), VAMP7 silencing did not prevent the transport of STxB and the CI-MPR from the EE to the TGN (Figure 6A,B). We next examined the role of these effectors on the endosomal distribution of internalized LDL and FC. Although the retromer complex interacts with Rab7 and regulates cargo sorting at the EE (16), silencing of Vps26 had no major effect on LDL and FC intracellular distribution. We could notice, however, that the distribution of endosomes was slightly different from control cells, probably reflecting the role of Vps26 in early endosomal sorting. Nevertheless, we did not observe the presence of FC or LDL into abnormally enlarged endosomes as shown under Rab7 silencing (Figure 6C,D). In the case of VAMP7-depleted cells, we noticed the presence of FC and LDL in enlarged endosomes (Figure 6C,D, arrowheads). These endosomes were positive for Lamp1 (unpublished data) in agreement with the role of this SNARE protein in regulating vesicular trafficking between LE and lysosomes (34). Again the phenotype was less severe than for Rab7 silencing. Cells silenced for STX16 presented quite a different pattern since FC and LDL was localized in smaller, dispersed endosomes (Figure 6C,D). Finally, we evaluated the functional impact of the Rab7 effectors on LDL processing by monitoring the reesterification of LDL-derived FC at the ER. We found that under STX16 knock-down, the fraction of CE specifically generated by ACAT was decreased two fold (Figure 6F). This is in agreement with a recent report involving STX16 in the retrograde transport of LDL-cholesterol (40). No measurable defects in FC re-esterification could be Traffic 2014; 15: 309–326

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Figure 6: Role of early and late Rab7 interacting proteins in LDL endosomal sorting. A-E) HeLa cells were transfected for 48 h with control, VAMP7, Vps26 and STX16 siRNAs as indicated. A) Cells were incubated for 40 min at 4◦ C with STxB-Cy3 and then chased for 45 min at 37◦ C before fixation. Scale bar, 10 μm. n = 2. B) CI-MPR intracellular distribution was observed by indirect immunofluorescence. Scale bar, 10 μm. n = 2. C and D) Cells were incubated for 3 h with 200 μg/mL LDL and stained with filipin (C) or incubated for 3 h with 200 μg/mL DiI-LDL and fixed (D). Examples of enlarged DiI-LDL-containing endosomes are indicated by arrowheads. Scale bar, 10 μm. n = 3. E) Efficiency of silencing was analyzed by immunoblotting with antibodies against VAMP7, Vps26 and STX16. The blot shows a representative experiment. n = 2. F) HeLa cells were transfected with VAMP7, Vps26, STX16 and control siRNA for 48 h, and incubated with 200 μg/mL LDL for 24 h. The amount of CE was quantified and expressed as the percent of the total amount of cell-associated cholesterol. The amount of ACAT-independent esters was measured in the presence of 10 μg/mL ACAT inhibitor (black bars). The results are expressed as means ± SD (error bars). n = 3, **p < 0.01, *p < 0.05 (Student’s t -test). Traffic 2014; 15: 309–326

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detected under VAMP7 or Vps26 silencing (Figure 6F). These data indicate that defects in late endosomal sorting do not significantly affect LDL delivery to the ER. This is in contrast to the functional impact of Rab7 LDL sorting at the EE.

Discussion Endosome biogenesis and dynamics play critical roles in signaling, cell growth, membrane turnover, pathogen survival and many other aspects of cellular homeostasis (1). Studies from the past 10 years have revealed that the endosomal network, long seen as a simple and passive continuum of the plasma membrane, is in fact a more complex, highly interconnected and dynamic system that is tightly coordinated with cargo uptake. This study further establishes this interplay with cargo uptake as we show that Rab7 controls endosomal sorting in a cargo-specific manner. Recently, new sorting machineries have been characterized on endosomes. The retromer complex, the ESCRT (endosome-associated complex required for transport) machinery, the sorting nexins and the WASH complex have been involved in the endosomal sorting of various molecules (35,41,42). Rab GTPases, as exemplified by Rab5 and Rab7, also play an important regulatory role on endosomal biogenesis and dynamics. There remains, however, a persistent controversy on the role of Rab7 in endosomal sorting. If most studies have established that Rab7 activity is required at later steps when cargo is transported from the LE to lysosomes, a few others have suggested that Rab7 may actually also control the transport of cargo from the EE to the LE. The first indications that Rab7 could be involved in EE sorting came from studies in cells expressing the constitutively inactivate form of Rab7. Thus, functional Rab7 was required for the transport of the vesicular stomatitis virus to LE, the cleavage of viral proteins in LE and for the delivery of newly synthesized CI-MPR and cathepsin D to late endosomal compartments (14–16). Similarly, Rab7 inactivation led to the sequestration of the Semliki Forest virus into EEA1 positive EE after its internalization by clathrin-coated pits (43). Further evidences came from a recent study focused on the recruitment of the retromer complex which consists of two sorting nexins and three vacuolar protein sorting 26/29/35 (4). It was shown that Rab7 acts in concert with Rab5 to recruit the Vps subunits 320

of the retromer complex to the EE. Then, Rab7 silencing blocked CI-MPR retrograde transport to the TGN, which accumulated into enlarged peripheral EE several hours after CI-MPR endocytosis (16). We confirmed this data in our study with the retrograde transport of STxB. In apparent contradiction with these studies, it was found that the EGF/EGF-R complex accumulated in endosomes characteristic of the late endocytic pathway in cells expressing the inactivate Rab7 mutant (12,13). Rab7 silencing further confirmed this data since the trafficking of the EGF/EGF-R complex through the EE to the LE/multivesicular body (MVB) was not affected but the exit of the complex from LE/MVB was blocked (12,13). Our results on EGF sorting and EGF-R degradation confirm these previous studies. Moreover, we found that LDL-R and Tfn recycling were only slightly affected in Rab7-silenced cells indicating that Rab7 may not be involved in this process. It is likely that distinct machineries regulated by Rab7 are involved in selective cargo recruitment and endosomal sorting. These interactions may be regulated by specific posttranslational modifications such as receptor ubiquitination and phosphorylation. Most known Rab7 effectors, however, have been identified at the late endosomal interface. At this stage, we can only speculate about the possible mechanisms that allow Rab7 to regulate endosomal sorting in a cargo-specific manner. The differential control of distinct early sorting events by Rab7 may reflect the existence of endosomal subpopulations or subdomains that differ from those involved in MVB formation and cargo degradation. Cargo segregation into endosomal subdomains could be mediated through specific lipid interactions. Different cargo-specific endosomal populations can be distinguished by their motility on microtubules (44). The existence of distinct subpopulations of signaling endosomes has also been documented for the EGF-R (45). Our findings that IFNAR1, but not EGF-R, requires Rab7 for early endosomal sorting further support this possibility. The precise kinetics of LDL uptake allowed us to functionally assess the respective contribution of early versus late endosomal sorting in LDL processing and transcriptional regulation of cholesterol homeostasis. Indeed, we found that Rab7 silencing led to the retention of most internalized LDL in EE at early times of uptake. We could functionally Traffic 2014; 15: 309–326

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confirm this effect by measuring a corresponding twofold increase in the intracellular amount of CE independently from ACAT activity. This increase reflects the lack of LDL delivery to the LE where LDL-derived CE hydrolysis normally occurs. It was unexpected to find a significant population of LE containing LDL and FC under Rab7 silencing since LDL transport from EE to LE was strongly impaired. While insufficient Rab7 silencing is unlikely, it is possible that other recycling pathways contribute to LE biogenesis independently from Rab7. Several of our findings support this possibility. First, the proportion of internalized LDL present in LE at late times of endocytosis was similar in control and Rab7 silenced cells. We also found that these endosomes conserve their ability to be acidified despite the loss of Rab7. Finally, when Rab9 was silenced simultaneously with Rab7, we observed that some of the enlarged endosomes were now positive for both EEA1 and Lamp1 indicating that LE biogenesis and maturation were impaired (unpublished data). Rab5 plays a key role in assembling the endosomal transport machineries on the EE membranes (46). In this context, RNA silencing in vivo against the three Rab5 isoforms resulted in almost the complete disappearance of EE, LE and lysosomes in mice liver (47). Thus, the persistence of a significant population of acidic LE under Rab7 silencing further supports our finding of a selective effect on Rab7 that depend on the type of cargo and/or sorting events. It also rules out an indirect effect due to an unspecific overall perturbation of the Rab5 dependent machinery. We report here the first functional role of Rab7 in cholesterol trafficking and transcriptional regulation of cholesterol homeostasis. Our data further establish the key role of LDL endosomal sorting in the cellular adaptation to variations in cholesterol concentration (48,49). Our results are also in line with a recent study showing that Hrs/Vps27, a member of the ESCRT machinery, is required for trafficking of LDL-derived cholesterol from EE to the ER (29). Rab9 has been classically involved in the regulation of transport between LE and the TGN (30). We found no effect of Rab9 silencing on endosome morphology and sterol-sensitive gene regulation, in agreement with published results (50). However, it was recently shown that RNAi-mediated silencing of the SNARE complex (VAMP4, syntaxin 6 and syntaxin 16) which controls the LE to TGN pathway led to a strong Traffic 2014; 15: 309–326

decrease on the amount of cholesterol reaching the ER (40). This SNARE complex, however, is also involved in EE to TGN transport (36). We could confirm this data in our system through syntaxin 16 down-modulation. Our results are not only note-worthy in the molecular understanding of intracellular trafficking but also in pathophysiology. The phenotype observed in Rab7silenced cells is reminiscent of the genetic Niemann-Pick disease type C phenotype where patient cells accumulate deleterious amounts of cholesterol and sphingolipids in the endolysosomal system (51,52). Interestingly, expressing Rab7 or Rab9 restored normal lipid trafficking from LE to the ER in NPC1 human skin fibroblasts (53). Another study has shown that Rab7 gene expression was elevated in response to hypercholesterolemic diet and was found in atherosclerotic plaques (54). High resolution live cell imaging allowed the Zerial group to show in 2005 that the EE to LE transformation is characterized by the progressive replacement of Rab5 by Rab7, a process called Rab conversion (10). This molecular switch in early-to-late endosomal maturation was recently confirmed in C. elegans with the identification of the Sand1/Mon1 complex as the complex controlling the Rab5 to Rab7 exchange. Thus, Rab5-GTP initiates the endosomal recruitment of the SAND-1/Mon1 complex and ccz1 that interacts with components of the HOPS (homotypic fusion and protein sorting) complex, which in turn recruits and activates Rab7. Rab5 is then inactivated by the SAND-1/Mon1 complex through the displacement of Rabex-5, a Rab5 guanine exchange factor (11). Dmon1, the Drosophila ortholog of mon1/sand1, was recently reported to play a similar function in endosome formation and maturation (55). Whether the Rab5 to Rab7 conversion occurs early or later during endosomal maturation and whether it is required for EE to LE cargo transport has not been addressed by these studies. Two groups have recently reported that the Rab5-Rab7 exchange is likely to occur during LE maturation (12,55). Our results reveal an early requirement for Rab7 activity in the transport of different cargos between the EE and the LE. In the absence of Rab7, Rab5 cannot be exchanged and remains abnormally activated resulting in increased recruitment of EEA1 and subsequently enlargement of the EE structure. This is particularly well illustrated in this study by IFNAR1, 321

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which is completely blocked at early times of endocytosis in enlarged endosomes that are enriched in EEA1. Indeed, the expression of the constitutively activate form of Rab5-Q79L led to the same phenotype with internalized LDL present in enlarged endosomes positive for EEA1 (Figure S4B). These data therefore indicate that the Rab5 to Rab7 conversion process is immediately required after cargo arrival in the EE to ensure further efficient sorting to the LE. In conclusion, our study confirms the intricate complexity of endosomal sorting and reveals that in addition to its established role in regulating cargo transport between LE and lysosomes, Rab7 activity is also required for earlier sorting events in a cargo-specific manner. It is likely that redundancy exists in cholesterol trafficking and that molecular machineries can be shared between EE and LE along the endocytic continuum. This has been well exemplified by the Rab5-Rab7 conversion process and more recently by the HOPS and CORVET (class C core vacuole-endosome transport) complexes in yeast (56). Indeed, the strong phenotype observed under Rab7 silencing on cholesterol trafficking and sterol-sensitive genes regulation is a likely consequence of the interaction of Rab7 with several actors of the endosomal sorting machinery both at the EE and LE levels. Most of the Rab7 effectors that have been identified recently are localized along the endolysosomal network (57). Our study calls for further efforts to identify new Rab7 interacting proteins at the early endosomal level and demonstrates the interest of studying the endocytosis of cargos other than the prototypical EGF-R and Tfn-R. This will undoubtedly contribute to a better molecular understanding of the endosomal circuitry and to the rational design of new therapeutics for the various diseases involving mutations on genes regulating endosomal machineries (58,59).

Materials and Methods

Antibodies and reagents Monoclonal or polyclonal antibodies against TfR (Sigma), Lamp-1 and EGF-R (BD Biosciences), Golgin 97 (Molecular probes), CI-MPR, Vps26 and VAMP7 (Abcam), IFNAR1 (Biogen), STX16 (Synaptic system), EEA1, Rab7, Rab9 and actin (Santa Cruz Biotechnology, Inc.) and Alexa488-, Alexa657-, Cy3-and Cy5-coupled secondary antibodies (Santa Cruz Biotechnology, Inc.) were purchased from the indicated suppliers. Polyclonal antibody against LDL-R was a kind gift from Dr. Joachim Herz (University of Texas Southwestern University). Alexa Fluor 633conjugated Tfn and Alexa Fluor 488-conjugated EGF were purchased from Invitrogen. STxB-Cy3 was prepared as previously described (22). DiI-LDL was prepared with DiI (1,1 -dioctadecyl-3,3,3 3 -tetramethylindocarbocyanine; Sigma-Aldrich) and LDL as previously described (49).

RNA interference ON-TARGET SMART pools (Dharmacon) composed of four different oligonucleotides, were used to silence Rab7, Rab9 and VAMP7. For Vps26 and STX16, oligonucleotides were used individually (22). Cells transfected with scramble siRNA were used as a control (siCtl). Vps26, STX16 and Ctl siRNAs were from Eurogentec. Cells were transfected with 25 nM siRNA using RNAiMAX transfection reagent (Invitrogen). Experiments were carried out 48 h after transfection.

Cell transfection Plasmids encoding GFP-Rab7-WT, GFP-Rab7-Q67L, GFP-Rab7-T22N and GFP-Rab5-Q79L were a gift from St´ephanie Miserey-Lenkei (Curie Institute, Paris, France). Transfection was performed using FuGENE HD (Roche) according to the manufacturer’s instructions. For each transfection, 2 μg/well of DNA was used in 6-well plates. Experiments were carried out 48 h after transfection.

RNA extraction, RT-PCR and quantitative real-time PCR Total RNA was extracted from cells using the RNeasy Mini kit (Qiagen). One microgram of total RNA was transcribed to cDNA using random hexamers (Amersham) and SuperScriptII reverse transcriptase (Invitrogen). Real-time quantitative PCR reactions were performed using ®

the Sybr Green reagents kit (Applied Biosystems) and the corresponding primers with an ABI PRISM 7900HT Sequence detector instrument (Applied Biosystems) according to the manufacturer’s instructions. Amplification was carried out in a final volume of 10 μL with 20 ng of cDNA, both sense and antisense primers (Eurogentec) in the Sybr Green PCR Master Mix (Eurogentec). HMGCoAR, LDL-R, SREBF-

HeLa cells were grown at 37◦ C and 5% CO2 in Dulbecco’s modified

2 and UBC primer sequences were described previously (49). Others primers used were: 5 -TGT CCA GCG CAG GTG TTT T (Rab7 F), 5 - TAC CTC TTG TCC CCA TAT GCA A (Rab7 R), 5 - TGG

Eagle’s medium (DMEM, Sigma-Aldrich) containing 10% heatinactivated fetal bovine serum (FBS, Invitrogen), 2 mM L-glutamine (Sigma-Aldrich), 100 UI/mL penicillin and 100 μg/mL streptomycin

CCG CGA GAC ACT CTT (Rab9 F) and 5 - ACT CAA ACG ACA CGG GAA AAG T (Rab9 R). Sample were heated for 15 min at 95◦ C and amplified in 40 cycles of 15 seconds at 95◦ C, and 1 min at 60◦ C.

(Eurobio). HeLa cells stably transfected with GFP-CI-MPR were cultured in complete DMEM medium in the presence of 0.5 mg/mL of geneticin (G418, Invitrogen) (22). RPE1 cells were grown in DMEM F12

Relative quantification for a given gene, expressed as fold-variation over control at T0, was calculated after normalization to a reference gene (UBC) and determination of the CT (cycle threshold) difference

(Invitrogen) complemented with 10% FBS.

between conditioned cells and control cells at T0 using the comparative

Cell culture

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CT method (60). Efficiencies of the target and control amplification were similar.

washed and resuspended in prewarmed complete DMEM medium. At the indicated times, aliquotes of cells were placed on ice and labeled with

Western blotting

secondary antibodies. Cells were pelleted and analyzed on a FACS Calibur system (Becton Dickinson). At least 10 000 cells were analyzed, and the mean Alexa-657 fluorescence was calculated. The values obtained at each

Cells were lysed in 1% triton X-100 in PBS containing protease inhibitor cocktail (Roche). Proteins (25 μg per lane) were resolved on precast Tris-acetate gels (NuPAGE 3–8%; Invitrogen) and transferred to PDVF membranes (Bio-Rad). Immunoblotting was performed by standard method. Blots were probed with specific antibodies or, as a loading control, an anti-actin antibody. Immunoreactive bands were visualized using ECL chemiluminescence kit (Amersham).

time point were expressed as a percentage of the value measured at t = 0.

Microscopy Images from Figures 1–4A, S1–S3A and S4 were obtained using an epifluorescent Leica microscope equipped with 63×/1.4 NA oil immersion objective and a coupled camera controlled by software (METAMolecular Devices). Confocal microscopy images (Figures 4D,E, S3C–E) were taken on a confocal laser-scanning microscope (Nikon).

MORPH,

Immunofluorescence Cells grown on glass coverslips were fixed with 4% paraformaldehyde in PBS for 15 min. Cells were permeabilized with 0.1% saponin PBS for 15 min. Cells were incubated with primary antibodies diluted in 1% BSA in PBS and 0.1% saponin for 1 h, followed by incubation with the appropriate conjugated secondary antibodies for 1 h. Cells were mounted in Mowiol (Beckman Coulter). Intracellular FC was visualized with freshly prepared 50 μg/mL filipin (Sigma-Aldrich) staining for 1 h at room temperature. LDL intracellular distribution was observed after 3 h incubation with 200 μg/mL DiI-LDL followed by fixation. Visualization of acidic organelles was performed by incubation of cells for 30 min with 200 nM Lysotracker DND-99 (Molecular Probes) prior to fixation.

LDL and EGF uptake HeLa cells grown on coverslips were incubated with DiI-LDL (200 μg/mL) in serum free medium for 40 min at 4◦ C and then shifted to 37◦ C for the indicated times, washed and fixed. For EGF uptake, cells were pulsed with 100 ng/mL Alexa Fluor 488-conjugated EG for 15 min at 37◦ C and then chased at 37◦ C for the indicated times. Cells were washed and fixed. After fixation, cells were processed for immunostaining.

Camera and acquisition software used were the standard Nis-elements system software and camera. The objective used was a 63×/1.4 NA oil immersion objective. Images presented are representative of each sample analyzed. Images were processed using IMAGEJ and ADOBE PHOTOSHOP. All the figures were produced using ADOBE ILLUSTRATOR.

Quantification of colocalization All images for a given experiment were acquired with the identical settings for the different channels. The Pearson’s correlation coefficient (PCC), which gives a measure of the overlap between two fluorescence signals, was calculated by analyzing 15–20 cells from at least two independent experiments using the JaCoP plugin (61) for IMAGEJ. The PCC was expressed as mean and standard deviations (SD). For colocalization quantification of the different cargoes with EEA1 or Lamp1, cargoes were outlined in the red channel whereas endosomal markers were outlined in the green channel. Background noise levels were subtracted for each picture in the red and the green channels. ROI (regions of interest) were defined for each channel. Finally, the number of pixels for each channel and overlapping channels (red-green colocalization) was quantified using an object-based colocalization finder. For each experimental condition 15 cells from at least two independent experiments were quantified.

STxB and IFNAR1 internalization assay About 1 μM Cy3-STxB was added to the cells for 40 min at 4◦ C. Cells were washed and incubated for 45 min at 37◦ C before fixation. For IFNAR1 internalization assay, cells were incubated 40 min at 4◦ C with IFNAR1 antibodies, washed and incubated 40 min at 37◦ C in the presence of 1000 U/mL IFN β. Cells were fixed and stained with secondary antibodies.

EGF-R degradation assay HeLa cells were preincubated for 1 h with cycloheximide (Sigma-Aldrich) at 100 μg/mL. Cells were detached, washed and incubated for indicated times at 37◦ C in the presence of 100 ng/mL EGF (Sigma-Aldrich). The cells were then placed on ice, washed and lysed. EGF-R levels were quantified by western blotting.

Tfn and Tfn-R binding HeLa cells were incubated 1 h at 4◦ C with Alexa Fluor 633-conjugated Tfn. For Tfn-R binding, HeLa cells were incubated 1 h at 4◦ C with antiTfn-R antibodies. Cells were then incubated with secondary antibodies

LDL-derived cholesterol delivery to the cell LDL (d = 1.019–1.063 g/mL) were separated from fresh human plasma

1 h at 4◦ C. Cells were kept at 4◦ C until analysis. At least 10 000 cells were analyzed on a FACS Calibur system (Becton Dickinson).

by sequential ultracentrifugation as described previously (48). HeLa cells deprived of sterols through incubation for 48 h in 10% LPDS medium were incubated for the indicated times with 200 μg/mL LDL.

LDL-R recycling

Measurement of cellular cholesterol

HeLa cells were detached, washed and incubated for 45 min at 37◦ C in the

Cells transfected were lysed in 0.2 M NaOH. Total cholesterol was

presence of antibodies against LDL-R. The cells were then placed on ice,

extracted with methanol (2.5 mL) followed by hexane (5 mL). 4.5 mL

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of the hexane phase was evaporated under vacuum and dissolved in mobile phase. Separation of FC and CE including cholesteryl docohexanoate (CDH), cholesteryl arachidonate (CA), cholesteryl linoleate (CL), cholesteryl myristate (CM), cholesteryl oleate (CO) and cholesteryl stearate (CS) was done by reverse phase HPLC on a C-18 column (25 × 0.46 cm length, 5-μm pore size, Sigma-Aldrich) by measuring the 205 nm absorbance after elution with acetonitrile/isopropanol (30/70, v/v). The values were normalized to the total cellular protein level, which was measured using the bicinchoninic acid (BCA) method. CE were expressed as the percentage of total cholesterol content.

ACAT activity The ACAT inhibitor Sandoz 58–035 (Sigma-Aldrich) (10 μg/mL) was added to the cholesterol loading medium. When ACAT activity is inhibited, CE are provided by preexisting pools such as endocytosed LDL. Therefore, the difference in cholesterol esterification measured by HPLC with and without Sandoz 58–035 represents the specific amount of cholesterol esterified by ACAT.

Statistical analysis Data were analyzed for statistical significance using Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.0001 in comparison with the control condition.

Acknowledgments We would like to thank Florence Niedergang for help with receptor recycling assays and St´ephanie Miserey-Lenkei for providing reagents. We are thankful to Sentil Arumugam for help with cell imaging quantification and to Maria Daniela Garcia-Castillo for carefully reading the manuscript. We are grateful to C´edric Blouin for his help with the graphical abstract. This work was supported by institutional funds from the Curie Institute and Agence Nationale de la Recherche to C.L. E.G. was supported by a doctoral fellowship from the Ministère de la Recherche and by FRM (Fondation pour la Recherche M´edicale). This work has been done by a team belonging to the labex CelTisPhyBio 11-LBX-0038. The authors declare that they have no conflict of interest.

Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1: Rab7 silencing causes the accumulation of LDL and FC in enlarged Lamp1 positive endosomes. A) HeLa cells were transfected with Rab7 siRNA for 48 h, incubated for 3 h with 200 μg/mL DiI-LDL, and fixed. Cells were processed for immunofluorescent labeling with anti-Lamp1 antibodies. Images are representative of three independent experiments. Scale bar, 10 μm. B) HeLa cells transfected with Rab7 siRNA for 48 h were loaded with 200 μg/mL LDL for 3 h, and stained with filipin. Cells were processed for immunofluorescent labeling with anti-Lamp1

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antibodies. Images are representative of three independent experiments. Scale bar, 10 μm. Associated with Figure 1C,D. Characterization of enlarged vesicles containing LDL and FC under Rab7 silencing. Figure S2: Rab7 is required for CI-MPR retrograde transport to the trans-Golgi network. A) HeLa cells stably expressing GFP-tagged CIMPR were transfected for 48 h with Rab7 or control siRNA. Cells were fixed and stained for Golgin 97. Scale bar, 10 μm. n = 2. B) HeLa cells were transfected with Rab7 or control siRNA for 48 h, and analyzed for the steady-state distribution of CI-MPR by indirect immunofluorescence. Scale bar, 10 μm. n = 3. Associated with Figure 4A. Impact of Rab7 silencing on the CI-MPR distribution. Figure S3: Rab7 knock-down impairs late endosomal transport of EGF and EGF-R degradation. HeLa cells were transfected for 48 h with Rab7 or control siRNA. A) Cells were incubated for 15 min with 100 ng/mL Alexa Fluor 484-conjugated EGF, washed, and incubated for the indicated times at 37◦ C before fixation. B) EGF-R degradation assay was performed in control and Rab7-depleted HeLa cells. The blot shows a representative experiment. n = 2. C–E) Control or Rab7 silenced RPE1 cells were pulsed for 15 min with 100 ng/mL Alexa Fluor 488-conjugated EGF. Cells were washed and chased for 20 min (C) or 60 min (D and E) at 37◦ C before fixation. Cells were then permeabilized and stained with an antibody against endogenous EEA1 and Lamp1. The mean values ± SD of the Pearson’s correlation coefficient (PCC) of 15–20 cells from two independent experiments are shown. Scale bar, 10 μm. Associated with Figure 4D. Impact of Rab7 silencing on EGF transport and degradation. Figure S4: Dominant Rab5 active mutant affects the intracellular distribution of internalized LDL and FC. HeLa cells were transfected for 48 h with a plasmid encoding the GFP-tagged active mutated form of Rab5. A) Cells were loaded for 3 h with 200 μg/mL DiI-LDL before fixation or incubated for 3 h with 200 μg/mL LDL and stained with filipin. B) Cells were incubated with 200 μg/mL DiI-LDL for 40 min at 4◦ C. Cells were washed, incubated at 37◦ C for 60 min and fixed. Cells were processed for immunofluorescent labeling with anti-EEA1 antibody. Arrowheads indicate enlarged EE loaded with DiI-LDL. Scale bar, 10 μm. n = 2. Associated with Figure 4E. Impact of dominant Rab5 active mutant expression on LDL and FC distribution.

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