correspondence and requests for materials should be ... · inhibition of autophagy progression by...

Inhibition of autophagy progression by TPC2

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TPC2 Signaling Inhibits Autophagosomal-Lysosomal Fusion by Alkalizing Lysosomal pH

Ying-Ying Lu1, Bai-Xia Hao1, Richard Graeff 1, Connie WM Wong2, Wu-Tian Wu2,3, Jianbo Yue1*

1Department of Physiology, University of Hong Kong, Hong Kong, China; 2Department of Anatomy

and State Key Laboratory of Brain and Cognitive Sciences, University of Hong Kong, Hong Kong, China; 3GHM Institute of CNS regeneration, Jinan University, Guangzhou, China.

Running title: Inhibition of autophagy progression by TPC2 Correspondence and requests for materials should be addressed to J Yue (e-mail: [email protected]) Keywords: Autophagy, lysosome, NAADP, Ca2+, pH, TPC2 Background: The role and mechanism of NAADP, an endogenous Ca2+ mobilizing nucleotide, in autophagic regulation remain to be determined. Results: Activation of NAADP/TPC2 signaling induced the accumulation of autophagosomes. Conclusion: The NAADP/TPC2 signaling inhibits autophagosomal-lysosomal fusion by alkalinizing lysosomal pH. Significance: Development of agonists or antagonists of NAADP should provide a novel approach to specifically manipulate autophagy. Autophagy is an evolutionarily conserved lysosomal degradation pathway, yet the underlying mechanisms remain poorly understood. Nicotinic acid adenine dinucleotide phosphate (NAADP), one of the most potent Ca2+ mobilizing messengers, elicits Ca2+ release from lysosomes via the two pore channel 2 (TPC2) in many cell types. Here we found that overexpression of TPC2 in HeLa or mouse embryonic stem (ES) cells inhibited autophagosomal-lysosomal fusion, thereby resulting in the accumulation of autophagosomes. Treatment of TPC2 expressing cells with a cell permeant-NAADP agonist, NAADP-AM, further induced autophagosome accumulation. On the other hand, TPC2 knockdown or treatment of cells with Ned-19, a NAADP antagonist, markedly decreased the accumulation of autophagosomes. TPC2-induced accumulation of autophagosomes was also markedly blocked by ATG5 knockdown. Interestingly, inhibiting mTOR activity failed to increase TPC2-induced autophagosome accumulation. Instead, we found that overexpression of TPC2 alkalinized lysosomal pH, and lysosomal re-acidification abolished

TPC2-induced autophagosome accumulation. In addition, TPC2 overexpression had no effect on general endosomal-lysosomal degradation but prevent the recruitment of Rab-7 to autophagosomes. Taken together, our data demonstrate that TPC2/NAADP/Ca2+ signaling alkalinizes lysosomal pH to specifically inhibit the later stage of basal autophagy progression. Macroautophagy (hereafter referred as autophagy) is an evolutionarily conserved catabolic degradation cellular process. Autophagy starts with sequestering of cytoplasmic material, such as misfolded proteins and/or damaged organelles, inside the phagophore, followed by the elongation and closure of the autophagosome, a double-membrane vesicle. The matured autophagosome then fuses with the lysosome to create an autolysosome, inside which the autophagosome inner membrane and its luminal contents are degraded by lysosomal enzymes for subsequent recycling of macromolecules. Autophagy can also be markedly induced by a wide variety of stresses, such as nutrient starvation, infection, and aging, for cell survival. Dysfunctional autophagy has been associated with wide ranging human diseases, e.g. cancer, neurodegenerative diseases, heart disease, diabetes, and infections (1).

Autophagy induction is controlled by the ULK1 and ULK2 complexes, and autophagosome formation requires class III phosphatidylinositol 3 kinase (PI3K) complexes. The key autophagy regulator is mTOR Ser/Thr kinase, which negatively regulates autophagy by inhibiting the ULK1/2 complex. Starvation, on the other hand, activates the AMP-activated protein kinase (AMPK) to inactivate mTOR,

http://www.jbc.org/cgi/doi/10.1074/jbc.M113.484253The latest version is at JBC Papers in Press. Published on July 8, 2013 as Manuscript M113.484253

Copyright 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

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thereby inducing autophagy. Another key step for autophagy induction is the activation of mammalian Vps34, a class III PI3K. Vps34 is activated by forming a multiprotein complex with beclin-1, UVRAG, and Vps15, to generate phosphatidylinositol-3-phosphate (PtdIns3P). Two ubiquitin-like conjugation systems, the LC3-II and Atg12–Atg5–Atg16L complex, are essential for the autophagosomal elongation process. The covalent conjugation of Atg12 to Atg5 is catalyzed by the E1-like enzyme Atg7 and the E2-like enzyme Atg10. The conjugation of phosphatidylethanolamine (PE) to LC3 is sequentially controlled by the protease Atg4, Atg7, and the E2-like enzyme Atg3. Lipidation of LC3 converts the cytosolic LC3 (LC3-I) to the autophagic vesicle-associated form (LC3-II). Notably, LC3-II is commonly used as a marker of autophagy because lipidated LC3-II exhibits a punctate staining pattern and has faster electrophoretic mobility compared with diffused LC3-I (1,2). Besides mTOR, other signaling pathways, e.g. JNK, Ras, and Ca2+, can modulate autophagy as well (3,4).

Even after extensive research, the regulation and mechanisms of autophagy induction, autophagosome formation and maturation, and especially autophagosomal-lysosomal fusion, remain elusive in mammalian cells. Although autophagosomal-lysosomal fusion is poorly understood, many factors and proteins, including lysosomal pH (5), Rab7 (6), SNAREs (7), the HOPS complex (8), TECPR1 (9), histone deacetylase-6 (10), Ubiquilins (11), Hrs (12), OATL1 (13), COP9 signalosome (14), presenilin 1 (15), the ESCRT III complex (16), LAMP1/2 (17), UVRAG (18), p97VCP (19), LRRK2 (20), and HSP70 (21) have been implicated in regulating this process. We have been studying the effects of lysosomal specific ion channels on autophagosomal- lysosomal fusion.

In vertebrates, three TPC genes, TPC1, 2, and 3, have been cloned. TPCs contain two putative pore-forming repeats, with each of the repeats containing six transmembrane domains and an intervening pore-forming loop. The transmembrane domain of TPCs is similar to that of voltage gated Ca2+/Na+ channels. Interestingly, human and rodent only encode TPC1 and TPC2. TPC2 predominantly localizes on lysosomes, whereas TPC1 is mainly on late endosomes and TPC3 is thought to be in the recycling endosomes. Ever since their identification, TPCs have become the prime

candidates for NAADP-mediated Ca2+ release from lysosome-related stores (22-24). NAADP is a metabolite of nicotinamide adenine dinucleotidephosphate (NADP) and is formed by a base-exchange reaction catalyzed by ADP-ribosyl cyclases, which replaces the nicotinamide moiety of NADP with nicotinic acid. NAADP mobilizes Ca2+ from acidic lysosome-related stores in a wide variety of cells, from plant to animal, including human (25). Ample evidence indicates that TPC2 forms NAADP sensitive Ca2+-permeable channels in many cell types. TPC2 overexpression promotes NAADP-induced Ca2+ release from lysosome-related stores, whereas ablating or knocking-down TPC2 expression blocks NAADP-induced Ca2+ release. Moreover, TPC2 knock-out abolishes NAADP-mediated smooth muscle contraction, a well established function of NAADP (26-33). Besides TPC2, other NAADP receptor candidates have been reported in different cell types, including TPC1 in SKBR3 human breast carcinoma (34,35), TRP mucolipin 1(TRPML1) in coronary arterial myocytes (36,37), and TRPM2 or Ryanodine receptors in T lymphocytes (38,39). Interestingly, several recent papers found that NAADP does not directly bind to TPC2, suggesting that NAADP first binds to accessory proteins, which subsequently activate TPC2 or other ion channels for Ca2+ mobilization depending upon the cell type (40-42).

Previously, two groups found that activation of NAADP/TPC2 signaling increased LC3-II levels (43), and another report found that downregulation of TPC2 by presenilin decreased LC3-II (44), thus concluding that TPC2 signaling mainly induces the initiation of autophagy. However, here we found that the increased LC3-II levels by TPC2 signaling are not due to the enhanced autophagy activity, but result from the inhibition of basal autophagy progression, which leads to the accumulation of autophagosomes. Inhibition of TPC2 signaling either by TPC2 knockdown or by treatment with a NAADP antagonist, on the other hand, facilitates autophagosomal-lysosomal fusion, thereby decreasing LC3-II levels. EXPERIMENTAL PROCEDURES Antibodies and reagents- LC3 antibody (antibody from MBL was used in study in the ES cells, and the one from Novus was used in study in HeLa cells; 1:500 for the immunofluorescence analysis (IF) and 1:1000


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for the Western blotting analysis (WB)), p62 antibody (Novus; 1:1000 WB), Cathepsin-L antibody (BD Bioscience; 1;250 WB), Lamp-1 and EGFR antibodies (Santa Cruz; 1:250 WB), Lamp-1 antibody (Cell signaling; 1:500 IF), phospho-mTOR and mTOR antibodies (Cell signaling; 1:500 WB), GAPDH antibody (Sigma; 1:5000 WB), and TPC2 antibody (a custom rabbit polyclonal antibody against rat TPC2; 1:500 IF and 1:1000 WB). Bafilomycin A1, rapamycin, and BAPTA-AM were all purchased from Sigma-Aldrich. NH4CL was purchased from Merck. Fura-2 AM, DQ-BSA green, lysosensor Green DND-189, and LysoSensor Yellow/Blue DND-160 were purchased from Invitrogen. Cell culture- HeLa cells (ATCC) were maintained in DMEM (Invitrogen) plus 10% fetal bovine serum (Invitrogen) and 100 units/ml of penicillin/streptomycin (Invitrogen) at 5% CO2 and 37 °C. ES cells, D3 from Prof. Tsang SY (Chinese University of Hong Kong), and 46C Sox1-GFP from Prof. Austin Smith (University of Cambridge), were normally maintained with feeders (mouse embryonic fibroblasts) in Dulbecco's Modified Eagle Medium plus 15% FBS (ES qualified, Invitrogen), 1% nonessential amino acids, 1% penicillin-streptomcyin, 0.2% 2-mercaptoethanol, 1000 U/ml leukemia inhibitory factor (LIF). Cells are passaged every two days. Prior to any experimental procedures, ES cells are cultured in feeder-free ES medium containing LIF on gelatin-coated plates for two passages. The pluripotency of ES cells are periodically assessed by alkaline phosphatase assay and Oct4 immunostaining. TPC2 shRNA, TPC2, GFP-LC3, RFP-GFP-LC3 lentivirus production and infection- Three optimal 21-mers were selected in the mouse TPC2 genes (Table S1). One 21-mer was selected in the GFP gene as a control. These sequences were then cloned into the pLKO.1 vector for expressing shRNA. Likewise, a rat TPC2, GFP-LC3, or RFP-GFP-LC3 (tfLC3) cDNA was cloned into pLenti-CMV-Puro-DEST (Addgene). The shRNA, TPC2, GFP-LC3, or tfLC3 lentivirus production was performed in 293T cells. For infection, ES cells were plated at a density of 3×105 cells/well in 6-well plates. On the next day, 100 µL pools of shRNAs lentivirus were added to the cells in fresh medium containing 8 µg/mL polybrene. Two days later, cells infected with shRNA lentiviruses were

selected in fresh medium containing puromycin (3 µg/mL) for 3–5 days. The puromycin- resistant cells were pooled and the knockdown efficiency was verified by both quantitative real-time RT-PCR and/or Western blot analyses. Intracellular Ca2+ measurement- Intracellular Ca2+ in HeLa cells were measured as described previously (45,46). Briefly, cells were cultured in 24-well plates at the density of 5 x104 cells /well in regular DMEM medium overnight. Before Ca2+ measurement, cells were incubated for 30 minutes in Hanks’ balanced salt solution (HBSS) containing 4 µM Fura-2 AM and 0.04% F127 in dark at room temperature. The cells were then washed with HBSS three times and incubated at room temperature for another 10 min. Cells were put on the stage of an Olympus epifluorescence microscope and visualized using a 20x objective. Fluorescence images were obtained by alternate excitation at 340 nm and 380 nm with emission set at 510 nm. Images were collected by a CCD camera every 3 seconds. Finally, the images were saved and analyzed by Cell R imaging software. Western blot analysis- Western blot analysis was performed as described previously (47,48). Briefly, cells were lyzed in an ice-cold EBC lysis buffer (50 mM HEPES at pH 7.5, 0.15 M NaCl, 1 mM EDTA, 1% NP-40, 150 µM PMSF, 10 mM NaF, 10 ng/ml leupeptin, 1 mM DTT, and 1 mM sodium vanadate) and passed through a 21-gauge needle several times to disperse any large aggregates. Protein concentrations of the cell lysates were determined by Bradford protein assay. 30-100 µg of protein per lane was diluted in the standard SDS-sample buffer and subjected to electrophoresis on SDS-PAGE gels. Proteins were then transferred to an Immobilon PVDF membrane (Millipore, Billerica, MA), blocked with 5% milk in TBST (20 mM Tris, 150 mM NaCl, pH 7.6), and incubated with the primary antibody overnight. After washing with TBST, the blots were probed with a secondary antibody (1:5000 dilution) for detection by chemiluminescence. RNA isolation and quantitative real-time RT-PCR- Total RNA was extracted from HeLa cells or mouse ES cells at various stages of neural differentiation using an RNA extraction kit (Life Technologies). The quantitative real-time RT-PCR using the SuperScript® III Platinum® One-Step Q-RT PCR Kit (Invitrogen Life Technologies) was performed in a MiniOpticonTM Real-time PCR Detection


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System (Bio-RAD) according to the manufacturer’s instructions. The primers for detecting tpc2, beta-actin, and Gapdh mRNAs are listed in Table S1. Relative gene expression was normalized to beta-actin or Gapdh expression. Immunofluorescence staining analysis- Immunofluorescence staining analysis was performed as described previously (49). Briefly, infected or transfected cells grown on coverslips were fixed for 20 min with 4% paraformaldehyde at room temperature, washed twice with PBS, and nuclei were labeled with DAPI to identify each individual cell. For immunostaining analysis, fixed cells were further blocked with 1% goat serum, 1% BSA, 0.1% Triton X-100 in PBS for 1 h. Thereafter, the cover glasses were incubated with primary antibodies for 2 h, washed and incubated with a secondary antibody (Alexa Fluor® 488 or 568 Goat Anti-Mouse or Rabbit IgG, Invitrogen; 1:1000 dilution) for 1 h. Images were captured under a Zeiss LSM710 confocal microscope with a Plan-Apochromot ×63/1.40 oil DIC objective. Images were processed and analyzed with Zeiss ZEN software. Transmission electron microscopy (TEM)- Cell preparation for TEM was performed as described previously (50). Briefly, cells were washed in PBS, and fixed in 2% formaldehyde and 2.5% glutaraldehyde for 2 hours. After fixation, cells were pelleted, transferred to glass slides, and allowed to dry on a hotplate at 37 °C before embedding in 2% agarose solution. Agarose embedded blocks were then fixed in the same fixative solution at 4 °C for 4 hours. Thereafter, the blocks were washed three times with PBS and post fixed with 1% osmium tetroxide solution in PBS at 4°C overnight. Blocks were subsequently dehydrated in graded ethanol, embedded in pure Epon, and kept at 60oC for 3 days. Ultrathin sections (~ 90 nm) were cut and stained with 8% uranyl acetate and lead citrate before observation under electron microscope.

For immunoelectron microscopy, cells were incubated with primary antibody (rabbit anti-LC3 antibody, 1:100) at 4°C overnight. After incubation in the primary antibody, the cells were incubated in biotinylated secondary antibody (biotinylated goat anti-rabbit IgG antibody, 1:200) for 40 min at room temperature. Cells were then incubated in ABC reagents (Vectastain ABC kit) for 60 min at room temperature, sequentially, followed by

incubating in 0.05% diaminobenzidine (DAB) and 0.03% H2O2 for 3-5 min until a brown reation product was observed. The stained cells were then processed to TEM study as described above. Neural differentiation- The in vitro neural differentiation of mouse ES cells was performed as described previously (51). Briefly, feeder free ES cells were plated onto 0.1% gelatin coated plates at a density of 0.8~1 x 104 cells/cm2 in N2B27 medium (1:1 mixture of DMEM/F12 and neurobasal medium supplemented with N2 and B27, plus 50 µg/ml bovine serum albumin (BSA) fraction V, and 20 µM beta-mercaptoethanol). The medium was changed every other day until day 8. Lysosomal pH measurement- LysoSensor Green DND-189 is commonly used to qualitatively measure the pH of acidic organelles, such as lysosomes, which become more fluorescent in acidic environments and less fluorescent in alkaline environments (52). Briefly, cells were loaded with 1 μM LysoSensor Green DND-189 in pre-warmed regular medium for 20 min at 37°C. Then the cells were washed twice with PBS and immediately analyzed by flow cytometry (collecting FL1 fluorescence and 10,000 cells were collected for each sample) or in a microplate reader (excitation/emission = 485/530 nm) in triplicates.

Quantification of lysosomal pH was performed using a ratiometric lysosomal pH dye LysoSensor Yellow/Blue DND-160. The pH calibration curve was generated as described previously (53). Briefly, cells were trypsinized and labeled with 2 µM Lysosensor Yellow/Blue DND-160 for 30 min at 37°C in regular medium, and excessive dye was washed out using PBS. The labeled cells were treated for 10 min with 10 µM monensin and 10 µM nigericin in 25mM 2-(N-morpholino)ethanesulfonic acid (MES) calibration buffer, pH 3.5–6.0, containing 5 mM NaCl, 115 mM KCl and 1.2 mM MgSO4. Quantitative comparisons were performed in a 96-well plate, and the fluorescence was measured with a microplate reader at 37°C. Light emitted at 440 and 535nm in response to excitation at 340 and 380nm were measured, respectively. The ratio of light emitted with 340 and 380nm excitation was plotted against the pH values in MES buffer, and the pH calibration curve for the fluorescence probe was generated from the plot using Microsoft Excel.


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NH4CL chase assay- Hela cells were seeded in regular DMEM medium overnight and then incubated with 100 µM NH4CL in regular DMEM medium without sodium bicarbonate for 20 min at 37°C. Thereafter, NH4CL was removed and the cells were washed with regular medium without sodium bicarbonate continuously and thoroughly. Samples were collected at the indicated time and finally analyzed by western blotting analyses or for pH measurement in a microplate reader. Statistical analysis- All data were presented as mean ± standard error of mean (SEM). Student’s T-test was performed to determine the differences among grouped data. * denotes statistically significant with P<0.05. RESULTS Reconstitution of NAADP competence in HeLa cells by TPC2 overexpression. HeLa cell line is an autophagy-competent cell line (Figure S1A and S1B). Yet HeLa cells are not responsive to NAADP-induced Ca2+ changes (Figure 1A), which might be due to the low expression level of endogenous TPC2 (Figure S1C). We thus first constructed a stable rat TPC2 overexpressing HeLa cell line by lentiviral infection. As shown in Figure 1B, TPC2 was overexpressed in the lysosome as indicated by its co-localization with the lysosomal marker Lamp1 in a subset of cellular puncta (the TPC2 positive vesicles were actually enlarged in some cells as compared to the control). Likewise, the HeLa cells expressing a rat TPC2 249L/P mutant, in which a highly conserved leucine residue within a putative pore region of rat TPC2 was mutated to proline (34), were constructed by lentiviral infection (Figure 1B). A cell permeant NAADP analogue, NAADP-AM (54), induced Ca2+ release only in wildtype TPC2 overexpressing HeLa cells and not in TPC2 249L/P mutant expressing cells (Figure 1A), and displayed a characteristic bell-shaped dose-response curve due to the activation of its receptor by low concentrations of NAADP and its desensitization by high concentrations of NAADP. The induced Ca2+ release was markedly inhibited by preincubation with Ned-19 (55), an NAADP antagonist (Figure 1C). These data indicated that Hela cells can be reconstituted to be NAADP competent via TPC2 overexpression. Inhibition of autophagic flux by TPC2 in Hela cells. Strikingly, under the electron microscope, large numbers of autophagic

vacuoles were observed in TPC2 overexpressing HeLa cells maintained in normal (nutrient rich) culture conditions, but not in control cells (Figure 2A). Similar to previous reports (43), lipidated LC3-II, a reliable autophagosome marker, was markedly increased in TPC2 overexpressing cells compared to that in TPC2 mutant expressing or control HeLa cells (Figure 2B). Interestingly, p62, an autophagic substrate (56), was also accumulated in TPC2 overexpressing cells compared to that in control HeLa cells, suggesting that TPC2 overexpression results in a defect of autophagic degradation instead of simply promoting autophagy (Figure 2B). Consistently, endogenous LC3-II puncta (Figure 2C) or GFP-LC3 puncta (Figures 2D and S1A) were greatly increased in TPC2 overexpressing cells and did not co-localize with Lamp1, which was similar to the cells treated with bafilomycin (BAF), an inhibitor of the vacuolar proton pump that blocks the fusion of autophagosomes with lysosomes (57). These data suggest that these LC3-II puncta present in TPC2 overexpressing cells are autophagosomes, not autolysosomes.

Since it is possible that the accumulation of LC3-II in TPC2 overexpressing cells is partly due to the increased early autophagic activity, we briefly (1h) treated control or TPC2 overexpressing HeLa cells with BAF in normal or serum-free medium. We reasoned that BAF treatment could further induce the accumulation of LC3-II and p62 if TPC2, like starvation, promotes early autophagic progression. However, BAF treatment (1h) only induced the accumulation of LC3-II and p62 in control cells, not in TPC2 overexpressing cells, regardless of whether cells were in normal or starvation states (Figures 3A and S1B). This suggests that TPC2 is not involved in early autophagy induction. Also notably, starvation not only induced the accumulation of LC3-II but also decreased p62 in both control and TPC2 overexpressing cells (comparing lane 5 and 6 in Figure 3A), suggesting that TPC2 overexpression only inhibits but does not abolish the fusion of autophagosomes with lysosomes. Following the initiation of the phagophores, autophagosomes undergo a stepwise maturation process from early to late autophagosomes, which ultimately fuse with lysosomes to form autolysosomes. Any missteps during the maturation process could lead to the accumulation of autophagosomes (1). Therefore, we next assessed whether TPC2 signaling


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compromises early autophagosome formation. To do so, GFP-LC3 or GFP-LC3/TPC2 expressing HeLa cells were transfected with RFP-ATG5. Under normal conditions (nutrient rich), ATG5 was diffuse throughout the cytoplasm in both control and TPC2 overexpressing HeLa cells, again suggesting that TPC2 does not induce autophagy. Starvation (60 min), on the other hand, markedly induced puncta formation of ATG5, some of which were not colocalized with LC3 in both cell types (Figure 3B). These data suggest that TPC2 signaling does not inhibit the early autophagosome(s) maturation. Subsequently, we tested whether the accumulated autophagsosomes in TPC2 overexpressing HeLa cells are the result of basal cellular autophagy activity. To do so, we knocked-down ATG5, which is required for the elongation and closure of autophagosomes, in both control and TPC2 overexpressing HeLa cells (Figure S2A). As expected, not only did TPC2 overexpression in ATG5 knockdown cells fail to induce autophagsosome(s) accumulation (Figures 3C and S2B), but also knockdown of ATG5 in TPC2 overexpressing cells markedly decreased the accumulated LC3-II and p62 (Figure 3D). These results place TPC2 downstream of ATG5 in autophagy regulation and again suggest that TPC2 overexpression does not completely block the fusion of autophagosomes with lysosomes.

p62, as a cargo receptor for some ubiquitinated proteins, is first incorporated into autophagosomes before being targeted to lysosomes for degradation (58). Therefore, it is also necessary to clarify whether accumulated p62 in TPC2 overexpressing cells is due to the defects in cargo incorporation into autophagososmes. As shown in Figure 3E, under normal culture conditions, p62 puncta were already markedly accumulated in TPC2 overexpressing cells compared to control cells, and most of them were co-localized with LC3-II. Moreover, similar to control cells, starvation further induced LC3-II puncta and p62 puncta in TPC2 overexpressing cells (Figure 3E). This indicates that TPC2 does not prevent p62 incorporation into autophagosomes, and suggests that accumulated p62 in TPC2 overexpressing cells is due to the defects in later stages of autophagy. Inhibition of the fusion between autophagosomes and autolysosomes by NAADP/TPC2 signaling in HeLa cells. To

further differentiate autophagosomes and autolysosomes during autophagy, we infected the control or TPC2 overexpressing HeLa cells with lentiviruses carrying expression cassettes that encode RFP-GFP-LC3 (tfLC3), in which RFP and GFP are tandem tagged to the N-terminal of LC3 (59). Before fusion with lysosomes, the LC3-II positive autophagosomes are shown by both GFP and RFP signals as yellow puncta, and after fusion, autolysosomes are shown by only RFP signals as red only puncta because GFP loses its fluorescence in acidic pH. As expected, yellow puncta (autophagosomes) were markedly increased with few red puncta signals (autolysosomes) in wildtype, not mutant, TPC2 overexpressing cells, which was similar to the effects of BAF in control cells. In contrast, starvation dramatically increased both yellow and red puncta in control cells (Figures 4A and 4B). We further performed immunoelectron microscopy analyses to distinguish autophagosomes from autolysosomes in control and TPC2 overexpressing HeLa cells (Figure 4C). The cells were incubated with anti-LC3 antibody followed by DAB staining, and were subsequently subjected to EM analyses. Two types of autophagic structures were quantified: autophagosomes whose surfaces are LC3 positive (Figure 4C1), and autolysosomes whose inside are LC3 positive (Figure 4C2). Consistently, the numbers of autophagosomes in TPC2 overexpressing cells were significantly more than those in control cells; whereas autolysosomes were hard to find in both control and TPC2 overexpressing cells (the bottom panel of Figure 4C). Thus, theses data clearly demonstrate that TPC2 overexpression inhibits the fusion between autophagosomes and autolysosomes, thereby resulting in a marked accumulation of autophagosomes. Since TPC2 is a NAADP sensitive Ca2+-permeable channel in lysosomes, we examined the effects of NAADP-AM on the fate of autophagosomes in TPC2 overexpressing cells. As shown in Figures 4D and S3A, NAADP-AM indeed further induced the accumulation of both LC3-II and p62 only in TPC2 overexpressing HeLa cells. On the other hand, treatment with Ned-19, a NAADP antagonist, decreased the levels of both LC3-II and p62 only in TPC2 overexpressing cells (Figures 4E, S3B, and S3C). Moreover, Ned-19 increased the red LC3 puncta (autolysosomes) while it decreased the yellow LC3 puncta


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(autophagosomes) in TPC2 /RFP-GFP-LC3 co-expressing HeLa cells (Figures 4A and 4B). Taken together, our results further demonstrate that the activation of NAADP/TPC2 signaling compromises autophagy progression, resulting in autophagosome accumulation. Inhibition of autophagic flux by endogenous TPC2 signaling in mouse embryonic stem cells. Upon withdrawal of self-renewal stimuli, mouse embryonic stem (ES) cells can spontaneously and robustly differentiate into neural progenitors in adherent monoculture (60). Interestingly, autophagy was markedly induced during neural differentiation of mouse ES cells (Figures 5A and S4A). TPC2 is detectable in mouse ES cells (Figure 5B) and quantitative RT-PCR analyses showed that the expression of TPC2 mRNA was dynamically regulated during neural differentiation of mouse ES cells: TPC2 was initially significantly downregulated, but was increased again during the late stages of differentiation (Figure S4B). Thus, we used lentivirus-mediated short hairpin RNA (shRNA) or cDNA to stably knockdown the expression of TPC2 (Figure 5B) or to overexpress TPC2 (Figure 5C), respectively, in mouse ES cells.

The control scramble-shRNA infected, TPC2-knockdown, or TPC2 overexpressing cells were then induced to differentiate into neural lineages following the monolayer culture protocol. Similar to HeLa cells, the LC3-II level was markedly accumulated in differentiated TPC2 overexpressing ES cells as compared to that in differentiated control ES cells, and BAF treatment only increased LC3-II levels in control but not in TPC2 overexpressing ES cells (Figures 5D and S4H). On the other hand, LC3-II levels (Figures 5E, S4C, and S4F) or LC3 puncta (Figures S4D and S4E) were significantly decreased in differentiated TPC2 knockdown ES cells compared to those in control ES cells. Two opposite mechanisms can lead to less LC3-II level or LC3 puncta in TPC2 knockdown cells: a decrease in autophagic induction causes less LC3-II lipidation; or an enhanced fusion between autophagosome and lysosome leads to a faster LC3-II degradation. Interestingly, BAF treatment further increased LC3-II in TPC2 knockdown cells to a level similar to that in control cells (Figures 5E and S4G), suggesting that the decreased LC3-II level in TPC2 knockdown cells during differentiation is not due to inhibition of autophagic induction but because of the enhanced autophagy progression leading to faster LC3-II degradation.

To further assess the possibility that TPC2 knockdown facilitates the fusion between autophagosome and lysosome to result in decreased LC3-II levels during differentiation, the RFP-GFP tandem-tagged LC3 (tfLC3) was again utilized to distinguish autophagosomes from autolysosomes in differentiated ES cells. Not surprisingly, in tfLC3 expressing ES cells during neural differentiation, more yellow LC3 puncta (autophagosomes) existed in TPC2 overexpressing cells, whereas more red only LC3 puncta (autolysosomes) appeared in TPC2 knockdown cells (Figure 5F), indicating that autophagosomal-lysosomal fusion is indeed facilitated by TPC2 knockdown but inhibited by TPC2 overexpression. Clearly, these data further demonstrate that the endogenous TPC2 signaling inhibits the fusion between autophagosomes and autolysosomes as well. In addition, we found that TPC2 knockdown facilitated mouse ES cells to differentiate into neural progenitors, whereas TPC2 overexpression inhibited mouse ES cells differentiated into neural progenitors (60). The effects of mTOR on TPC2-induced autophagy inhibition. The mTOR kinase is one of the key regulators for autophagy, and the activation of mTOR complex 1 occurs at the surface of lysosomes and requires v-ATPase activity (61-63). Moreover, increased mTOR activity is found to be essential for termination of autophagy and reformation of lysosomes (64), and it has been recently shown that ATP activates mTOR to inhibit TPC2 gating (65). We, therefore, assessed whether TPC2 inhibits the fusion between autophagosomes and autolysosomes via mTOR. Unexpectedly, TPC2 overexpression in HeLa cells not only failed to affect mTOR activity but also had no effect on the ability of starvation to inhibit mTOR (Figures 6A and S5B). Notably, inhibition of mTOR induced by starvation in TPC2 overexpressing cells was accompanied by not only the increase of LC3-II but also the decrease of p62, suggesting that mTOR inhibition does not increase the ability of TPC2 to inhibit autophagy (Figure 6A). Similarly, treatment of cells with rapamycin (50 µM), an mTOR inhibitor, for 3 hr increased LC3-II levels and decreased p62 levels in both control and TPC2 overexpressing cells (Figure 6B). Therefore, these data suggest that TPC2 signaling suppresses autophagy maturation independent of mTOR.


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The effects of Ca2+ on TPC2-induced autophagy inhibition. NAADP can induce Ca2+ release from lysosomes (25), and we found that NAADP treatment actually inhibited autophagosomal-lysosomal fusion (Figures 4C and S3A). Thus we next examined whether NAADP/TPC2 signaling suppresses autophagy progression via Ca2+ release from lysosomes. As shown in Figure 6C, after 1 or 2 h of treatment of cells with BAPTA-AM, a Ca2+ chelator, both LC3-II and p62 were markedly decreased in TPC2 overexpressing HeLa cells, but not in control cells. Similarly, BAPTA-AM treatment for 1 h significantly decreased LC3-II puncta in TPC2 overexpressing HeLa cells (Figure 6D). Collectively, these data suggest that the inhibition of autophagy progression by NAADP/TPC2 is dependent on Ca2+. TPC2 on lysosomal pH in HeLa and mouse ES cells. Interestingly, brief treatment of cells with BAF (~1 h) failed to further induce the accumulation of LC3-II and p62 in TPC2 overexpressing cells (Figures 3A and S5A), yet longer treatment with BAF (> 3 h) actually further induced the accumulation of these two proteins (Figures S5A and S5B). Considering that BAF blocks autophagosomal-lysosomal fusion by raising lysosomal pH via inhibiting the V-proton ATPase and that it has been previously shown that NAADP increases the pH of acidic Ca2+ stores in sea urchin egg (66,67), we speculated that TPC2 overexpression might increase lysosomal pH as well. We first applied LysoSensor Green DND-189 (pKa = ~ 5.2) to qualitatively measure lysosomal pH (52). LysoSensor Green DND-189 permeates cell membranes and accumulates in acidic intracellular organelles, and its fluorescence increases or decreases in acidic or alkaline environments, respectively. As shown in Figures 7A, S5C, and S5D, TPC2 overexpression indeed raised lysosomal pH in HeLa cells, which was similar to that by 1 h of BAF treatment but was less than that by 3 h of BAF treatment. This also explains why only 3 h, not 1 h, of BAF treatment further induced the accumulation of LC3-II and p62 in TPC2 overexpressing HeLa cells (Figures S5A and S5B). We further quantified lysosomal pH by dual-emission ratio imaging of LysoSensor Yellow/Blue DND-160 stained cells (Figure S5E), and found that lysosomal pH in TPC2 overexpressing HeLa cells was increased from pH 4.9 in control cells to pH 5.2 (Figure 7B).

Similar results were observed in mouse ES cells (Figures 7C and 7D). In addition, NAADP-AM transiently raised lysosomal pH only in TPC2 overexpressing HeLa cells (Figure S5F), which correlated with its effects on LC3-II accumulation (Figure 3D). Next, we assessed whether reacidifying lysosomes in TPC2 overexpressing HeLa cells could relieve the fusion blockage of autophagosomes and lysosomes. An established ammonium chloride (NH4Cl) pulse-wash technique was adopted to artificially acidify lysosomal pH (68). To do so, cells were initially incubated with NH4Cl. The accumulation of ammonia in lysosomes resulted in an increase of lysosomal pH, which was indicated by a drop of LysoSensor Green DND-189 fluorescence in NH4Cl pulsing cells compared to that in cells without NH4Cl pulsing (time 0 in Figure 7E). When NH4Cl was removed from the medium by thorough washing, the efflux of ammonia caused the pH in the lysosomes to drop rapidly and transiently reach a value lower than that before the ammonium chloride was added, which was indicated by the increases of LysoSensor Green DND-189 fluorescence in NH4Cl pulsed cells after washing (Figures 7E). The transient reacidified lysosomal pH, produced after washing out ammonium chloride, mitigated the fusion blockage in TPC2 overexpressing cells (Figure 7F). Taken together, these data clearly demonstrate that TPC2 signaling suppresses autophagosomal-lysosomal fusion by alkalizing the lysosomal pH. TPC2 on Rab7 recruitment to autophagosomes. Rab7, a small GTPase, is required for autophagosome-lysosome fusion (6). We, thus, assessed the effects of TPC2 overexpression on the localization of Rab7 by transfecting RFP-Rab7 into GFP-LC3 or GFP-LC3/TPC2-overexpressing HeLa cells. We found that starvation induced the co-localization of Rab7 with both GFP-LC3 and Lamp1 in control cells (Figure 8A). On the other hand, RFP-Rab7 puncta were colocalized with Lamp1, but few of them were colocalized with LC3-GFP in TPC2 overexpressing cells (Figure 8B). Next, we assessed whether transient reacidified lysosomal pH by NH4Cl pulse-wash in TPC2 overexpressing HeLa cells restores the association of Rab7 with autophagosomes. As shown in Figure 8C, in majority (2/3) of TPC2 overexpressing HeLa cells, reacidified lysosomal pH (10 min after NH4Cl washing) markedly decreased LC3 puncta, which is


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consistent with the fact that re-acidification decreased LC3-II levels in TPC2 overexpressing cells as shown in Figure 7F. Interestingly, Rab7 failed to co-localize with the remaining LC3 puncta in these cells. We speculate that lysosomal reacidification in majority of TPC2 overexpressing cells quickly relieves the fusion blockage of autophagosomes and lysosomes to form autolysosomes, thereby leading to the degradation of LC3-II and the quenching of GFP fluorescence. The remaining LC3 puncta in these cells likely represent the immature autophagosomes induced by the repetitive post NH4Cl washing. However, around 1/3 of TPC2 overexpressing cells still contained large number of LC3 puncta after removing NH4Cl from medium, and Rab7 puncta were indeed co-localized with LC3-II in these cells (Figure 8D). We reason that lysosomal reacidification in these cells might be slow and just start to recruit Rab7 to the autophagosomes but not yet induce the fusion of autophagosomes with lysosomes. Taken together, these results suggest that TPC2 signaling prevent the recruitment of Rab-7 to autophagosomes by alkalizing lysosomal pH to inhibit autophagosomal-lysosomal fusion. TPC2 on general endosomal-lysosomal degradation. Since the increase of lysosomal pH normally compromises the lysosomal activity, we assessed whether TPC2 signaling simply disrupts the general lysosomal functions to inhibit autophagy maturation. The processing of cathepsin L from the precursor form to its mature form has been commonly used as a marker for lysosomal activity, yet we found that the processing of cathepsin L was only slightly compromised in TPC2 overexpressing HeLa cells compared to that in control cells (Figure 9A), which might be due to the fact that TPC2 overexpression just marginally increased lysosomal pH (Figure 7B). Next, an epidermal growth factor receptor (EGFR) degradation assay was performed to examine whether the TPC2 signaling also affects the general endosomal-lysosomal pathway. In this assay, both control and TPC2 overexpressing HeLa cells were treated with EGF. In principle, after EGF binds to its receptors (EGFR), the receptor complex undergoes endocytosis and is targeted to lysosomes where it is ultimately degraded. As shown in Figures 9B and S6A, BAF inhibited EGF-triggered EGFR degradation in a dose-dependent manner, whereas TPC2 overexpression had little effect on it, which was similar to that in control cells. Finally, a

DQ-BSA-green degradation assay was applied to measure the general endosomal-lysosomal degradation. DQ-BSA-green is a BSA labeled with a self-quenching fluorescent dye. After DQ-BSA-green is delivered to lysosomes via endocytosis, it is hydrolyzed into single dye-labeled peptides by lysosomal proteases, thereby relieving self-quenching and the fluorescence can subsequently be monitored by flow cytometry. As shown in Figures 9C and S6B, TPC2 overexpression also had little effect on BSA degradation whereas BAF markedly inhibited it. Collectively, these results document that TPC2 signaling does not inhibit the general endosomal-lysosomal degradation. DISCUSSION Here, we provide definitive evidence that NAADP/TPC2 signaling induced the accumulation of matured autophagosomes by inhibiting autophagosomal-lysosomal fusion (Figures 2-5). Perturbing TPC2/NAADP signaling by either TPC2 knockdown or addition of an NAADP antagonist, on the other hand, facilitated the progression of autophagy by promoting the formation of autolysosomes (Figures 3-5). Instead of affecting or functioning downstream of mTOR (Figures 6A and 6B), TPC2 signaling actually alkalinized lysosomal pH to suppress autophagosomal-lysosomal fusion (Figure 7), likely by preventing the recruitment of Rab-7 to autophagosomes (Figure 8). Yet, TPC2 signaling had little effects on general endosomal-lysosomal degradation (Figure 9).

The acidic milieu in lysosomes is generated by the V-ATPase, a multi-subunit protein complex, which pumps protons into the lysosomal lumen against an electrochemical gradient at the expense of ATP hydrolysis (69). The influx of protons into the lysosomal lumen also creates a lumenpositive membrane potential, which, reciprocally, mitigates the ability of the V-ATPase to continue pumping protons. Thus, transporters for cation efflux, or anion influx, or both, must be activated to maintain the balance of acidic pH and membrane potential inside lysosomes (70). Although Cl- influx, possible via ClC-7 or CFTR, has been shown to dissipate the restrictive electrical gradient for lysosomal acidification (71,72), whether Cl- is the main counter ion is debatable and the identity of Cl- transporters in lysosomes also remains controversial (70). Likewise, Ca2+, whose


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concentration in lysosomes is high and is partially dependent on H+ gradient (73), has been proposed to constitute another counter ion pathway. TPC2, TRPM2, and TPML1 have all been shown to mediate Ca2+ release from lysosomes triggered by NAADP or ADPR (24,36). Yet, here we found that NAADP treatment or TPC2 overexpression increased lysosomal pH to inhibit autophagosomal- lysosomal fusion (Figure 7), and chelating Ca2+ by BAPTA-AM (≥ 1 hr treatment) relieved TPC2-induced accumulation of both LC3-II and p62 (Figures 6C and 6D), indicating that Ca2+ is required for NAADP/TPC2-induced lysosomal alkalinization and subsequent fusion blockage. These data further suggest that Ca2+ is not the counter ion for maintaining high H+ in lysosomes and the search for real counter ion pathway must continue.

Regarding how NAADP/TPC2-triggered Ca2+ release raises lysosomal pH, we speculate that either a putative Ca2+/H+ exchanger (74), or an unidentified lysosomal Ca2+ ATPase (75), similar to SERCA, or the coupling of several lysosomal proton-cation counter-transporters, such as sequential action of Ca2+/Na+ exchangers and Na+/H+ exchangers, can be activated to refill the lysosomal Ca2+ pools at the expense of proton efflux upon Ca2+ release from lysosomes via TPC2. Obviously, deciphering how Ca2+ enters lysosomes is the key to resolving this mystery. However, other than the V-ATPase, ion channels or transporters responsible for the formation of the unique ionic environment, e.g. Na+, Cl-, K+, and Ca2+, inside lysosomes remain unknown (74). A future lysosomal proteomics study should help in identifying the respective transporters.

Here we found that the HeLa cell line, an NAADP incompetent line, can be reconstituted to be NAADP competent by overexpressing a wildtype, but not a pore-mutant, TPC2 (Figures 1A and 1C), and treatment of TPC2 overexpressing cells with Ned-19, a NAADP antagonist, markedly inhibited NAADP-induced Ca2+ release (Figure 1C). Thus, these data provide unambiguous evidence that NAADP can activate TPC2 for Ca2+ release and echo the previous finding that TPC2 is an NAADP effector (26-31). Yet, we also found that TPC2 overexpression in HeLa cells, in the absence of exogenous NAADP treatment, already alkalinized lysosomal pH (Figures 7A-7D) and inhibited autophagy maturation (Figures 2-5). Although the effects of TPC2 overexpression

might be due to endogenous NAADP, the effects of NAADP-AM on Ca2+ release (Figures 1A and 1C) and autophagy inhibition (Figures 4D and S3A) in TPC2 overexpressing cells were transient. It has recently been shown that NAADP does not directly bind to TPC2, arguing that NAADP might first bind to accessory proteins within the TPC2 complex, and subsequently activate TPC2 (40-42). Therefore, the effects of TPC2 overexpression in HeLa or mouse ES cells might be caused by accessory proteins in the TPC2 complex, and NAADP, induced by extracellular stimuli or cellular stress (25,76), could bind to these accessory proteins to further activate TPC2. However, at this stage, we still could not exclude the possibility that the effects of TPC2 overexpression are due to endogenous NAADP, as the transient effects of NAADP-AM might be due to its instability in aqueous solution (54).

Two recent papers found that TPC2 forms a Na+ channel in lysosomes, which can be potentiated by PI(3,5)P2, an endolysosome- specific phosphoinositide (PIP), and inhibited by ATP (65,77). It was proposed that high intracellular ATP levels enable mTOR to phosphorylate TPC2, thereby keeping it at a closed state. On the other hand, cell starvation results in lower ATP levels and dissociation of mTOR from TPC2, thus activating TPC2 to allow Na+ release from lysosomes (65). However, here we found that overexpression of a wildtype, not a pore-mutant, TPC2 in cells maintained in nutrient rich medium already alkalinized lysosomal pH and suppressed autophagosome-lysosome fusion (Figures 2-5), suggesting that TPC2 in high ATP levels is already open. Starvation promoted autophagy, e.g. LC3-II induction and p62 degradation, in both control and TPC2 overexpressing cells (Figures 3A and 6A), arguing that nutrient deprivation does not further activate TPC2 to inhibit autophagy progression. Thus these data suggested that TPC2 suppresses autophagosome-lysosome fusion independent of mTOR or ATP levels. However, the role of intracellular PI(3,5)P2 or Na+ release from lysosome on TPC2-mediated autophagy inhibition remains to be determined.

Also notably, these two papers found that NAADP cannot activate TPC2 for Ca2+ release from lysosomes mainly by performing patch clamp study on enlarged endolysosomes treated with vacuolin-1 (65,77), which is contrary to some of results presented in this study and a


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body of work published previously (26-31). Further investigations are needed to resolve the controversy and a recent study suggests that a GFP tagged on the N-terminal of TPC1 disrupts NAADP action (78).

Interestingly, mutation of TPC2, M484L or G734E, has been associated with blond versus brown hair by genome-wide association studies for pigmentation (79). The melanosome, a lysosome-related organelle, is the site of synthesis and transport of melanin pigments, thereby providing tissues with color and photoprotection. In addition, autophagy and autophagy regulators have been shown to play roles in both the biogenesis of melanosomes and melanosome destruction (80). Therefore, it is of interest to assess whether and how the two identified TPC2 mutants affect melanosome biogenesis or destruction via autophagy related processes. Without doubt, TPC2 signaling could play roles in other autophagic related cellular

processes. Given that autophagy plays important roles in a wide variety of cellular processes and dysfunctional autophagy has been associated with many human diseases, it is important to further dissect the TPC2 signaling to identify novel regulators in this pathway. Development of novel reageants targeting TPC2 signaling, e,g. NAADP analogues, to manipulate autophagy should provide alternative pharmaceutical intervention in treatment of autophagy-related human disorders. Along this line, Niemann-Pick disease, a human neurodegenerative lysosomal storage disorder, has been associated with abnormal lysosomal Ca2+ homeostasis, and its cellular phenotype is similar to that in TPC2 overexpressing cells (Figure 2A) (29,81). Whether TPC2-mediated autophagic regulation contributes to this disorder is, therefore, of great interest to pursue.

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FOOTNOTES We thank Grant Churchill for providing us several batches of NAADP-AM and Ned-19, Connie Lam and Yong-Juan Zhao in Prof. Hon-Cheung Lee’s lab for sharing reagents, King-Ho Cheung, Hon-Cheung Lee, and members of Yue lab for advice on the manuscript. Confocal imaging and FACS analyses were performed in Faculty of Medicine Core Facility at HKU. This work was supported by Research Grant Council (RGC) grants (HKU 782709M and HKU 785911M), and a Special Fellow Award from the Leukemia and Lymphoma Society of America (J. Y).

The abbreviations used are: NAADP, Nicotinic acid adenine dinucleotide phosphate; TPC2, two pore

channel 2; tfLC3, RFP-GFP tandem-tagged LC3; BAF, bafilomycin; EGFR, epidermal growth factor

receptor; TEM, transmission electron microscopy; BSA, bovine serum albumin.

FIGURE LEGENDS Figure 1. Reconstitution of NAADP-competent HeLa cells by overexpressing TPC2. (A)

NAADP-AM (100 nM) only induced Ca2+ release in wildtype, but not in an inactive mutant, TPC2

overexpressing HeLa cells. (B) Localization of Lamp1-RFP, TPC2, and TPC2 249L/G in HeLa cells.

A rabbit polyclonal TPC2 antibody was used to detect overexpressing TPC2. Scale bar = 5 µm. (C)

NAADP-AM induced Ca2+ release in TPC2 overexpressing HeLa cells, which was markedly inhibited

by Ned-19 (10 µM). Note the bell shaped concentration dependence curve for NAADP-AM. Quantification of the [Ca2+]i peak induced by drug treatment in (A) and (C) is expressed as mean ±

S.E., n= 30-40 cells. The * symbols indicate the results of t Test analysis, p < 0.05.

Figure 2. TPC2 overexpression induced a defect in autophagic degradation in HeLa cells. (A) Large

numbers of autophagic vacuoles were observed in TPC2 overexpressing cells, but not in control cells,

under electron microscope. (B) Wildtype, not an inactive mutant, TPC2 overexpression induced the

accumulation of both LC3-II and p62. (C) Wildtype, not an inactive mutant, TPC2 overexpression

markedly induced endogenous LC3-II puncta (green) in HeLa cells, which were not colocalized with

RFP-Lamp1. Scale bar = 5 µm. Quantification of LC3 puncta is expressed as mean ± S.E., n= ~ 80

cells. The * symbols indicate the results of t Test analysis, p < 0.05. (D) Starvation induced LC3

puncta, some of which were colocalized with LAMP1 puncta, whereas the LC3 puncta induced by

bafilomycin or TPC2 expression failed to co-localize with LAMP1 in RFP-LAMP1/GFP-LC3 HeLa

cells. Scale bar = 10 µm.


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Figure 3. TPC2 overexpression did not affect the early autophagic progression in HeLa cells. (A)

Bafilomycin (10 nM, 1 h) only induced the accumulation of LC3-II and p62 in control, not TPC2

overexpressing, HeLa cells, whereas starvation induced autophagic progression in both cells. The

quantification of four boxed lanes is shown. (B) Starvation (60 min) induced RFP-ATG5 puncta, most

of which were not colocalized with GFP-LC3 puncta, in both control and TPC2 overexpressing HeLa

cells. (C) TPC2 failed to induce LC3-II and p62 in ATG5 knockdown cells. (D) ATG5 knockdown

markedly inhibited TPC2-induced accumulation of LC3-II and p62 in HeLa cells. (E) TPC2

overexpression did not affect p62 incorporation into autophagosome in HeLa cells. Scale bar = 10 µm.

Figure 4. TPC2 signaling inhibited the progression of autophagy in HeLa cells. (A) HeLa cells were

transfected with a RFP-GFP tandem fluorescent tagged LC3 (tfLC3) plasmid. TPC2 overexpression

induced yellow puncta with few red only puncta signals in cells. In contrast, starvation dramatically

increased both yellow and red puncta in control cells. Scale bar = 10 µm. (B) Quantitation of % of

yellow puncta or red only puncta/per cell of (A) are expressed as mean ± S.E., n= ~ 80 cells. The * symbols indicate the results of t Test analysis, p < 0.05. (C) Control and TPC2 overexpressing cells

were processed for immunoelectron microscopy analyses. C1: representative autophagosomal

structures, whose surfaces are LC3-positive, in TPC2 overexpressing HeLa cells, C2: representative

autolysosomal structures, whose inside are LC3 positive, in TPC2 overexpressing HeLa cells.

Quantitation of autophagosomes and autolysosomes per cell are expressed as mean ± S.E., n= ~ 40

cells (bottom panel). (D) NAADP-AM (100 nM) increased the accumulation of both LC3-II and p62

only in TPC2 overexpressing, not the control, HeLa cells. (E) Ned-19 (10 µM) decreased the levels of both LC3-II and p62 in TPC2 overexpressing, but not the control, HeLa cells.

Figure 5. TPC2 signaling inhibited the progression of autophagy during neural differentiation of

mouse ES cells. (A) In vitro neural differentiation of mouse ES cells initiated by monolayer adherent

culture markedly induced LC3-II in D3 mouse ES cells. (B) TPC2 knockdown by shRNA in ES cells

was determined by TPC2 immunoblot analyses. (C) TPC2 is expressed in lysosome-related organelles

as it is co-localized with LysoTracker (red). Scale bar = 50 µm. (D) TPC2 overexpression induced accumulation of LC3-II in ES cells during neural differentiation. Treatment with bafilomycin (100

nM) only induced the accumulation of LC3-II in control cells, but not in TPC2 overexpressing cells.

(E) TPC2 knockdown decreased the accumulation of LC3-II in ES cells during neural differentiation.

Treatment with bafilomycin (100 nM) induced the accumulation of LC3-II in both control and TPC2

knockdown cells. (F) Mouse D3 ES cells were infected with RFP-GFP tandem fluorescent tagged

LC3 (tfLC3) lentiviruses. TPC2 overexpression induced yellow puncta with few red only puncta

signals, whereas TPC2 knockdown increased the red only puncta in ES cells after 4 days of neural

differentiation. Quantification of at least 50 cells from three independent experiments is expressed as


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mean ± S.E. and shown in the right columns. The * symbols indicate the results of t Test analysis, p < 0.05. Scale bar = 10 µm.

Figure 6. Ca2+, not mTOR, was involved in TPC2-induced autophagosome accumulation in HeLa

cells. (A) Starvation induced LC3-II levels and promoted the degradation of p62 in both control and

TPC2 overexpressing HeLa cells, accompanied with the inactivation of mTOR. TPC2-induced

accumulation of LC3-II and p62 was decreased by Ned-19 (10 µM). (B) Treatment of cells with

rapamycin (50 µM) for 3 hr promoted autophagy in both control and TPC2 overexpressing cells. (C)

and (D) BAPTA-AM (20 µM) decreased the levels of LC3-II and p62 (C) and LC3-II puncta (D) in TPC2 overexpressing HeLa cells. Quantification of LC3 puncta is expressed as mean ± S.E., n= ~ 50

cells. The * symbols indicate the results of t Test analysis, p < 0.05. Scale bar = 10 µm.

Figure 7. TPC2 signaling increased lysosomal pH to suppress autophagy progression. (A) TPC2

overexpression or bafilomycin (BAF) treatment induced an increase of lysosomal pH in HeLa cells as

determined by FACS analyses of Lysosensor DND-189 stained cells. (B) Quantification of lysosomal

pH in control and TPC2 overexpressing HeLa cells as determined by dual-emission ratio imaging of

LysoSensor Yellow/Blue DND-160 stained cells. (C) and (D) TPC2 overexpression or BAF treatment

induced an increase of lysosomal pH in D3 ES cells as determined by FACS analyses (C) and

microplate reader measurement (D) of Lysosensor DND-189 stained cells. (E) Washing out NH4Cl in

both control and TPC2 overexpressing HeLa cells after pulsing with NH4Cl (100 µM) for 20 min transiently reacidified lysosomal pH. The data are expressed as mean ± S.D., n = 4. (F) Washing out

NH4Cl decreased the accumulation of LC3-II and p62 in TPC2 overexpressing cells after been pulsing

with NH4Cl (100 µM) for 20 min. The first lanes show samples without NH4Cl pulsing. The graph in (B) and (D) represents data from three independent experiments, and the data are expressed as mean ±

S.D., n = 3. The * symbols indicate the results of t Test analysis, p < 0.05.

Figure 8. TPC2 prevent the recruitment of Rab-7 to autophagosomes in HeLa cells. (A) Starvation (60

min) induced RFP-Rab7 puncta, which were colocalized with GFP-LC3 puncta (left panel) or Lamp1

puncta (right panel), in control HeLa cells. (B) RFP-Rab7 puncta were colocalized with Lamp1 (right

panel), but not with GFP-LC3 puncta in TPC2 overexpressing HeLa cells. (C) Removing NH4Cl (10

min) from medium decreased LC3 puncta in majority (2/3) of TPC2 overexpressing cells pulsed with

NH4Cl, and RFP-Rab7 failed to colocalize to the remaining LC3 puncta. (D) After washing out

NH4Cl for 10 min, 1/3 of TPC2 overexpressing cells still contained large number of LC3 puncta,

which were co-localized with RFP-Rab7. Scale bar = 10 µm.

Figure 9. TPC2 did not inhibit general endosomal-lysosomal degradation in HeLa cells. (A) The

processing of cathepsin L from the precursor form to its mature form in control or TPC2

overexpressing HeLa cells treated with or without bafilomycin for 1 or 3 h. (B) TPC2 overexpression


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failed to change EGF-induced EGFR degradation, whereas BAF (100 nM) completely abolished it in

HeLa cells. (C) TPC2 overexpression did not inhibit the degradation of DQ-BSA-green in HeLa cells,

while BAF (100 nM) markedly inhibited it.


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Jianbo YueYing-Ying Lu, Bai-Xia Hao, Richard Graeff, Connie W. M. Wong, Wu-Tian Wu and

Lysosomal pHTPC2 Signaling Inhibits Autophagosomal-Lysosomal Fusion by Alkalizing

published online July 8, 2013J. Biol. Chem.

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