Autophagosome-Lysosome Fusion Depends on the pH in Acidic ...

[Autophagy 3:2, 154-157, March/April 2007]; ©2007 Landes Bioscience

Addendum

Autophagosome-Lysosome Fusion Depends on the pH in Acidic Compartments in CHO Cells Akinori Kawai Hiromi Uchiyama Syuichi Takano† Nobuhiro Nakamura* Shoji Ohkuma

Abstract

Original manuscript submitted: 10/24/06 Manuscript accepted: 11/29/06 Previously published online as an Autophagy E-publication: http://www.landesbioscience.com/journals/autophagy/abstract.php?id=3634

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*Correspondence to: Nobuhiro Nakamura; Division of Life Sciences; Graduate School of Natural Science and Technology; Kanazawa University; Kakuma, Kanazawa, Ishikawa 920-1192, Japan; Tel.: 81.76.234.4464; Fax: 81.76.234.4466; Email: [email protected]

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Address: University of Texas Health Science Center at San Antonio; San Antonio, Texas USA

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†Present

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Division of Life Sciences; Graduate School of Natural Science and Technology; Kanazawa University; Kakuma, Kanazawa, Ishikawa Japan

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Autophagy is the bulk degradation of cytoplasmic constituents in response to starvation and other environmental or intracellular cues. During this process, most of the cytoplasm is sequestered into autophagosomes, which then fuse with lysosomes where the degradation of the sequestered material proceeds. We investigated the relationship between autophagosome‑lysosome fusion and the pH in acidic compartments by visualizing the fusion process using fluorescence in CHO cells. In this experiment, mitochondria were labeled with GFP by transfecting CHO cells with the presequence of ornithine transcarbamylase, and lysosomes were labeled with Texas Red Dextran; any fusion was identified by the colocalization of mitochondria (in autophagosomes) and lysosomes using fluorescence microscopy. When CHO cells were treated with rapamycin or starvation medium to induce autophagy, the colocalization of fluorescence was observed. Whereas when they were treated with 3‑MA, an inhibitor of autophagy, the colocalization disappeared. We conclude that the colocalization reflects the fusion of autophagosomes and lysosomes. Moreover, when the CHO cells were treated with drugs that increase the pH of acidic compartments, the colocalization disappeared. This suggests that the autophagosome‑lysosome fusion is inhibited by increasing pH in acidic compartments independently of V‑ATPase activity in CHO cells.

Introduction

macroautophagy, fusion, pH, acidic compartment, mitochondria

Autophagy is a system of degradation involving lysosomes wherein various cellular components including organelles are broken down in response to starvation and other environmental or intracellular cues. Although macroautophagy had been observed by electron microscopy in mammalian cells, its mechanism was unclear. However, with the discovery that autophagy occurs in yeast, the mechanism involved, especially ubiquitin‑like reactions, has come to be clarified.1,2 In this process, portions of the cytoplasm are nonselectively sequestered into double membrane vesicles called autophagosomes. The PAS (preautophagosomal structure) identified in yeast cells is required for the de novo formation of autophagosomes.3 The autophagosomes subsequently fuse with endosomes4‑7 and with lysosomes, forming autolysosomes, the contents of which are degraded by hydrolases therein. But precisely how autolysosomes form including the fusion between autophagosomes and lysosomes is not yet clear. Bafilomycin A1, a potent selective inhibitor of V‑ATPase, inhibits the internal acidification of organella containing V‑ATPase (lysosomes and endosomes etc.).8 In mammalian cells, the inhibition of V‑ATPase prevents autophagosomes from fusing with lysosomes or endosomes, resulting in the accumulation of autophagosomes in the cytosol and a decrease in the number of autolysosomes. This means that inhibition of internal acidification in acidic organelles disturbs autophagosome‑lysosome fusion, preventing autophagy.6 However, there is no evidence that directly traces fusion to degradation or that the internal pH of organelles regulates fusion. Therefore we have tried to construct an index of “the fusion between autophagosomes and lysosomes”, to directly demonstrate that such fusion is related to the pH of acidic compartments. This is possible by labeling mitochondria with GFP by transfecting CHO cells with the presequence of ornithine transcarbamylase and by labeling lysosomes with Texas Red Dextran, and determining the fusion based on the colocalization of mitochondria (in autophagosomes) and lysosomes using fluorescence microscopy.

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Acknowledgements

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We are indebted to Prof. Mori M. (Kumamoto University, Japan) for generously providing pCAGGS-pOTC-GFP. We thank Ms. Nagami Yamashita for her supports. Prof. Ohkuma died on November 5, 2006 by myocardial infarction at his home. We greatly miss him as a scientist and a friend. We offer sincere thanks to all the friends, colleagues and former collaborators of Prof. Ohkuma who showed him kindness during his lifetime.

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Addendum to:

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Quantitative Monitoring of Autophagic Degradation

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Akinori Kawai, Syuichi Takano, Nakamura and Shoji Ohkuma

Nobuhiro

Biochem Biophys Res Commun 2006; 351:71-7

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Autophagosome-Lysosome Fusion Requires Internal Acidity

Figure 1. Autophagy and the colocalization of mitochondria and lysosomes. mtGFP‑CHO cells were incubated overnight with 500 mg/ml of Texas Red Dextran. They were then incubated in F‑12 medium containing 100 nM rapamycin and protease inhibitors (A), or in F‑12 medium deprived of amino acids and serum but containing 10 mM 3‑MA and protease inhibitors (B) at 37˚C for 12 h or 6 h, and washed three times with PBS. After fixation with 4% paraformaldehyde, they were observed under a laser scanning confocal microscope. (a) Texas Red Dextran (lysosome; red), (b) GFP (mitochondria, green), (c) phase contrast micrographs, (d) merged images of (a and b). Yellow exhibits colocalization of GFP and Texas Red Dextran. An appropriate slice of confocal sections is shown. Bar=10 mm.

In this paper we show that the fusion between autophagosomes and lysosomes in CHO cells is inhibited by increasing the pH in acidic compartments independently of V‑ATPase as well as by inhibiting V‑ATPase. The results suggest that autophagosome‑lysosome fusion is regulated by the internal pH in acidic organelles.

Results Autophagosome‑lysosome fusion in autophagy. In autophagy, mitochondria are thought to be surrounded by double membrane structures termed autophagosomes which fuse with lysosomes. We established an index that directly determines whether mitochondria have reached the stage of degradation by lysosomes in the autophagic process. Cells in which both mitochondria and lysosomes were labeled fluorescently were used, and the fusion was determined by their colocalization. To label mitochondria, a construct expressing GFP fused with the presequence of mitochondrial ornithine transcarbamylase was introduced into CHO‑K1 cells (mtGFP‑CHO cells).9 mtGFP‑CHO cells were incubated overnight with Texas Red Dextran to label lysosomes. In this case, cells were incubated in medium containing protease inhibitors to inhibit the degradation of mitochondria. After fixation, they were observed under a laser scanning confocal microscope. Some mitochondria colocalized with lysosomes and the colocalization depended on starvation.21 The colocalization may reflect the state of autolysosomes in the autophagic pathway. Moreover, 3D images of the colocalization were constructed by confocal laser scanning microscopy. It was found that granular mtGFP was completely surrounded by acidic compartments.21 In addition, we tried to identify the condition for the induction or inhibition of autophagy; we used rapamycin to induce autophagy or 3‑methyladenine (3‑MA)10 to inhibit autophagy in starved cells. When mtGFP‑CHO cells were treated with rapamycin,11‑12 the colocalization was observed. The colocalization was also confirmed by 3D imaging (data not shown). When the cells were treated with 3‑MA, no colocalization was apparent. Therefore, we conclude that the colocalization reflects the fusion of autophagosomes with lysosomes (Fig. 1). Inhibition of autophagosome‑lysosome fusion by a Rise of pH of acidic compartments. Bafilomycin A1 is a selective inhibitor of V‑ATPase. It is also used to inhibit the fusion between autophagosomes and lysosomes. We used basic drugs (ammonium chloride and chroloquine diphosphate) and acidic ionophore (nigericin) to raise the pH in acidic compartments independently of V‑ATPase activity.13 We confirmed that these drugs raised the lysosomal pH under the above conditions (data not shown). When the pH in acidic compartments was increased, no colocalization of lysosomes and mitochondria was observed (Fig. 2). These drugs induce vacuolization. We examined whether the colocalization between lysosomes and mitochondria is affected by www.landesbioscience.com

vacuolization. To induce vacuolization without increasing the pH in acidic compartments, starved cells were treated with 20 mM sucrose. Under these conditions, the colocalization of lysosomes and mitochondria was observed. Therefore, autophagosome‑lysosome fusion is not affected by vacuolization (Fig. 3). These results suggest that the autophagosome‑lysosome fusion is inhibited by increasing the pH in acidic compartments independently of V‑ATPase. The pH in acidic compartments may bear a key role in the fusion between autophagosomes and lysosomes.

Discussion We show that autophagosome‑lysosome fusion is inhibited by increasing the pH in acidic compartments independently of V‑ATPase as well as by treatment with bafilomycin A1. These results are consistent with reports that the internal acidification of vesicles is essential to vesicular transport.14‑18 Furthermore, the activity of V‑ATPase is required for the maturation of endosomes

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Figure 3. The colocalization of mitochondria and lysosomes is independent of vacuolization. (A) The colocalization was analyzed as in (Fig. 1). Cells cultured in F‑12 medium deprived of amino acids and serum but containing 20 mM sucrose were observed under a laser scanning confocal microscope. (a) Texas Red Dextran (lysosome; red), (b) GFP (mitochondria, green), (c) phase contrast micrographs, (d) merged images of (a) and (b). Yellow exhibits colocalization of GFP and Texas Red Dextran. Bar=10 mm. (B) Areas marked by white squares in merged pictures of (A) were enlarged and presented with sliced images of the z‑axis of the corresponding areas (numbered). Top; x‑z images, bottom left; x‑y images, bottom right; y‑z images. Green, blue and red lines in the panels indicate the planes for x‑z, x‑y and y‑z images. An appropriate slice of confocal sections is shown. Bar = 2 mm.

Figure 2. Inhibition of the colocalization of mitochondria and lysosomes by a basic substance or acidic ionophore. The colocalization was analyzed as in Figure 1. Cells were cultured in F‑12 medium deprived of amino acids and serum but containing 10 mM ammonium chloride (a and b), 25 mM chroloquine diphosphate (c and d), 2 mg/ml nigericin (e and f) or 100 nM bafilomycin A1 (g and h). Fluorescence micrographs (a, c, e and g) and phase contrast micrographs (b, d, f and h) were taken with a laser scanning confocal microscope. Red is Texas Red Dextran (lysosomes) and green is GFP (mitochondria). Yellow exhibits colocalization of GFP and Texas Red Dextran. An appropriate slice of confocal sections is shown. Bar = 10 mm.

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and fusion between endosomes and lysosomes.14,16 The results raise the possibility that autophagosome‑lysosome fusion is also regulated by the internal pH in acidic organella. Autophagosomes fuse with endosomes before they fuse with lysosomes.7 Bafilomycin A1 does not inhibit the fusion between autophagosomes and endosomes.17 The internal pH of acidic compartments may be important in the fusion of lysosomes. But it is not clear if the internal pH of lysosomes or of autophagosomes regulates the autophagosome‑lysosome fusion, partly because the internal pH of autophagosomes is not known. When lysosomes are not acidified, the fusion between autophagosomes and lysosomes is not efficient because the contents are not degraded in autolysosomes. Therefore, autophagosome‑lysosome fusion may be regulated by pH to avoid unnecessary fusion.

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However, in yeast the fusion of autophagosomes with vacuoles was not disturbed by an inhibitor of V‑ATPase, although the inhibition of V‑ATPase causes autophagic bodies to accumulate in vacuoles upon starvation.1,18 The regulation of autophagosome‑lysosome fusion by the internal pH of acidic compartments may be specific to higher organisms. References 1. Takeshige K, Baba M, Tsuboi S, Noda T, Ohsumi Y. Autophagy in yeast demonstrated with protease‑deficient mutants and conditions for its induction. J Cell Biol 1992; 119:301‑11. 2. Mizushima N, Noda T, Yoshimori T, Tanaka Y, Ishii T, George MD, Klionsky DJ, Ohsumi M, Ohsumi Y. A protein conjugation system essential for autophagy. Nature 1998; 395:395‑8. 3. Suzuki K, Kamada Y, Ohsumi Y. Studies of cargo delivery to the vacuole mediated by autophagosomes in Saccharomyces cerevisiae. Dev Cell 2002; 3:815‑24. 4. Tooze J, Hollinshead M, Ludwig T, Howell K, Hoflack B, Kern H. In exocrine pancreas, the basolateral endocytosis pathway converges with the autophagic pathway immediately after the early endosome. J Cell Biol 1990; 111:329‑45. 5. Liou W, Geuze HJ, Geelen MJH, Slot JW. The autophagic and endocytic pathways converge at the nascent autophagic vocuoles. J Cell Biol 1997; 136:61‑70. 6. Yamamoto A, Tagawa Y, Yoshimori T, Moriyama Y, Masaki R, Tashiro Y. Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosome and lysosome in rat hepatoma cell line, H‑4‑E cells. Cell Struct Funct 1998; 23:33‑42. 7. Berg TO, Fengsrud M, Stromhaug PE, Berg T, Seglen PO. Isolation and characterization of rat liver amphisomes: Evidence for fusion of autophagosomes with both early and late endosomes. J Biol Chem 1998; 273:21883‑92. 8. Bowman EJ, Siebers A, Altendorf K. Bafilomycins: A class of inhibitors of membrane ATPase from microorganisms, animal cells, and plant cells. Proc Natl Acid Sci USA 1988; 85:7972‑6. 9. Yano M, Kanazawa M, Terada K, Namchai C, Yammaizumi M, Hanson B, Hoogenraad N, Mori M. Visualization of Mitochondrial protein import in cultured mammalian cells with green fluorescent protein and effects of overexpression of the human import receptor Tom20. J Biol Chem 1997; 272:8459‑65. 10. Seglen PO, Gordon PB. 3‑methyladenine: Specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proc Natl Acad Sci USA 1982; 79:1889‑92. 11. Blommaart EF, Luiken JJ, Blommaart PJ, Woerkom GM, Meijer AJ. Phosphorylation of ribosomal protein S6 is inhibitory for autophagy in isolated rat hepatocytes. J Biol Chem 1995; 270:2320‑6. 12. Schmelzle T, Hall MN. TOR, a central controller of cell growth. Cell 2000; 103:253‑262. 13. Ohkuma S, Poole B. Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents. Proc Natl Acad Sci USA 1978; 75:3327‑31. 14. Clague MJ, Urbe S, Anient F, Gruenberg J. Vacuolar ATPase activity is required for endosomal carrier vesicles formation. J Biol Chem 1994; 269:21‑4. 15. Presley JF, Mayor S, McGraw TE, Dunn KW, Maxfield FR. Bafilomycin A1, treatment retards transferring receptor recycling more than bulk membrane recycling. J Biol Chem 1997; 272:13929‑36. 16. Van Weert AWM, Dunn KW, Geuze HJ, Maxfield FR, Stoorvogel W. Transport from late endosomes to lysosomes, but not sorting of integral membrane proteins in endosomes, depends on vacuolar proton pump. J Cell Biol 1995; 130:821‑34. 17. Reaves B, Banting G. VacuolarATPase inactivation block recycling to the trans‑Goldi network from the plasma membrane. FEBS Lett 1994; 345:61‑6. 18. Oda K, Nishimura Y, Ikehara Y, Kato K. Bafilomycin A1 inhibits the targeting of lysosomal acid hydolases in cultured hepatocytes. Biochem Biophy Res Commun 1991; 178:369‑77. 19. Nara A, Mizushima N, Yamamoto A, Kabeya Y, Ohsumi Y, Yoshimori T. SKD1 AAA ATPase‑dependent endosomal transport is involved in autolysosome formation. Cell Struct Funct 2002; 27:29‑37. 20. Nakamura N, Matsuura A, Wada Y, Ohsumi Y. Acidification of vacuoles is required for autophagic degradation in the yeast, Saccharomyces cerevisiae. J Biochem (Tokyo) 1997; 121:338‑344. 21. Kawai A, Takano S, Nakamura N, Ohkuma S. Quantitative monitoring of autophagic degradation. Biochem Biophys Res Commun 2006; 351:71‑77.

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