Bafilomycin A1

Does bafilomycin A1 block the fusion of autophagosomes with lysosomes?
Daniel J. Klionsky, Zvulun Elazar, Per O. Seglen & David C. Rubinsztein
Published online: 22 Aug 2008.


[Autophagy 4:7, 849-850; 1 October 2008]; ©2008 Landes Bioscience

Editor’s Corner
Does bafilomycin A1 block the fusion of autophagosomes with lysosomes?

Daniel J. Klionsky,1,* Zvulun Elazar,2 Per O. Seglen3 and David C. Rubinsztein4
1Life Sciences Institute; University of Michigan; Ann Arbor, Michigan USA; 2Department of Biological Chemistry; The Weizmann Institute of Science; Rehovot Israel; 3Department
of Cell Biology; Institute for Cancer Research; Rikshospitalet HF and Department of Molecular Biosciences; University of Oslo; Oslo Norway; 4Department of Medical Genetics; Cambridge Institute for Medical Research; Cambridge UK
Key words: acidification, amphisome, autophagy, autolysosome

Bafilomycin A1 is a specific inhibitor of the vacuolar type H+-ATPase (V-ATPase) in cells, and inhibits the acidification of organelles containing this enzyme, such as lysosomes and endo- somes. Recently, while editing and reviewing chapters on autophagy for Methods in Enzymology, we noticed repeated references to the effect of bafilomycin A1 in blocking the fusion of autophagosomes with lysosomes. Of course we have seen this in various research papers as well, but reading this routinely in chapters written by various people over a short period of time really caused this to stand out. Every one of these chapters referred to the paper by Yamamoto et al.1 In that paper, treatment with 100 nM bafilomycin A1 for 1 h blocks the fusion of autophagosomes with lysosomes in the rat hepatoma H-4-II-E cell line, based on data from electron micros- copy. However, data from one of our labs noted an apparently different result in a relatively recent manuscript.2 Therefore, we decided to look into this more carefully.

A far from exhaustive search on the Web indicated that indeed most papers where the researchers use bafilomycin A1 cite the 1998 paper1 as conclusive evidence that this drug inhibits fusion. In fact, we only found two other papers, from one of our labs3 and from Kawai et al.,4 which examines this directly, in normal rat kidney and Chinese hamster ovary cells, and reached a similar conclusion. In these studies, extended treatments (12–24 h) with 100 nM or 400 nM bafilomycin A1 decrease the percent of vesicles that display colocalization of mCherry-LC3 and lgp120 (LAMP-1)-GFP, or the colocalization of mitochondria and lysosomes. On the other hand, as stated above, the data in Fass et al.,2 demonstrate that 2 h of bafilomycin A1 treatment in Chinese hamster ovary cells blocks lysosome acidification (in agreement with the paper by Yamamoto et al.,1 and many other studies), but did not result in an obvious separation between LC3-labelled vesicles and LAMP-1-stained vesicles. Similarly, a paper examining the effects of short incubations (1 h) of bafilomycin A1 and the related compound concanamycin A in hepatocytes finds that the main effect is inhibition of lysosome

acidification and suppression of endocytosis. Although there is an inhibition of autophagic fusion that causes accumulation of early autophagic vacuoles, a high autophagic flux into the lysosome is still maintained.5
What about fusion between autophagosomes and endosomes, or between amphisomes and lysosomes? Yamamoto et al.1 find that bafilomycin A1 prevents the delivery of endocytosed horseradish peroxidase to autophagosomes. Similarly, Jahreiss et al.3 find decreased fusion of endosomes and autophagosomes (i.e., formation of amphi- somes), Fass et al.2 do not detect the early endosome marker EEA1 colocalizing with GFP-LC3 in the presence of bafilomycin A1, and Mousavi et al.5 find that bafilomycin A1 blocks fusion between late endosomes and lysosomes. The papers by Jahreiss et al.3 and Mousavi et al.5 note the difficulty of examining endocytic convergence with the lysosome, because interference with endosomal-lysosomal fusion may affect lysosome biogenesis. Furthermore, Mousavi et al.5 note previous studies suggesting that the effects of bafilomycin A1 on endocytic transport may be cell-dependent, in some cases affecting transport from early to late endosomes,6 and in others from late endo- somes to lysosomes.7,8 The inhibition of fusion between endocytic compartments and the lysosome presents an additional complication because there is a potential for errors in interpretation of the electron microscopy data. For example, in the analysis by Yamamoto et al.,1 without the use of additional markers it is not possible to determine whether the accumulated structures are autophagosomes or amphi- somes (which are difficult to identify when endocytosis is blocked), and it is likely that bafilomycin A1 inhibits fusion of the latter with lysosomes (Brech A, unpublished observations).
What is to be concluded from these various studies? First, as apparently different results were obtained with the same cell lines and similar concentrations of bafilomycin A1,2,4 it appears that the
treatment time influences the outcome. In fact, it is likely that the
obvious fusion block is an indirect result of the acidification defect, the latter being the immediate result of bafilomycin A1 treatment; extended time in the presence of the drug appears to interfere with fusion in a range of cell types (unpublished observations). Indeed, a number of agents that affect this process in a V-ATPase-independent fashion (e.g., ammonium chloride, chloroquine diphsophate, nige- ricin) appear to block autophagosome-lysosome fusion in the same

way as bafilomycin A1 (after 6–12 h).4 In some cell types there is
morphological evidence for fusion defects at early time points, but these may be difficult to detect by fluorescence methods because more than 50% of the GFP-LC3 vesicles in normal media in a Autophagy 849

Does bafilomycin A1 block the fusion of autophagosomes with lysosomes?

wide range of cell types are also LAMP-1-positive in the absence of bafilomycin A1.3 Furthermore, the removal of GFP-LC3 from autolysosomes will also be delayed after perturbing lysosomal acidifi- cation with this drug, further limiting the ability to detect an obvious separation of LC3-positive and LAMP-1-positive compartments at early time points (1-2 h) after bafilomycin A1 treatment—the “phenotype” of impaired autophagosome-lysosome fusion. While this issue could theoretically be resolved by real-time analysis of fusion rates of LC3-positive/LAMP1-negative vesicles with lysosomes after short periods of bafilomycin A1 treatment, this experiment is very difficult due to the rarity of such fusion events and the 20–30 minute half-lives of autophagosomes.3 The issue may be further complicated as bafilomycin A1 does appear to block transport within the endocytic pathway potentially including the convergence with autophagy, although again it is not clear that this is the direct effect. Finally, there is the issue of partial effects from bafilomycin A1 treat- ment that are exacerbated in conditions of high flux. For example, in rat hepatocytes or H-4-II-E hepatoma cells that have a high rate of autophagic flux, early autophagic vacuoles accumulate rapidly during bafilomycin A1 treatment despite a considerable rate of delivery to lysosomes, and autolysosome formation.5 Thus, the time window used to examine the effect of drug treatment can be critical.
It is likely that the way(s) that bafilomycin A1 impairs LC3-II degradation does differ at different times of treatment. At early time-points, the drug probably has its major effects on slowing the degradation of LC3-II within existing autolysosomes. At later time- points, the defective lysosomal acidification (and possibly endosomal or amphisomal acidification) impairs the fusion of autophagosomes with both late endosomes and lysosomes.3 Nonetheless, treatment with bafilomycin A1 can still be used to assess autophagic flux because inhibition of either fusion with the lysosome or hydrolase activity will impair degradation of LC3-II; however, it is important that researchers determine the dose of the drug that is saturating for LC3-II accumulation in their experimental system. If one is aiming to separate autophagosomes that have not fused with late endosomes or lysosomes, from autolysosomes, then it is important to examine the effect of bafilomycin A1 in each system to identify conditions and time-points that induce overt separation of these compartments, or to use a different inhibitor that has less complicated effects on the endocytic and autophagic systems. In addition, we recommend the use of more than one assay to monitor autophagic flux, whenever possible.

1. Yamamoto A, Tagawa Y, Yoshimori T, Moriyama Y, Masaki R, Tashiro Y. Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells. Cell Struct Funct 1998; 23:33-42.
2. Fass E, Shvets E, Degani I, Hirschberg K, Elazar Z. Microtubules support production of starvation-induced autophagosomes but not their targeting and fusion with lysosomes. J Biol Chem 2006; 281:36303-16.
3. Jahreiss L, Menzies FM, Rubinsztein DC. The itinerary of autophagosomes: from peripheral formation to kiss-and-run fusion with lysosomes. Traffic 2008; 9:574-87.
4. Kawai A, Uchiyama H, Takano S, Nakamura N, Ohkuma S. Autophagosome-lysosome fusion depends on the pH in acidic compartments in CHO cells. Autophagy 2007; 3:154-7.
5. Mousavi SA, Kjeken R, Berg TO, Seglen PO, Berg T, Brech A. Effects of inhibitors of the vacuolar proton pump on hepatic heterophagy and autophagy. Biochim Biophy Acta 2001; 1510:243-57.
6. Clague MJ, Urbe S, Aniento F, Gruenberg J. Vacuolar ATPase activity is required for endo- somal carrier vesicle formation. J Biol Chem 1994; 269:21-4.
7. van Deurs B, Holm PK, Sandvig K. Inhibition of the vacuolar H+-ATPase with bafilomycin reduces delivery of internalized molecules from mature multivesicular endosomes to lyso- somes in HEp-2 cells. Eur J Cell Biol 1996; 69:343-50.
8. 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 the vacuolar proton pump. J Cell Biol 1995; 130:821-34.

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