Were the autophagosome-lysosome/vacuole fusion models illustrated correctly in the literature?

ABSTRACT

Exploration of autophagy in different species has become a hotspot in cell biology in the past decades. Macroautophagy (hereafter, autophagy) is the most widely studied type. One of the hallmarks of autophagy is the fusion of the outer membrane (OM) of a closed double-membrane mature autophagosome (AP) with the lysosomal/vacuolar single membrane. Most researchers in the autophagy field agree upon this description. However, AP-lysosome/vacuole fusion models that do not follow this description frequently appear in the literature, even published in some prestigious journals until now. Some of the misrepresented models are from autophagy laboratories with brilliant publication records. These flaws should be addressed as a public announcement in the autophagy field to avoid spreading misinformation. The editors and reviewers are the guardians to ensure correct models.

Abbreviations: AP: autophagosome; IM: inner membrane; OM: outer membrane.

The description of the autophagy process can be easily found in autophagy papers or heard from autophagy oral presentations at conferences. In the late stages of autophagy, outer membranes (OMs) of mature double-membrane autophagosomes (APs) fuse with the single membrane of lysosomes or vacuoles. These conclusions are mainly based on the high-resolution images from electron microscopy. The emerging cryo-focused ion beam and cryo-electron tomography techniques further verify the double-membrane feature of APs and the single-membrane feature of lysosomes or vacuoles [1]. However, many models of AP-lysosome/vacuole fusion do not illustrate the membrane features correctly in the literature, presenting double-membrane lysosomes and single-membrane APs. Furthermore, even if double-membrane APs and single-membrane lysosomes are presented in fusion models, the fusion contact sites are not illustrated correctly. I summarize the types of fusion models in the literature in Figure 1, with one AP and one lysosome/vacuole excluding amphisomes for simplicity and brevity, where a phospholipid bilayer is simplified as a single line, to focus on the fusion and immediate post-fusion steps.

Figure 1.

Summary of the representative two-dimensional/2D illustrations of an autophagosome (AP) fusing with a lysosome/vacuole in the literature. For simplicity, the amphisome structure and its fusion steps are omitted. Only membranes and lumenal contents of one AP and one lysosome/vacuole are drawn with a circular line to represent the phospholipid layer. (A) in animal cells (i-iii) either the double-membrane AP is illustrated as a single-membrane AP or the single-membrane lysosome (Lys) is illustrated as a double-membrane lysosome. Therefore, the fusion and post-fusion patterns are incorrect. (iv-vi) the illustrated fusion patterns of double-membrane APs with single-membrane lysosomes will not actually occur. (vii) the relatively correct pattern to describe the fusion between a double-membrane AP with a single-membrane lysosome. (B) in yeast and plant cells, the relatively correct pattern to describe the fusion between a double-membrane AP with a single-membrane vacuole. Different colors, numbers of line, line styles and thicknesses are applied to show the differences. The symbol “X” is put in the upper location (to avoid covering information below) of the first specific inaccuracy. The symbol “√” is put in the upper location of the correct fusion process. The symbol “?” is used to question the way the image was drawn regarding the composition of the lumenal content or membrane.

Most of the questionable fusion models are regarding AP-lysosome fusion in animal cells (Figure 1A). They can be divided into three categories. In category one, including Ai-iii, APs or lysosomes are illustrated incorrectly in membrane features. In Ai the AP is incorrectly illustrated as having a single membrane. Therefore, the assumed single membrane of the AP fuses with the single membrane of the lysosome so that the lysosomal contents digest AP cargoes to be lysosomal content-like. In Aii the AP is incorrectly illustrated as a phospholipid monolayer. The following fusion and post-fusion steps are illustrated strangely as the assumed single-membrane monolayer AP becomes a double-membrane bilayer AP so that the single membrane of the lysosome fuses with the assumed OM of the double-membrane AP. Subsequently, the lysosomal contents enter the AP lumen to degrade the AP inner membrane (IM) and its cargoes to be lysosomal content-like. In Aiii the lysosome is incorrectly illustrated as having a double membrane. The double-membrane AP fuses with the assumed double-membrane lysosome, with the OM and IM of one connecting with the OM and IM of the other so that the lysosomal contents also digest AP cargoes to be lysosomal content-like. This model once became popular and spread widely for a while when the incorrect autophagy template was provided by the scientific image and illustration software BioRender and the user could not distinguish the accuracy. Fortunately, this mistake was pointed out by the Autophagy editor Daniel Klionsky in time and BioRender corrected the wrong template and has issued a recall of their autophagy template [2].

In category two, including Aiv-vi, APs and lysosomes are illustrated correctly in membrane features, but the membrane fusion at the contact site is not described correctly. The single membrane of the lysosome overlaps with the OM (Aiv) or both membranes (Av) of the AP without fusion. These situations will not really occur because in three dimensions the membranes would only contact and not overlap if there were no fusions. Therefore, the lysosomal contents would not enter the AP lumen and the autophagy process would not reach completion. In Avi, the single membrane of the lysosome is illustrated to fuse with both membranes of the AP. However, each membrane is a phospholipid bilayer with two faces, which are usually defined as the protoplasmic (P)- and extracellular (E)-faces, with the P-face facing the cytoplasm [3]. When the lysosomal phospholipid bilayer membrane fuses with the AP outer phospholipid bilayer membrane, the fused phospholipid bilayer still has P- and E-faces on the same side as the original membranes. If the fused membrane further invaginated, I hypothesized that the E-face of the fused membrane would face and meet the P-face of the AP IM first. It is unknown whether these two different faces could fuse. If they fuse, the remnant P-face of the originally fused membrane might fuse with the remnant E-face of the AP IM; then, a hole would be generated to release the AP lumenal contents. The autophagy process would not reach completion. In addition, according to biophysical principles, there is no chance for the lysosomal phospholipid bilayer to simultaneously fuse with the two AP phospholipid bilayers in the same pattern of faces, represented as Avi.

In category three, represented as Avii, the single membrane of the lysosome fuses with the OM of the double-membrane AP so that the lysosomal contents enter the intermembrane space between the outer and inner membranes of the AP. The lysosomal hydrolases destroy the AP IM and its cargoes then the lysosomal contents fill in the AP lumen. This is the most plausible AP-lysosome fusion model.

In yeast and plant cells, the relative size of vacuole to AP is different from that of lysosome to AP in animal cells [4]. The AP-vacuole fusion models are illustrated correctly with the single-membrane vacuole fusing with the OM of the double-membrane AP as described in the literature. Like Avii, the vacuolar contents enter the intermembrane space between the outer and inner membranes of the AP, then degrade the AP IM and its contents. The vacuolar contents fill in the AP lumen (Figure 1B).

However, even in the most plausible models described in Avii or B, there are still many details which are not described accurately and clearly (Table 1). For example, the compositions of lysosomal/vacuolar membranes are definitely different from those of AP OMs. When these two types of membranes fuse, what is the composition of the hybrid membrane? Similarly, the compositions of the lysosomal/vacuolar lumen are definitely different from the AP IM and its contents. After the AP-lysosome/vacuole fusion, the small volume of lysosomal/vacuolar contents will be diluted in the hybrid structure, although these will be less significant in yeast and plant cells because of the relatively large vacuolar volume. Furthermore, in the actual autophagy process, multiple lysosomes fuse with the same AP ([5], Movie 1) and multiple APs fuse with the same vacuole ([6], Movie S1). In mammalian cells, lysosomes are reformed from autolysosomes by the autolysosome reformation process [5], and AP components are recycled by the autolysosome components recycling complex [7,8]. The site selection for fusion and reformation, and the spatiotemporal changes in membrane surface area and hybrid structure volume during and after AP-lysosome/vacuole fusion are far beyond our current understanding. All these situations make the description for the AP-lysosome/vacuole post-fusion process more complex. Therefore, the membranes and lumenal contents should not be illustrated arbitrarily as the corresponding parts of lysosomes/vacuoles or APs, which can mislead the field. With a further deep understanding of AP-lysosome/vacuole fusion and post-fusion processes, these models will be more accurately described in the future.

Table 1.

Limitations of the AP-lysosome/vacuole fusion models in Figure 1Avii and B.

#	Items	Limitations
1	Membrane composition	The autolysosomal (hybrid structure) membrane should not be drawn as either the lysosomal/vacuolar membrane or AP membrane because the membrane composition of AP, lysosome/vacuole and autolysosome (hybrid structure) are different.
2	Lumenal contents	The lumenal contents of the autolysosome (hybrid structure) should not be drawn as either those of the lysosome/vacuole or AP because the lumenal contents of the AP, lysosome/vacuole and autolysosome (hybrid structure) are different.
3	Spatiotemporal regulation of membrane fusion and regeneration	The simple 2D models did not reflect the spatiotemporal situations of highly dynamic changes of membranes during and after fusion, including fusion and regeneration processes.
4	Site selection for fusion and reformation	The locations for fusion and reformation are unknown when multiple lysosomes fuse with the AP or multiple lysosomes are reformed from the autolysosome. Furthermore, whether these locations will be reused is also unknown.
5	Autolysosomal (hybrid structure) membrane surface area and volume	The effects of the involvement of the number of fused lysosomes (with APs) and the speed of autolysosomal reformation (vacuolar reformation) on the autolysosomal (hybrid structure) membrane surface area and volume are unknown.

The frequent occurrence of incorrect AP-lysosome fusion models in the literature indicates that many authors, readers, reviewers and editors in the autophagy field were not understanding the fusion process accurately or paying enough attention to the details of this process. This situation needs to end through public announcement. The Autophagy journal is the best matched journal to take on this responsibility.

Funding Statement

This work was supported by the Natural Science Foundation of China [92354302, 91954125], and the Priority Academic Program Development of Jiangsu Higher Education Institutions [680-809007].

Disclosure statement

No potential conflict of interest was reported by the author(s).