bCd Abnormal motor performance in mice at 10-weeks. show behavioral defects, mainly as a result of cerebellar neuronal loss. The accumulation of iron and ceruloplasmin is also found in the neuronal cells. These abnormalities are suppressed by the expression of Dram1, which is another crucial molecule for alternative autophagy. Although Atg7to be such a gene, together with either or yeast cells. In cells lacking Pep4, a vacuolar protease, to avoid the degradation of autophagic body-like Nrp1 structures inside vacuoles9. Consistently, from the aspect of proteolysis, AmphoB-induced degradation of green fluorescent protein (GFP)-fused pho8yeast cells, but not in cells, Golgi stacking (b, arrow), autophagosome (AP)-like structures (c) and autophagic body (AB)-like structures (d) were observed. In values cannot be described since the value is too small [MEFs) (Supplementary Fig.?1a, b) and induced alternative autophagy by the addition of etoposide, a DNA-damaging reagent. As described previously5, the ultrastructural analysis demonstrated the etoposide-induced formation of autophagosomes (double-membrane structures) and autolysosomes (single-membrane structures digesting subcellular constituents) in MEFs (Fig.?2a, Supplementary Fig.?2a). In contrast, such autophagic structures were not observed in etoposide-treated MEFs (Fig.?2b, Supplementary Fig.?2b), and the exogenous expression of Wipi3 (Supplementary Fig.?1b) restored the induction of autophagic structures (Fig.?2c, Supplementary Fig.?2c). These data suggested that Wipi3 is required for the induction of etoposide-induced alternative autophagy in mammals, as observed for Hsv2 in yeast cells (Fig.?1). Open in a separate window Fig. 2 Wipi3 is essential for etoposide-induced alternative autophagy.aCc Electron micrographs of the indicated MEFs treated with etoposide (10?M) for 18?h. Arrows indicate autolysosomes. Bars?=?2?m. A magnified image of the dashed square is shown in Supplementary Fig.?2b. Quick freeze-substitution images of autophagosome (AP) and autolysosome (AL) are shown on the right. Bars?=?0.5?m. d, e The mRFP-GFP tandem protein assay showed the essential role of Wipi3 in alternative autophagy. The indicated MEFs expressing a mRFP-GFP protein were left untreated or were treated with etoposide (10?M), and were immunostained with an anti-Lamp2 antibody. d Representative images at 18?h are shown. Bars?=?10?m. Red puncta indicate acidic compartments. e The populations of cells with red puncta (>1?m) are shown (MEFs (12?h: MEFs (MEFs (MEFs (Supplementary Fig.?4aCc), confirming the correct detection of autolysosomes. When this tandem protein was expressed within the cytoplasm, only a few red puncta were observed in nontreated MEFs, whereas etoposide-treated MEFs demonstrated many large red puncta that colocalized with the lysosomal protein Lamp2 (Fig.?2d). Red puncta were frequently observed in MEFs and Wipi3-expressing MEFs, but not in MEFs (Fig.?2d, Supplementary Fig.?3b). Quantitative analysis of cells with more than one red puncta (>1?m) confirmed these findings (Fig.?2e), showing the essential role of Wipi3 in etoposide-induced alternative autophagy. Autophagy can be more simply assessed by the formation of large Lamp2 puncta, as shown Senexin A in Fig.?2d Lamp2, because most red puncta from mRFP-GFP are included into the large Lamp2 puncta (Fig.?2d merge). The identity of the large Lamp2 puncta has been confirmed to be autolysosomes by CLEM analysis5,10. Consistent with the results of the mRFP-GFP assay, the large Lamp2 puncta assay showed the induction of alternative autophagy in MEFs and Wipi3-expressing MEFs, but not in MEFs upon etoposide treatment (Supplementary Fig.?5). Wipi3 is crucial for alternative autophagy-dependent Senexin A proteolysis We also analyzed whether Wipi3 contributes to alternative autophagy-dependent proteolysis. To this end, we analyzed Senexin A the degradation of the mCherry-Rab9 fusion protein, because we previously showed the existence of Rab9 on autophagic vacuoles5. When we treated mCherry-Rab9-expressing MEFs with etoposide, cleavage of mCherry-Rab9 and its inhibition by bafilomycin A1, an inhibitor of autolysosomal degradation, was observed (Fig.?2f), demonstrating that mCherry-Rab9 is a substrate of alternative autophagy. Importantly, this cleavage was not observed in etoposide-treated MEFs, and was recovered by the expression of Wipi3 (Fig.?2g, h), indicating the important role of Wipi3 in alternative autophagy-dependent proteolysis. Because the Rab9-fusion protein is degraded in autolysosomes, we visualized this degradation using mRFP-GFP-fused Rab9. We found that mRFP-GFP-Rab9 was localized in the cytoplasm as small yellow puncta in untreated MEFs (Fig.?2i: NT), which became big red puncta (owing to the autolysosomal quenching of the GFP fluorescence) surrounded by Lamp2 immunofluorescence after etoposide treatment (Fig.?2i: etoposide). Such puncta did not appear in etoposide-treated MEFs (Fig.?2j), indicating the Wipi3-dependent engulfment of mRFP-GFP-Rab9 into autolysosomes. The essential role of Wipi3 in alternative autophagy was confirmed by the treatment of MEFs with etoposide (Supplementary Fig.?6), and when we used a different alternative autophagy inducer, 1,3-cyclohexanebis (methylamine), which is an inhibitor of Golgi trafficking9,22 (Supplementary Fig.?7). All these data indicated that Wipi3 is required for alternative autophagy. Note that apoptosis was induced by etoposide23 concomitantly with alternative autophagy, but Wipi3 showed minimal effect on apoptosis (Supplementary Fig.?8a, b). Furthermore, the autophagic response was not affected by the inhibition of apoptosis with the pan-caspase inhibitor q-VD-OPh (Supplementary Fig.?8cCf). Wipi3 localizes on the Golgi membrane upon.