Time-course RNA-seq of the DIOM remodeling in wild-type Drosophila

(A) A timeline of DIOM remodeling. Samples were collected at five time points: third instar larva (3IL) and 1 to 4 days after puparium formation (APF). (B) The scheme for sample preparation of DIOMs. Red-colored rectangles indicate single DIOMs. (C) DESeq2 principal component analysis (PCA) of all mRNA-seq libraries. The first four principal components are shown. PC1-2, left; PC3-4, right. The dotted arrow in the PC1-2 plot represents the direction of the transcription dynamics. (D) Heat map of fuzzy c-means cluster core expression profiles. All genes were categorized into twelve clusters. (E) Factor loadings of each cluster to PC1-2 and PC3-4. The circles show r=0.8 (PC1-2) or r=0.7 (PC3-4). (F) GO enrichment at each time point during metamorphosis. The width of each line represents the expression level of the clusters indicated.

Transcriptional dynamics during DIOM remodeling is independent of autophagy

(A) A schematic of DIOM remodeling in wild-type and autophagy-deficient conditions. Autophagy-dependent muscle atrophy starts at 12-14 h APF. (B) Genotypes and time points of the comparative RNA-seq analysis (top) and a diagram of autophagosome formation (bottom). FIP200 or Atg18a RNAi blocks autophagosome formation. Stx17 RNAi blocks the autophagosome-lysosome fusion. (C) DESeq2 PCA of all mRNA-seq libraries. PC1-2, left; PC3-4, right. A total of 20 samples were analyzed. (D and E) DIOM volume changes in control or FIP200 RNAi from 3IL to 4 d APF. GFP expression indicates DIOMs. Projected images of XY and XZ planes are shown (D). (E) Relative DIOM cell volume for each genotype normalized to 3IL (set to 1).

Loss of BNIP3 results in accumulation of mitochondria in DIOMs

(A) A schematic of 4 d APF DIOMs in wild-type, FIP200 RNAi, or Stx17 RNAi. (B) TEM image of Stx17 RNAi DIOM at 4 d APF. White arrowheads indicate autophagosome structures. (C) TEM image of wild-type DIOM at 1 d APF. White arrows indicate mitophagosome membrane structure. (D) The expression level of mitophagy regulators in DIOMs at 1 d APF. N=4. Normalized counts in RNA-seq are shown. (E) The expression level of BNIP3 and Fbxl4 in DIOMs. N=4. (F) A diagram illustrating the BNIP3 knockout strategy, where all exons were deleted using two gRNAs and replaced with a 3xP3-RFP marker. (G) Loss of BNIP3 phenotype on mitochondria and myofibrils in DIOM at 4 d APF.

BNIP3 is required for mitophagosome formation

(A and B) TEM images of DIOM transverse sections at 4 d APF in wild-type (A) or BNIP3 KO (B). Mitochondria are shown in green. (C and D) TEM images of DIOM transverse sections at 4 d APF in Stx17 RNAi (C) or a combination of Stx17 RNAi and BNIP3 KO (D). Mitophagosomes, pink; Mitochondria, green; autophagosomes, blue. (E) The number of mitophagosomes and mitochondria per unit area in the indicated genotypes. (F) The number of total autophagosomes, including mitophagosomes, per unit area in the indicated genotypes. Stx17 RNAi, N = 20; Stx17 RNAi and BNIP3 KO, N = 17 (Mann-Whitney test) (E and F).

The LIR and MER motifs are required for BNIP3-mediated mitochondrial clearance

(A) Schematics of Drosophila BNIP3 and its mutants. (B) GFP pulldown experiment between GFP-BNIP3 and HA-Atg18a in S2 cells. (C) The structure of the BNIP3-Atg18a complex predicted by AlphaFold 3. Top, overview: Atg18a is depicted in white, featuring a β-propeller structure consisting of seven blades, with blades 2 and 3 highlighted in green and pink, respectively. For BNIP3, only residues 29–74 are shown in blue for clarity, with the α-helix spanning residues 42–53 highlighted in yellow. Bottom, close-up view: Amino acids positioned to form intramolecular contacts through their side chains are labeled and represented as sticks, with potential hydrogen bonds shown as dashed lines. (D and D’) BNIP3 rescue experiment in DIOMs at 4 d APF. The indicated GFP-tagged BNIP3 constructs and tdTomato-Mito were co-expressed in BNIP3 KO flies using the GAL4/UAS system (D). A minimum of 50 DIOMs were imaged for quantification (D’). (E) The amount of GFP-BNIP3 in WT and Atg101 KO muscles. The GFP-BNIP3 constructs were expressed by Mef2-GAL4 in WT or Atg101 KO flies. Larval fillets were lysed and subjected to western blotting for GFP.

BNIP3-mediated mitophagy eliminates larval muscle mitochondria during muscle remodeling

(A and A’) Time course microscopy of Mito-GFP and F-actin in control or BNIP3 KO during DIOM remodeling (A). Mitochondria area per total cell area. For 3IL, control, N = 18; BNIP3 KO, N = 17. For 1 d APF, control, N = 20; BNIP3 KO, N= 18 (Mann-Whitney test) (A’). (B) Mitophagy assay using Mito-QC in DIOMs at 1 d APF. Pixel intensity correlation profiles and Spearman’s correlation coefficients (R values) are shown. (C, D, and D’) Scheme of the use of GAL80 temperature-sensitive mutants (GAL80ts). The animals were raised at 29°C (Restrictive) to induce Mito-GFP expression until mid-3IL, then shifted to 18°C (Permissive) to block expression (C). Mito-GFP and ATP5A immunostaining (total mitochondria) signals in muscles at 3IL or 4 d APF (D). Mito-GFP intensities in muscles at each time point normalized to control (set to 1). For 3IL, control, N = 16; BNIP3 KO, N = 15. For 1 d APF, control, N = 15; BNIP3 KO, N= 15 (Mann-Whitney test) (D’). (E) A model of muscle remodeling with or without BNIP3. Loss of BNIP3 leads to mitochondrial accumulation, which disrupts muscle remodeling.