Figure 4.
dMtmr6 and MTMR8 affect autolysosome size.(A and B) Representative images of fed L3 fat body expressing EGFP-mCherry-Atg8a and either control Lucif-RNAi (A) or dMtmr6-RNAi (B). Tissues were stained with Hoechst (blue) to detect nuclei. (C) Quantification of the number of Atg8a puncta expressing both GFP and mCherry (AP, autophagosomes) or mCherry only (ALY, autolysosomes) in Lucif-RNAi and dMtmr6-RNAi fat body as represented in A and B. (D and E) Electron micrographs of Drosophila larval midgut cells during developmental autophagy (2 h APF) from control w1118 (D) or dMtmr6 knockdown (E) tissues. Whole-cell sections on left, yellow box indicates area shown at high magnification in panels on right. Arrows indicate lysosomes (LY, orange), autophagosomes (AP, yellow), and autolysosomes (ALY, white). (F) Quantification of the ratio of the indicated organelles area relative to the total area of cytoplasm. (G) Quantification of the diameter of indicated organelles. (H and I) Electron micrographs of serum-fed COS7 cells transfected with siRNA targeting scrambled NC control (H) or MTMR8 (I). (J) Quantification of the ratio of the indicated organelles area (μm2) relative to the total area of cytoplasm. (K) Quantification of the diameter of indicated organelles. (L and M) Representative maximum intensity projection of z-stack image from serum-fed HeLa cells siRNA-transfected to target NC control (L) or MTMR8 (M) stained with Hoechst to detect nuclei (blue) and antibodies to detect LAMP1 (green) and LC3B (magenta), indicative of lysosomes and autophagosomes, respectively. Autolysosomes contain both LAMP1 and LC3B. (N–R) Quantification of the size, number, and colocalization of cellular LAMP1 and LC3B puncta as represented in L and M. Data are presented as mean ± SD, n ≥ 5 random images taken across three samples from each genotype (C); and as mean ± minimum to maximum (F, G, J, K, and N–R), with n ≥ 10 random images taken across samples from three biological replicates (F and G), n = 15 images per genotype, and one experimental replicate in (J and K), or n = 10 images per genotype and three experimental replicates (O–S). Asterisks denote statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001) using one-way ANOVA (C, F, G, J, and K) and unpaired, two-tailed t test with Welch’s correlation (O–S). Scale bars represent 20 µm (A, B, L, and M), 10 µm (D and E, left panels), 1 µm (D [zoom] and E [zoom], right panels), and 2 µm (H and I).

dMtmr6 and MTMR8 affect autolysosome size. (A and B) Representative images of fed L3 fat body expressing EGFP-mCherry-Atg8a and either control Lucif-RNAi (A) or dMtmr6-RNAi (B). Tissues were stained with Hoechst (blue) to detect nuclei. (C) Quantification of the number of Atg8a puncta expressing both GFP and mCherry (AP, autophagosomes) or mCherry only (ALY, autolysosomes) in Lucif-RNAi and dMtmr6-RNAi fat body as represented in A and B. (D and E) Electron micrographs of Drosophila larval midgut cells during developmental autophagy (2 h APF) from control w1118 (D) or dMtmr6 knockdown (E) tissues. Whole-cell sections on left, yellow box indicates area shown at high magnification in panels on right. Arrows indicate lysosomes (LY, orange), autophagosomes (AP, yellow), and autolysosomes (ALY, white). (F) Quantification of the ratio of the indicated organelles area relative to the total area of cytoplasm. (G) Quantification of the diameter of indicated organelles. (H and I) Electron micrographs of serum-fed COS7 cells transfected with siRNA targeting scrambled NC control (H) or MTMR8 (I). (J) Quantification of the ratio of the indicated organelles area (μm2) relative to the total area of cytoplasm. (K) Quantification of the diameter of indicated organelles. (L and M) Representative maximum intensity projection of z-stack image from serum-fed HeLa cells siRNA-transfected to target NC control (L) or MTMR8 (M) stained with Hoechst to detect nuclei (blue) and antibodies to detect LAMP1 (green) and LC3B (magenta), indicative of lysosomes and autophagosomes, respectively. Autolysosomes contain both LAMP1 and LC3B. (N–R) Quantification of the size, number, and colocalization of cellular LAMP1 and LC3B puncta as represented in L and M. Data are presented as mean ± SD, n ≥ 5 random images taken across three samples from each genotype (C); and as mean ± minimum to maximum (F, G, J, K, and N–R), with n ≥ 10 random images taken across samples from three biological replicates (F and G), n = 15 images per genotype, and one experimental replicate in (J and K), or n = 10 images per genotype and three experimental replicates (O–S). Asterisks denote statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001) using one-way ANOVA (C, F, G, J, and K) and unpaired, two-tailed t test with Welch’s correlation (O–S). Scale bars represent 20 µm (A, B, L, and M), 10 µm (D and E, left panels), 1 µm (D [zoom] and E [zoom], right panels), and 2 µm (H and I).

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