Atl (atlastin) regulates mTor signaling and autophagy in Drosophila muscle through alteration of the lysosomal network

Abstract

atl atlastin; ALR autophagic lysosome reformation; ER endoplasmic reticulum; GFP green fluorescent protein; HSP hereditary spastic paraplegia; Lamp1 lysosomal associated membrane protein 1 PolyUB polyubiquitin; RFP red fluorescent protein; spin spinster; mTor mechanistic Target of rapamycin; VCP valosin containing protein.

Keywords: ATG8; Hereditary Spastic Paraplegia; LAMP1; Neurodegeneration; endosome; protein aggregates; rab7.

Figure 1.

Loss of atl causes polyUB accumulation that is marked for degradation by autophagy with ref(2)P. (A) Phalloidin stain of a wild-type (w¹¹¹⁸) third instar Drosophila larva. (B) Schematic representation of one segment (indicated by a white dashed line in A) of larval musculature with muscles labeled in the right hemisegment. The body wall muscles studied in this work include the ventral oblique muscles (VO, also called chevrons, labeled 15,16, and 17, colored tan) and ventral lateral longitudinal muscles (VL, labeled 6, and 7, colored light blue). (C-N) confocal imaging (maximum intensity z-projections) of fixed third instar larval musculature. Scale bar: 50 µm. (C-H) antibodies against polyubiquitin (polyUB, green, C, F, I, and L) and ref(2)P (red, D, G, J, and M) in wild-type control larva (attP2, C-E) and the atl null mutant (atl², F-H). (I-N) muscle RNAi knockdown of atl (L-N). Panels E, H, K, and N represent overlayed images of polyUB (green) and ref(2)P (red). The inset in H and N are enlarged images of the region indicated by the dashed white box. (O-P). Quantification of ref(2)P and polyUB colocalization. (O) Imaris-generated surfaces of polyUB that lies within surfaced generated by ref(2)P, similar to Manders M1 coefficient. (P) Imaris-generated surfaces of ref(2)P that lies within surfaced generated by polyUB, similar to Manders M2 coefficient. (Q). Quantification of total cytoplasmic polyUB aggregates within the volume examined in panels C, F, I and L. (R). Quantification of total ref(2)P puncta number within the volume examined in panels D, G, J and M. Imaging data was quantified using Imaris v9.7.2. Quantitative data are presented as mean ± S.E.M with each individual sample represented in the scatter plot. Data shown are representative of at least five different experiments (n = 5).

Figure 2.

Loss of atl decreases the volume and complexity of the apical lysosomal network marked by spin-RFP. (A-F) Live imaging (maximum intensity z-projections) of larval body wall muscle 6 expressing the lysosomal/endosomal marker spin-RFP in wild-type control animals (Mhc>spin-GFP, A), atl² (B), and an atl deficiency (atl² /Df(atl), C). Scale bar: 5 µm. Imaris surface modeling of the spin-RFP surface of the indicated genotypes are shown in (D, E, and G) Discontinuous surfaces are represented as different colors. (H-M) Live imaging (maximum intensity z-projections) of larval body wall muscle 6 expressing the lysosomal/endosomal marker spin-RFP in wild-type control animals (24B>spin-RFP, H) and muscle RNAi knockdown of atl (24B>spin-RFP, atlRNAi, I). (J and K) Imaris surface modeling of the spin-RFP surface of the indicated genotypes are shown in (H, and I). (L and M) LysoTracker staining in wild-type (L) and atl knockdown larvae (M). Quantification of endolysosomal volume marked by spin-RFP in wild type, atl², and an atl deficiency (atl² /Df(atl), (G) and muscle RNAi knockdown (N).

Figure 3.

Loss of atl decreases conventional lysosome numbers.

Figure 4.

Loss of atl results in a blockage of autophagic flux.

Figure 5.

Loss of atl results in the accumulation of undegraded cargo in Rab7 positive compartments.

Figure 6.

Autophagic cargo does not reach Lamp1 positive lysosomes without atl.

Figure 7.

Visualization of autophagic structures with electron microscopy.

Figure 8.

Loss of atl results in decrease mTor activity in muscle.

Figure 9.

Reduction in atl, but not mTor, increases polyubiquitin aggregate accumulation.

Figure 10.

Models of mTor signaling and basal autophagy.