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. 2024 Oct 8;4(10):100875. doi: 10.1016/j.crmeth.2024.100875

Figure 5.

Figure 5

Organelle-targeted sensors of proteostasis indicate that distinct endocrine signaling factors differentially impact subcellular proteostasis

(A–C) Western blot analyses of detergent-soluble and insoluble fractions from control whole flies (Mhc>+, gray) and isogenic flies with muscle-specific overexpression of Akh (Mhc>Akh, blue), the Drosophila functional homolog of glucagon and related peptides. Western blotting with anti-GFP antibodies indicates the levels of FlucDM-EGFP sensors, whereas Ponceau staining and β-actin are used as normalization controls. Akh differentially impacts the detergent-insoluble levels of FlucDM-EGFP variants targeted to distinct cell compartments during aging. Akh reduces the detergent-insoluble levels of nuclearly localized NLS-FlucDM at all ages (C), whereas it does not substantially impact the detergent-insoluble levels of cytoplasmic FlucDM (A) and mitochondrial mito-FlucDM (B). The ages analyzed for each genotype are 10, 30, and 60 days. n = 3 (biological replicates) with the mean ± SD indicated; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (unpaired two-tailed t test).

(D–F) Western blot analysis of detergent-soluble and insoluble fractions from head extracts (enriched for the CNS) from control flies (Mhc>+, gray) and isogenic flies with muscle-specific overexpression of Amyrel (Mhc>Amyrel, green), which preserves proteostasis via maltose/SLC45 signaling. Amyrel reduces the detergent-insoluble levels of the untargeted (i.e., cytoplasmic) FlucDM (D) but not of mito-FlucDM (E) or NLS-FlucDM (F). Altogether, these findings indicate that muscle-derived Amyrel improves cytoplasmic proteostasis in the CNS. The ages analyzed for each genotype are 10, 30, and 45 days. n = 3 (biological replicates) with the mean ± SD indicated; ∗p < 0.05, ∗∗p < 0.01 (unpaired two-tailed t test).