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

Figure 4.

Figure 4

Use of compartment-targeted FlucDM as sensors of subcellular proteostasis

FlucDM are misfolding-prone proteins and, therefore, can be used to determine whether an experimental intervention modulates proteostasis compared to a control intervention. Specifically, FlucDM protein misfolding leads to its detergent insolubility, which indicates defects in protein quality control.

(A) Testing compartment-targeted FlucDM with thermal stress (red) versus controls (gray). Western blots of detergent-soluble and -insoluble fractions from whole flies with anti-GFP antibodies detect the EGFP-tagged FlucDM variants targeted to the mitochondria (mito-FlucDM) and the nucleus (NLS-FlucDM) and the untargeted FlucDM that localizes primarily to the cytoplasm. Ponceau staining and β-actin are shown as normalization controls, and the graphs refer to the GFP levels normalized by β-actin. Heat shock significantly decreases the detergent-soluble levels of all compartment-targeted FlucDM. This corresponds to an increase in the detergent-insoluble levels of the untargeted (i.e., cytoplasmic) FlucDM, NLS-FlucDM, and, to a lower extent, mito-FlucDM. Altogether, these findings indicate that thermal stress compromises proteostasis across the cell but more prominently in the cytoplasm and nucleus compared to the mitochondria. n = 3 (biological replicates) with the mean ± SD indicated; ∗p < 0.05 and ∗∗∗p < 0.001 (unpaired two-tailed t test).

(B) Testing compartment-targeted FlucDM with cold shock (blue) versus controls (gray). Shown are western blots of detergent-soluble and -insoluble fractions from whole flies with anti-GFP and anti-β-actin antibodies. Cold shock reduces the detergent-insoluble levels of the untargeted (i.e., cytoplasmic) FlucDM, whereas there are no changes in the detergent-soluble levels and in the detergent-insoluble levels of mito-FlucDM and NLS-FlucDM. Altogether, these findings indicate that cold shock improves cytoplasmic proteostasis but does not impact protein quality control in the nucleus and mitochondria. n = 3 (biological replicates) with the mean ± SD indicated; ∗∗p < 0.01 (unpaired two-tailed t test).