Stress-induced Cdk5 activity enhances cytoprotective basal autophagy in Drosophila melanogaster by phosphorylating acinus at serine⁴³⁷

Essential revisions:

1) in vitro kinase assays with purified proteins to show that Cdk5 kinase can directly phosphorylate Acinus.

We performed two sets of experiments to address this concern.

First, we prepared GST proteins fused to Acn^402-527 (WT or S437A) and exposed them to phosphorylation with commercially available Cdk5/p35 complexes (Σ; 14-477). We observed Cdk5/p35-dependent S437-phosphorylation, that was not present in S437A controls (new Figure 6J). Of note, we tried several other GST-Acn fusions for these experiments, but none was more stable against degradation than the GST-Acn-402-527 fusions.

Second, we over-expressed Strep-tag and Myc-tagged full-length Acinus proteins (WT or S437A) in S2 cells, immobilized and purified the proteins on Strep-Tactin beads before incubation with commercial Cdk5/p35. These experiments also showed Cdk5/p35-dependent S437-Acn phosphorylation (new Figure 6K).

In combination, we believe these experiments convincingly address the reviewers’ concerns to show that Cdk5 kinase can directly phosphorylate Acinus at S437.

2) The current data does not sufficiently support the conclusion that autophagy activity is induced by S437-Acn. The authors showed that the number of Atg8a puncta and LysoTracker-positive structures is increased in Acn^S437D larval fat bodies. The autolysosomes in Acn^S437D larval fat bodies are much larger and more abundant compared to Acn^WT. The mean diameter of lysosomal/autolysosome structures is even increased more than 8-fold! Inhibition of autophagy at the step of autophagosome maturation and/or degradation results in the same phenotype. Does starvation-induced autophagy also cause accumulation of enlarged autolysosomes in fat bodies?

Starvation-induced autophagy in fat bodies has been extensively studied by multiple labs. Examples include: (Scott et al., 2004) (Takats et al., 2014) (Takats et al., 2013). Neither of these papers quantified the changes in size from basal to starvations-induced autolysosomes. Importantly, however, all of these papers showed examples of electron micrographs from starved tissues containing large autolysosomes exceeding 1 µm or even 2 µm (e.g. Figure 2B-D in (Scott et al., 2004); Figure 2I in (Takats et al., 2013); or Figure 4A,B in (Takats et al., 2014).

Additional characterization of autophagy flux would strengthen the interpretations of autophagy induction induced by Acn-pS437. This could be achieved with assays using the tandem fluorescent Atg8 reporter and/or Atg8-II levels in animals fed BafilomycinA.

To address this concern, we performed experiments in which we blocked lysosomal acidification and degradation in larval fat bodies by feeding chloroquine to larvae following a well-established protocol to measure autophagic flux in larval fat bodies (Low et al., 2013; Mauvezin et al., 2014; Lorincz et al., 2017). When staining these chloroquine-treated fat bodies for endogenous ATG8, we observed significant increases in ATG8a punctae and ATG8a signal intensity for wild-type starved or for fed S437D phospho-mimetic mutants consistent with increased autophagic flux (new Figure 2E-L and Figure 2—figure supplement 1).

To keep this figure manageable, we have split the previous Figure 2 and separated the analysis of the effects of S437A and S437D mutants on starvation-resistance and longevity into the new Figure 3.

In addition, we like to point out that the beneficial effects endowed by the S437D phospho-mimetic mutation on starvation-resistance (new Figure 3A), longevity (new Figures 3B, 7M), and suppression of polyQ protein accumulation (new Figure 8) would be hard to explain as a consequence of defects in lysosomal degradation or reduced autophagosome maturation. By contrast, these effects are entirely consistent with beneficial effects of increased autophagy in the context of longevity and proteinopathies previously reported by several labs (some of many examples: (Sarkar et al., 2007; Simonsen et al., 2008; Nandi et al., 2014; Menzies et al., 2015; Gelino et al., 2016). We have now addressed this issue in the first paragraph of the Discussion.