(C) PLOS One
This story was originally published by PLOS One and is unaltered.
. . . . . . . . . .



The Rab GTPase activating protein TBC-2 regulates endosomal localization of DAF-16 FOXO and lifespan [1]

['İçten Meraş', 'Department Of Anatomy', 'Cell Biology', 'Mcgill University', 'Montreal', 'Division Of Endocrinology', 'Metabolism', 'Department Of Medicine', 'Metabolic Disorders', 'Complications Program']

Date: 2022-10

Endosome trafficking and signal transduction are intimately linked processes regulating signal propagation, specificity and attenuation [9,56]. However, there remains a large knowledge gap in the spatial regulation of cell signaling and where downstream transcription factors are regulated. We identified a previously unknown localization for the DAF-16 FOXO transcription factor on endosomes in C. elegans. Endosome localization is limited by the TBC-2 Rab GAP. Loss of C. elegans TBC-2 results in increased endosomal localization of DAF-16 at the expense of nuclear localization. As such, C. elegans TBC-2 is partly required for several daf-2 IGFR mutant phenotypes including lifespan extension, increased fat storage, and increased DAF-16 target gene expression that result from DAF-16 nuclear translocation. DAF-16 endosome localization is largely dependent on IIS consistent with this being a phosphorylated, inactive pool of DAF-16. Together our data show a role for the TBC-2 Rab GAP in regulating the balance of nuclear versus endosomal localization of the DAF-16 transcription factor.

DAF-16 localizes to endosomes

We were surprised to find that DAF-16 FOXO localizes to a subset of RAB-5 and RAB-7 endosomes in wild-type animals. Many studies have used DAF-16 cytoplasmic versus nuclear localization to assess IIS activity under various conditions. We assume that DAF-16 positive endosomes were not discovered earlier as nuclear translocation can be assessed at low magnification where DAF-16 positive endosomes are not apparent, and DAF-16 positive endosomes are not present in every cell or every animal. Furthermore, we first took notice of DAF-16 positive endosomes in the tbc-2 mutant background where they are more prominent. These are likely not an artefactual consequence of overexpression, as we see these vesicles in the endogenously tagged daf-16::GFP and we do not see similar vesicles in a GFP overexpression strain. DAF-16::mNG and DAF-16::mK2 also localize to vesicles indicating that membrane localization is unlikely to be an artifact of the GFP tag. Additionally, DAF-16-vesicles are regulated by IIS.

Many components of the IIS pathway localize to endosomes in mammalian cells including active insulin receptor and downstream signaling components such as PI3K, Akt, PTEN and 14-3-3 proteins [9,11,15,57–61]. In the case of Akt and PTEN, both have demonstrated roles in regulation of endosome trafficking independent of IIS [12,62–64]. On the other hand, the PI3Ks are Rab5 effectors and Rab5 has been shown to promote Akt activity on endosomes [60,61,65–67]. While endosomal localization of FOXO proteins has not been reported to the best of our knowledge, knockdown of Rab5 in mouse liver results in a strong increase in phosphorylated FOXO1 [68]. This is contrary to the finding that Rab5 promotes Akt phosphorylation [61,66], which could be a consequence of indirect regulation or suggest tissue-specific regulation. The fact that TBC-2 is a RAB-5 GAP is consistent with increased RAB-5 activity promoting DAF-16 localization on endosomes. This is further supported by the fact that rab-5 and rab-7 RNAi knockdown reduces the number of animals with DAF-16 vesicles in both wild type and tbc-2 mutants. Given the importance of RAB-5 for endosome trafficking, it is difficult to parse whether RAB-5 is promoting a platform for DAF-16 localization or if it also has a role in IIS.

We demonstrated that DAF-16 localized to a subset of RAB-5 and RAB-7 positive endosomes. Since RAB-5 and RAB-7 promote trafficking to the lysosome and promote receptor tyrosine kinase degradation, it is possible that DAF-16-positive endosomes are signaling endosomes, in which case we would expect other upstream signaling components might be present. Consistent with that hypothesis, knockdown of IIS reduces the number of animals with DAF-16 vesicles. However, when analyzing DAF-16 localization in daf-2(e1370) mutants at 15°C vs. 25°C, we find that while there is a reduction in the number of DAF-16 endosomes, these endosomes are noticeably fainter at 25°C. This suggests that there are not necessarily less endosomes being generated, but rather less DAF-16 on the vesicles which may be inconsistent with these being signaling endosomes derived from DAF-2 internalization at the plasma membrane. On the other hand, the fact that Akt and 14-3-3 can localize to endosomes in mammalian cells [15,59,61] and that AKT-1 and FTT-2 promote DAF-16 localization to endosomes, suggests that these proteins might recruit DAF-16 onto endosomes rather than DAF-16 interacting directly with membranes. Future studies should test whether DAF-2 IGFR and downstream IIS components actively recruit DAF-16 to endosomes or whether IIS has a passive role. IIS inhibition of nuclear DAF-16 could result in increased DAF-16 in the cytoplasm where it can bind endosomes.

If these are not signaling endosomes, then what are the DAF-16 endosomes? Since RAB-5 and RAB-7 are also regulators of autophagy, so it is possible that these endosomes contribute to degradation of inactive excess DAF-16. There is precedent for selective autophagy in the degradation of the GATA4 transcription factor [69]. Alternatively, these endosomes could serve as a reservoir of inactive DAF-16 that can be quickly mobilized if environmental stress is encountered. For example, we found that acute starvation is a potent regulator of DAF-16 endosome localization, even in the tbc-2 mutant background.

We find it interesting that there is such variability within the population, and amongst the intestinal cells in a given animal, as to whether there will be DAF-16 positive endosomes or not. It suggests that each intestinal cell autonomously senses changes in IIS, or possibly other nutrient and stress sensing pathways, to regulate DAF-16 localization. Then, why is endosomal DAF-16 more prominent in tbc-2 mutants? One explaination would be that the expansion of endosomal membranes in a tbc-2 mutant create more storage space for inactive DAF-16. Another would be that IIS or other pathways are more active in tbc-2 mutants, or some combination of the two. The fact that loss of IIS does not eliminate DAF-16 localization from endosomes suggests that additional signaling pathways could regulate DAF-16 endosome localization. GLP-1/Notch signaling in the germline regulates longevity in a DAF-16-dependent manner as well as DAF-16 nuclear translocation [70], and IIS post-translationally regulates GLP-1 signaling [71], thus it would be interesting to determine if DAF-16 localization to endosomes are regulated by GLP-1/Notch signaling and if TBC-2 regulates GLP-1 to DAF-16 target gene expression [72]. Additionally, AMPK, JNK and LET-363/mTor signaling regulate DAF-16 and could regulate DAF-16 localization to endosomes or be subject to regulation by TBC-2, particularly mTor which localizes to lysosomes [70,73–77].

[END]
---
[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1010328

Published and (C) by PLOS One
Content appears here under this condition or license: Creative Commons - Attribution BY 4.0.

via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/

You are viewing proxied material from magical.fish. The copyright of proxied material belongs to its original authors. Any comments or complaints in relation to proxied material should be directed to the original authors of the content concerned. Please see the disclaimer for more details.