The effect of AMPK on MERCs regulation is mediated by MFF

In order to understand the mechanism of how AMPK regulates MERCs, we reasoned that an AMPK target must play a crucial role in MERCs. Here, we found out that MFF fits such a target since it was the only protein that scored after crossing published publicly available data sets of AMPK targets (Egan et al., 2011b; Gwinn et al., 2008) and MAM proteome (Hung et al., 2017).

MFF localizes to mitochondria, where it functions as a receptor for DRP1, which catalyzes membrane scission during the process of mitochondrial fission (Losón et al., 2013; Otera et al., 2010; Shen et al., 2014; Toyama et al., 2016). In fact, in our subcellular fractionation results, we confirmed the presence of MFF in mitochondrial fractions. Interestingly in ‘pure mitochondrial’

fractions its amount was robustly decreased while increased levels were observed in the cytoplasm in AMPK-DKO cells. Altogether, this suggests that MFF phosphorylation by AMPK is required for its localization to mitochondria. Consistently, it was proved that the participation of MFF in mitochondrial fission is highly regulated by AMPK (Toyama et al., 2016). Notably, we found an enrichment of MERCs in cells lacking MFF-KO, providing evidence that MFF indeed is involved in repressing MERCs formation. Remarkably, the average mitochondrial perimeter of those involved in MERCs was not changed.

The elegant study from Toyama and colleagues demonstrated the role of AMPK on mitochondrial fission via its activity on MFF (Toyama et al., 2016). We took advantage of the remarkable manipulation of MFF phosphorylation in this study to elucidate how AMPK activity is coupled to MFF in remodeling MERCs, by expressing WT MFF, a non-phosphorylatable Ser155Ala-Ser172Ala (SA2) mutant MFF, as well as, a phospho-mimetic Ser155Asp-Ser172Asp mutant (SD2) in AMPK-DKO cells. Consistent with the previous study, the SD2 MFF mutant displayed gain-of-function activity when introduced into AMPK-DKO MEFs, displaying shortened mitochondria comparable with those expressing WT MFF (Toyama et al., 2016).

In the context of MERCs, our EM data shows that the phospho-mimetic isoform of MFF (SD2 MFF) normalizes the mitochondria-ER contact sites in cells lacking AMPK activity, suggesting that the effect of AMPK on MERCs is mediated by MFF. This result is consistent with the idea that the increased number of MERCs observed due to genetic absence of AMPK or absence of AMPK activity, has more a structural role rather than functional. Since, our data show that increasing

MFF activity prevents the increase in contacts observed in AMPK-DKO MEFs, this effect might suggest that the re-activation of the mitochondrial fission process by more interaction of DRP1 with MFF completes the mitochondrial fission, thus minimizing the requirement for ER contacts to mark constriction sites on mitochondria.

Previously, the role of MERCs on mitochondrial fission was shown, in which ER tubules wrap around mitochondria to mark the fission sites (Friedman et al., 2011; Murley et al., 2013).

Remarkably, MFF also localizes in these constriction regions to mark the sites where DRP1 will be recruited in order to trigger membrane scission (Friedman et al., 2011). From our data, we conclude that MFF seems to be involved in both mitochondrial fission and contact sites. In the absence of or diminished AMPK signaling, or when MFF is removed, mitochondrial fission is repressed, resulting in an increased proportion of long mitochondria. It seems the AMPK-MFF axis tightly regulates to linked processes: mitochondrial fission and from our data mitochondria-ER contact sites. Notwithstanding, this study could not establish a direct role of AMPK as a structural tether between both organelles. However, it is also important to keep in mind that it is rather difficult to appreciate the role of a single tethering pair or molecule in remodeling MERCs. This is likely due to the large list of tethering proteins, which have been discovered to be associated with MERCs (Hung et al., 2017) and further justified by the findings that it is virtually impossible to eliminate a contact just by deleting any tether pair (Scorrano et al., 2019). Despite these limitations, here we demonstrated that signaling pathways are able to regulate MERCs, in particular AMPK via MFF.

Taken together, it seems that the association of mitochondria with the ER is another aspect of mitochondrial biology that is regulated by AMPK. The importance of AMPK in cell metabolism is very well characterized. For instance, AMPK is known to play a role in the upregulation of genes involved in mitochondrial biogenesis in the presence of mitochondrial dysfunction (Garcia-Roves et al., 2008; Herzig and Shaw, 2018; Jager et al., 2007) or to promote mitochondrial quality control via the removal of damaged mitochondrial through its regulation of the autophagy process (Egan et al., 2011a; Mihaylova and Shaw, 2011). However, in our model of chronic mitochondrial malfunction AMPK activity is repressed, and thus unable to activate adaptive mechanisms to improve mitochondrial quality. Therefore, it seems rational that the lack of AMPK will be couple to the regulation of a mechanism of increasing MERCs in the chronic mitochondrial model, as “backup” mechanism to increase mitochondria performance.

Consistently, the acute mitochondrial stress model, which activates AMPK signaling, is likely able to engage pathways that trigger mitochondrial biogenesis and/or mitophagy, and would not

require this “backup” mechanism, leading to its suppression. In a recent paper from our group, using the same model of chronic mitochondrial malfunction, defects in lysosomal function and consequently impaired autophagy as a consequence of mitochondrial dysfunction (Fernandez-Mosquera et al., 2019) were observed. These findings suggest that maintenance of mitochondrial quality via mitophagy was impaired and highlight the crucial need for a mechanism to increase mitochondrial function to support survival. The increase of MERCs via the AMPK-MFF axis seems to be the missing ‘wild card’.