| dc.description.abstracteng | In eukaryotic cells, inter-organelle communication is crucial for several cellular functions, as well as for several cell signaling mechanisms. Mitochondria and the endoplasmic reticulum (ER), for example, form tight contact sites that are implicated in many aspects of cell physiology, and whose disruption has been associated with pathology, particularly neurodegenerative diseases. Although the contacts between these organelles are one of the most studied and most stable, the basic understanding of their regulation, as well as the communication of these two organelles via signaling pathways remains unclear.
Here, we explored the regulatory environment of the mitochondria-ER contact sites (MERCs) in various mammalian cells with mitochondrial malfunction. To identify the mechanisms regulating mitochondria and ER crosstalk, we used cellular models of acute and chronic mitochondrial dysfunction, which were generated by chemical inhibition of the respiratory chain and by silencing the expression of UQCRC1 in HeLa cells or preparing murine embryonic fibroblasts from the Ndufs4 KO mouse.
Interestingly, we found distinct effects of mitochondrial malfunction on MERCs formation depending on whether the mitochondrial defect was acute or chronic. Electron microscopy (EM) analysis showed a high number of MERCs in both UQCRC1 KD and Ndufs4 KO models of chronic mitochondrial malfunction. Opposed to these data, acute mitochondrial stress seems to repress MERCs formation, since fewer contact sites were observed. Importantly, we found differential response of AMPK to chronic and acute mitochondrial dysfunction, which negatively correlated with MERCs formation. Indeed, while chronic mitochondrial malfunction resulted in the repression of AMPK, acute mitochondrial stress activated AMPK signaling. Consistently, in AMPK depleted cells, EM and confocal analyses confirmed the increased mitochondria-ER juxtaposition, which was observed in the chronic mitochondrial malfunction models.
We also identified mitochondrial fission factor (MFF), which is involved in mitochondrial fission, as an AMPK target that localized to the MERCs. Indeed, we showed that MFF is involved in the regulation of MERCs, since a robust increase in the number of MERCs was observed in cells lacking MFF. Furthermore, a phospho-mimetic isoform of MFF normalized the number of MERCs to WT levels in cells lacking AMPK activity, suggesting that AMPK mediates the repression of mitochondria-ER contact sites via phosphorylation of MFF.
Taken together, our data identified a cellular signaling pathway downstream of a key kinase, AMPK that regulates the number of MERCs by modulating MFF activity. Moreover, our results highlight that the association of mitochondria with ER is yet another aspect of mitochondrial biology under the control of AMPK. This study contributed to a better understanding of MERCs by elucidating their response to mitochondrial respiratory chain malfunction in a defined manner, thereby providing a better understanding of pathological conditions linked to MERCs. | de |