c-FLIPL localizes at the endoplasmic reticulum and mitochondria-associated membranes and regulates organelle morphology and ER-mitochondria crosstalk

Cellular FLICE-inhibitory proteins (c-FLIPs) regulate Death Receptor (DR)-induced apoptotic as well as non-apoptotic pathways, by modulating caspase-8 activation. Here we showed that the long isoform of c-FLIP unexpectedly controls the Endoplasmic Reticulum (ER) morphology and the physical and functional crosstalk between ER and mitochondria. Besides its previously described cytosolic localization, we observed that c-FLIPL is also retrieved at the ER and Mitochondria-associated membranes (MAMs), ER subdomains involved in the ER-to-mitochondria Ca2+ transport, lipid metabolism and ER stress-induced apoptosis. We demonstrated that the ablation of c-FLIPL in mouse embryonic fibroblasts (MEFs) specifically alters ER morphology, inducing ER-sheets proliferation and altering the luminal contiguity of this organelle. Re-introduction of c-FLIPL in c-FLIP-/- cells partially recovers these structural defects, therefore confirming the role of c-FLIPL in modulation of ER morphology. In agreement, we also reported that c-FLIP-/- MEFs show reduced expression of the ER shaping protein reticulon4 (RTN4), that is mainly known as regulator of ER biogenesis and dynamics. Furthermore, we observed that c-FLIPL ablation loosens ER-mitochondria juxtaposition. We demonstrated that functionally, c-FLIPL ablation lowers the cytosolic Ca2+-increase evoked either by agonist stimulation or passive ER discharge. Furthermore, c-FLIP-/- cells are more resistant than their WT counterpart to cell death induced by the ER stress inducers thapsigargin and tunicamycin. Taken together, our findings suggest a novel role for c-FLIP as regulator of ER function and ER-mitochondria crosstalk.