Esplorazione della modulazione dei siti di contatto tra mitocondri e altri organelli attraverso l'uso delle sonde fluorescenti SPLICS: dalla malattia di Parkinson al tumore al seno

Inter-organelle membrane contact sites (MCSs) are crucial for regulating cellular homeostasis and coordinating signaling pathways between subcellular compartments. In this Ph.D. thesis, split green fluorescence protein (GFP)-based probes, known as SPLICS, were utilized to monitor MCSs and investigate their rewiring under different conditions, such as upon dysregulations of Parkinson's disease (PD)-related proteins and in breast cancer. We expanded the existing set of SPLICS probes by developing novel sensors designed to detect mitochondria interactions with actin (SPLICSactin-MT), lysosomes (SPLICSLY-MT), and lipid droplets (LDs) (SPLICSLD-MT). These probes were validated by assessing their ability to detect alterations in the number of MCSs in response to different metabolic stimuli and through modulation of different tethering protein expression. Furthermore, this project explored the role of PD-related proteins α-synuclein and DJ-1 in MCSs remodeling. Overexpression of both α-synuclein and DJ-1 resulted in an increase in ER-mitochondria interactions and a decrease in lysosome-mitochondria contacts. Additionally, we demonstrated that this specific rearrangement of MCSs significantly impacts mitochondrial calcium homeostasis and TFEB nuclear translocation. Our study also revealed that DJ-1-dependent rewiring of MCSs differentially affects the nucleus and LD-mitochondria MCSs. Mutants of DJ-1 were generated to further characterize these effects, and interestingly, the pathological variant DJ-1 M26I induced an opposite MCS rewiring pattern compared to the wild-type protein. Moreover, we explored the relationship between MCS rearrangement and cancer progression in breast cancer cell lines, revealing an increase in ER-mitochondria MCSs that correlated with tumor aggressiveness. Rewiring of ER-mitochondria and lysosome-mitochondria MCSs was also observed in response to metabolic modulators (2DG, Etomoxir, and UK5099) in a cell line-dependent manner. Overall, these findings provide critical insights into the role of inter-organelle communication in cellular homeostasis, pathogenesis of PD, and cancer progression. Moreover, they underscore the utility of SPLICS probes as powerful tools for investigating MCSs under a variety of physiological and pathological conditions.