Design of a Genetically Encoded Tool to Study the Function of Mitochondria–Lysosome Interactions

Abstract

Membrane contact sites (MCS) are regions of close proximity between organelles that mediate crucial organelle functions. However, the list of effective biological tools to study MCS function still needs expansion. Mitochondria and lysosomes are organelles whose dysfunction is closely associated with Parkinson’s disease (PD) pathology, and some PD-associated genes has been shown to play a role in mitochondria-lysosome contact regulation. Our work presents a novel biosynthetic system for reversible induction of interactions between mitochondria and lysosomes, leveraging abscisic-acid (ABA)-mediated heterodimerization of ABI and PYL protein domains from Arabidopsis thaliana. We engineered combinations of genetically-encoded protein heterodimer constructs each containing the ABI or PYL domain, a fluorescent protein tag (EGFP, mCherry, iRFP, or BFP), and a transmembrane sequence targeting the lysosomal membrane or outer mitochondrial membrane. Using confocal microscopy to visualize organelle colocalization, we show efficient and reversible induction of mitochondria-lysosome contacts with our ABA-responsive system. Our findings indicate that mitochondrial constructs tagged with mCherry and iRFP effectively mediate ABA-induced mitochondria-lysosome colocalization when paired with lysosomal constructs tagged with EGFP/BFP or lacking a fluorescent tag. In live-cell imaging experiments, we show that mitochondria-lysosome contact durations are tunable with this system, increasing proportionally with ABA concentration used. Overall, our system of ABA-induced reversible organelle interactions is positioned as a powerful biological tool for studying organelle dynamics and MCS functions, with relevance to therapeutic strategies in neurodegenerative disorders and organelle dysfunction.