Grant: Discovery Grants Program - Individual

Mitochondria are specialized organelles that play numerous vital roles in eukaryotic cells. As such, the quantity and the quality of mitochondria are strictly regulated to ensure they optimally fulfill cellular needs. This is achieved through a tight balance between mitochondrial biogenesis and mitochondrial degradation through mitochondrial Quality Control (mQC). One of the best described mQC mechanism involves selective degradation of mitochondria through an autophagy-mediated mechanism (i.e. mitophagy). Our lab and others have recently uncovered a novel mQC pathway in which mitochondria shed-off small, mitochondrial-derived vesicles (MDVs) from their membranes to deliver mitochondrial contents to the multivesicular bodies (MVB) for lysosomal degradation. However, the content of MDVs, their various fates, and the mechanisms involved in their production remain poorly understood. The overarching objective of this program is thus to gain a better understanding of this vesicular process in the mitochondria-rich striated muscle. For the upcoming term, three research aims will be pursued: 1: Define the proteome of MDVs under baseline and stress conditions, and identify putative regulators of MDV formation: An in vitro MDV budding assay will be used in isolated mitochondria to map the proteome of vesicles generated under baseline and stress conditions, and identify putative regulators involved in their formation. Global cysteine profiling will also be employed to quantify thiol reactivity in proteins released in MDVs. This will allow to determine whether: i) the nature of cargo incorporated in MDVs varies according to the type of stress, ii) proximity to a source of free radicals promotes the incorporation of selected proteins into MDVs and iii) susceptibility to oxidative modifications promote incorporation of proteins into MDVs. 2: Determine the fate and function of MDVs in muscle cells: Cultured, myoblasts will be used to track the intracellular fate of MDVs under various conditions (i.e. baseline, specific mitochondrial stressors, differentiation), and gene silencing strategies will be used to knockdown/knockout regulators of MDV formation. This will allow to determine the impact of interfering with MDV production on other mQC pathways (mitophagy), mitochondrial morphology/functions, and cell fate (i.e. survival, proliferation, differentiation). Overall, this work will provide a comprehensive and mechanistic portrait of mQC in muscle cells. 3: Determine whether MDVs support the release of mitochondrial material in extracellular vesicles (EVs): Cultured myoblasts exposed to the above-mentioned conditions will be used to determine whether, under some circumstances, MDVs can escape the lysosomal pathway and support the release of mitochondrial proteins in EVs (exosomes, microparticles) for mitochondrial stress signaling. This will provide novel insights on the potential role of MDVs as an inter-cellular communication mechanism.