Mitochondrial quality control in striated muscle

横纹肌线粒体质量控制

• 批准号: RGPIN-2022-03229
• 负责人: Burelle, Yan
• 金额: $ 2.91万
• 依托单位: University of Ottawa
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=757075
• 关键词: Mitochondrial; quality; control; striated; muscle

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.

线粒体是在真核细胞中发挥许多重要作用的特化细胞器。因此，线粒体的数量和质量受到严格调节，以确保它们最佳地满足细胞需求。这是通过线粒体质量控制（mQC）在线粒体生物发生和线粒体降解之间的紧密平衡来实现的。最好描述的mQC机制之一涉及通过自噬介导的机制（即线粒体自噬）选择性降解线粒体。我们的实验室和其他实验室最近发现了一种新的mQC途径，其中线粒体从其膜上脱落小的、神经源性囊泡（MDV），将线粒体内容物递送到多泡体（MVB）进行溶酶体降解。然而，MDV的内容，它们的各种命运，以及参与其生产的机制仍然知之甚少。因此，该计划的首要目标是更好地了解这种囊泡过程中的横纹肌丰富的神经。对于即将到来的学期，将追求三个研究目标：1：定义基线和应激条件下MDV的蛋白质组，并确定MDV形成的推定调节因子：将在分离的线粒体中使用体外MDV出芽测定来绘制基线和应激条件下产生的囊泡的蛋白质组，并确定参与其形成的推定调节因子。还将采用全局半胱氨酸分析来量化MDV中释放的蛋白质中的巯基反应性。这将允许确定：i）掺入MDV中的货物的性质是否根据应激类型而变化，ii）接近自由基源是否促进所选蛋白质掺入MDV中，以及iii）对氧化修饰的敏感性是否促进蛋白质掺入MDV中。第二章：确定MDV在肌细胞中的命运和功能：培养的成肌细胞将用于跟踪MDV在各种条件下（即基线、特异性线粒体应激源、分化）的细胞内命运，并且基因沉默策略将用于敲低/敲除MDV形成的调节因子。这将允许确定干扰MDV生产对其他mQC途径（线粒体自噬）、线粒体形态/功能和细胞命运（即存活、增殖、分化）的影响。总的来说，这项工作将提供肌肉细胞中mQC的全面和机械的画像。第三章：确定MDV是否支持细胞外囊泡（EV）中线粒体物质的释放：暴露于上述条件的培养成肌细胞将用于确定在某些情况下MDV是否可以逃避溶酶体途径并支持EV（外来体、微粒）中线粒体蛋白的释放，用于线粒体应激信号传导。这将为MDV作为细胞间通信机制的潜在作用提供新的见解。