Mitochondrial quality control in striated muscle

线粒体是一种特殊的细胞器，在真核细胞中发挥着重要的作用。因此，线粒体的数量和质量受到严格监管，以确保它们以最佳方式满足细胞需求。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实现紧密平衡而实现的。最好的MQC机制之一涉及到通过自噬介导的机制(即有丝分裂)选择性地降解线粒体。我们的实验室和其他人最近发现了一种新的MQC途径，在这种途径中，线粒体从膜上脱落小的线粒体衍生小泡(MDV)，将线粒体内容物运送到多泡小体(MVB)进行溶酶体降解。然而，MDV的含量、它们的各种命运以及它们的生产机制仍然知之甚少。因此，这个项目的首要目标是更好地了解富含线粒体的横纹肌中的这一囊泡过程。在即将到来的学期中，将追求三个研究目标：1：确定MDV在基线和应激条件下的蛋白质组，并确定MDV形成的可能调控因素：体外MDV发芽试验将用于分离的线粒体，以绘制在基线和胁迫条件下产生的囊泡的蛋白质组图，并确定参与其形成的可能调控因素。全球半胱氨酸图谱也将被用来量化MDV释放的蛋白质中的硫醇反应活性。这将有助于确定：i)MDV中结合的货物的性质是否因应激类型的不同而不同，ii)接近自由基源促进选定的蛋白质纳入MDV，以及iii)氧化修饰的敏感性促进蛋白质纳入MDV。2：确定MDV在肌肉细胞中的命运和功能：培养的成肌细胞将被用来追踪MDV在不同条件下(即基线、特定线粒体应激源、分化)的细胞内命运，基因沉默策略将用于敲除/敲除MDV形成的调节因子。这将有助于确定干扰MDV产生对其他MQC途径(有丝分裂)、线粒体形态/功能和细胞命运(即生存、增殖、分化)的影响。总体而言，这项工作将为肌肉细胞中的MQC提供一个全面和机械的肖像。3：确定MDV是否支持细胞外小泡(EVS)中线粒体物质的释放：将培养的成肌细胞暴露在上述条件下，将被用来确定在某些情况下，MDV是否能够避开溶酶体途径，支持EVS中线粒体蛋白(外切体、微粒)的释放，以传递线粒体应激信号。这将为MDV作为细胞间通信机制的潜在作用提供新的见解。

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.