Characterization of molecular mechanisms regulating the formation of mitochondria-derived vesicles and their roles

线粒体不仅仅是细胞的动力源。它们是高度动态的细胞器，构成了一个不断被聚变和裂变事件重塑的网状结构。此外，线粒体从细菌祖先那里遗传了释放称为线粒体衍生小泡(MDV)的小泡的能力，这些小泡在各种应激条件下释放，从氧化应激、热应激到暴露于各种药物，如脂多糖(LPS)和thapsigargin。这些囊泡的功能仍然知之甚少。我们已经证明，MDV的形成至少受到两种蛋白质的积极抑制，PINK1和Parkin是一种线粒体蛋白和一种与线粒体短暂结合的细胞质蛋白。在没有MDV的情况下，MDV被释放，线粒体成分被输送到溶酶体，在那里它们被加工成线粒体抗原递呈(MITAP)。这是一种新的抗原提呈途径。因此，了解调控这一过程的分子机制是至关重要的。我们项目的目的是研究参与MDV形成的蛋白质及其在先天免疫反应中的作用。为了确定MDV的特征，细胞将用MDV释放的诱导剂(热应激，LPS)处理。然后，我们将使用细胞分离方法来纯化MDV结构，并与高通量蛋白质组学方法相联系，以确定它们的构成蛋白。将对数据进行生物信息学分析，以选择感兴趣的蛋白质。我们将使用shRNA方法下调选定蛋白质的表达，以建立稳定的巨噬细胞系，并确定这些蛋白质是否在MDV-MITAP途径中发挥作用。为了破译MDV在先天免疫反应中的作用，我们将测量内毒素刺激后对照细胞或MDV生物发生相关基因(如Snx9、Rab9、Rab7)缺陷的细胞中促炎细胞因子的产生水平。我们还将测量含有线粒体DNA的MDV的生产水平，以及它们在先天性免疫反应中的意义。该项目将能够识别MDV-Mitap途径中涉及的关键蛋白质和分子机器，为调节MDV的生物发生和功能特性的分子机制提供有价值的线索。

Mitochondria are more than just the powerhouse of the cell. They are highly dynamic organelles constituting a reticulum constantly remodeled by fusion and fission events. Furthermore, mitochondria have inherited from their bacterial ancestors the ability to shed small vesicles called mitochondria-derived vesicles (MDVs), which are release in various stress conditions ranging from oxidative stress, heat stress, and exposure to a variety of drugs such as lipopolysaccharide (LPS) and thapsigargin. The function of these vesicles is still poorly understood. We have shown that the formation of MDVs is actively repressed by at least two proteins, PINK1 and Parkin, a mitochondrial protein and a cytoplasmic protein that transiently associate with mitochondria. In their absence, MDVs are released and mitochondrial components are delivered to lysosomes where they are processed for mitochondrial antigen presentation (MitAP). This is a new antigen presentation pathway. It is thus of prime importance to understand the molecular mechanisms regulating this process. The aim of our project is to characterize the proteins involved in the formation of MDVs and the role of this compartment in the innate immune response.To characterize MDVs, cells will be treated with inducers of MDVs release (heat stress, LPS). We will then use cell fractionation methods, to purify MDVs structures, linked with a high-throughput proteomics approach to identify their constituting proteins. Bioinformatics analyses will be performed on the data to select proteins of interest. We will knock-down the expression of selected proteins, using a shRNA approach, to generate stable macrophage cell lines and determine whether these proteins play a role along the MDVs-MitAP pathway. To decipher the role of MDVs in the innate immune response, we will measure the level of pro-inflammatory cytokines production after LPS stimulation in control cells or in cells deficient in genes implicated in MDVs biogenesis (e.g. Snx9, Rab9, Rab7). We will also measure the production level of MDVs containing mitochondrial DNA, and their implication in the innate immune response. This project will allow the identification of key proteins and molecular machines involved in the MDV-MitAP pathway, providing valuable hints into the molecular mechanisms regulating the biogenesis and functional properties of MDVs.