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

点击翻译按钮获取中文摘要

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.