A multifunctional mitochondrial genetic probe to understand mitochondrial biology.

Stagi实验室已经开发了一套可转基因/可转导的探针，可以在生化、蛋白质组和生理应用中研究线粒体。简而言之，该探针是遗传编码的，并被合并到线粒体中。该集合包括：不同颜色的线粒体标记通过局部生物素化系统招募到外膜或内膜，以进行无自旋分离。与此相结合的是带有钙离子、pH和膜电位传感器的探针，可以进行实时成像，并进行FACS分类，以便分离具有特定特征的线粒体。这使得活体成像和蛋白质组学在细胞系中的应用相结合。该项目将包括优化分离和研究线粒体的技术，使用最先进的成像、生物化学、酶学和生理学技术，为特定的有丝分裂蛋白质组学/代谢组学研究开发可靠的分析方法。我们将编制用这一创新的生化系统分离的线粒体的蛋白质组学分析。如果这套工具的测试和完成成功，必将在研究线粒体生理学方面有很大的应用，直接在细胞器中进行，不受污染。

The Stagi lab has developed a set of transfectable/transducable probes which allow the study of mitochondria in biochemical, proteomic and physiological applications. Briefly, the probe is genetically encoded and incorporated in the mitochondria. The set is composed of: Mitochondrial markers in different colours recruited to outer or inner membranes with localized biotinylation systems for spin-free isolation. Coupled to this are probes with Ca2+, pH and membrane potential sensors which allow live imaging, and FACS sorting so that mitochondria with specific characteristics can be isolated. This allows coupled live imaging and proteomic applications in cell lines. The project will involve optimising techniques to isolate and study mitochondria, using the state of the art of imaging, biochemistry enzymology and physiology, to develop robust assays for specific mito-proteomics/metabolomics studies. We will compile proteomic assays of mitochondria isolated with this new innovative biochemical system. If the testing and completion of this set of tools will be successful is certainly have a large application in study the physiology of mitochondria directly in the organelle without contamination.