Mitotrap: a new tool for studying mitochondrial-nuclear communication

In addition to producing the majority of cellular ATP, mitochondria are central to apoptosis, signalling and several metabolic processes. It is not a surprise therefore, that mitochondria developed a number of mechanisms that allow to coordinate their actions to those of other cellular organelles such as the Endoplasmic Reticulum (ER) and the nucleus. Although, it is well established that the mitochondria-ER communication depends on direct physical interactions between the two organelles, the existence of specific contact sites that guarantee the functional link between mitochondria and Nucleus remains equivocal. In line with this hypothesis, recent findings suggest the existence of physical contacts between mitochondria and nucleus, however, several aspects of this relationship remain to be defined. To better understand the means through which mito-nuclear communication occurs, we developed a molecular tool, able to manipulate the subcellular distribution of mitochondria. In this thesis, as proof of principle generate “Mito-Trap” a molecular anchor able to relocate mitochondria to the nuclear envelope (NE). Mitotrap contains two domains, its amino-terminus is designed to link the tool to the outer mitochondrial membrane, while its carbossi-terminus contains a peptide able to recognise and engage a nuclear pore component. Using a combination of confocal imaging, biochemical analysis and FRET-based live cell imaging, we performed a structural and functional validation of Mitotrap. We demonstrate that Mitotrap effectively drives the repositioning of mitochondria on the NE, and that this relocation has no major effects on mitochondrial morphology and protein expression. As expected, functional analysis demonstrated that relocation of mitochondria affects the intracellular Ca2+ handling. Our experiments identify Mitotrap as a valid tool for studying several aspects of mitochondrial-nuclear communication. In addition, we envision that Mitotrap will find important application in the study of the functional significance of mitochondrial distribution in stem cell differentiation.