Elucidate the regulatory cascade controlling mitochondrial fission in mitosis and its impact on mitotic progression and chromosome segregation

• Applicant: Valentina Piano, Ph.D.
• Subject Area: Cell Biology ; Biochemistry
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 512633796

Mitochondria are the energy factories of eukaryotic life. It is of paramount importance that during cell division, a functional population of mitochondria is transmitted to the offspring. Improper reorganization and partitioning of mitochondria during mitosis endangers chromosome inheritance, increasing the risk of tissue degeneration and cancer. We have however still little understanding of the molecular mechanisms that regulate mitochondrial segregation during cell division. The central aim of this proposal is to elucidate the signalling cascade coordinated by mitotic kinases that culminates with the timely mitochondrial fission and redistribution into the two nascent daughter cells, and how defects in these mechanisms can eventually affect mitotic progression and chromosome segregation. We will achieve this aim by integrating my knowledge of the mitotic cell signalling with my unique expertise in in vitro reconstitution. We will focus on three mains objectives: 1) determine how the key fission factor Dynamin-related protein 1 (DRP1) is recruited at mitochondria in mitosis; 2) expand the interactome of DRP1 mitotic regulators; 3) describe the phenotypes resulting from perturbing mitochondrial fission in mitosis, by introducing newly identified mutations that precisely disrupt the mitotic regulation of DRP1. In particular, we will first reconstruct in vitro the regulatory cascade that induces DRP1 assembly at fission sites. We will probe the protein-protein interactions and the post-translational modifications necessary for DRP1 activity in mitosis, using a combination of binding assays and cross-linking mass-spectrometry analysis, and we will structurally characterize the resulting protein complexes. We will then uncover new mitotic regulators of fission, by delivering in living cells chemically modified/engineered recombinant proteins using protein electroporation and analysing their specific interactors at mitochondria. Finally, we will integrate our observations in vitro with cellular assays to: i) provide the first thorough analysis of the mitotic phenotypes resulting from mitosis-specific perturbation of DRP1 function; ii) determine possible mechanisms that link mitochondria and chromosome segregation and iii) whether mitochondrial dynamics is also monitored by the mitotic checkpoint during cell division. This work will dramatically advance our knowledge of the mechanisms controlling fragmentation and reshuffling of mitochondria in the cytoplasm during cell mitosis. A molecular understanding of this processes will uncover the interplay between the organelles and chromosome segregation machineries and shed the light on how dividing cells ensure that both complete their functions error-free.

• DFG Programme: Research Grants