Tour-de-Force

Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007DiscussionBy performing the proposed research, the understanding of several basic cell biological issues, such ascell cycle arrest, cell cycle progression and oxidative phosphorylation will be enriched. Through Mfn2 alink might be found between cell cycle progression and mitochondrial activity. However, this link to the cellcycle is not the only reason the proposed research is relevant. Mfn2 is mutated in variousneurodegenerative diseases, of which Charcot-Marie-Tooth disease is the most prevalently studied.Moreover, since decreased levels of cytosolic Mfn2 have been noticed in hyper-proliferative diseases,insight into this protein might help to develop new medical therapies.However, research is a dynamic process, and thus the proposed research does not provide a completepicture. Even if all our hypotheses are indicated to be true, further research has to be done. The secondhypothesis will gain insight into the function of the Mfn2-Stoml2 complex. It could, however, still beexplored what the binding site of Stoml2 to Mfn2 is. In addition, this research doesn’t go into the exactpathway through which Mfn2 is able to regulate OXPHOS. Further research could investigate throughwhat mechanism this occurs.Hypothesis 3 will provide insight into the formation of cytosolic Mfn2. It is suggested to conductadditional experiments that explore the conditions under which mitochondrial Mfn2 is cleaved to formcytosolic Mfn2. As proposed in the introduction, when cell cycle arrest is needed, there might be amechanism that cleaves the mitochondrial isoform of Mfn2. This will result in a decrease of mitochondrialMfn2, as well as an increase in cytosolic Mfn2 levels. Therefore, a bi-functional mechanism will work toachieve cell cycle arrest or apoptosis. Although this research will point out whether cleavage occurs, moreresearch will have to be done to determine whether this proposed mechanism is true. It could beinvestigated where cleavage occurs, as this might occur either in the cytosol or after insertion into themitochondrial membrane. Furthermore, it can be investigated which protein(s) are involved in this processof cleavage. Additionally, it can be explored under which cellular conditions cleavage is upregulated.The fourth hypothesis focuses on the relation between Ras, cytosolic Mfn2 and cell cycle arrest.This research will show under what conditions the binding of Mfn2 to Ras causes cell cycle arrest,however it doesn’t go into the binding mechanisms. Further research could investigate this.If hypothesis 5 proves to be true, thus, if there is a correlation between mitochondrial or cytosolicMfn2 levels and cyclin D1 and cyclin E, further research is needed to see whether this correlationindicates causality. As a follow up experiment, the first step would be to look at the activity of the cyclins,since this depends on their interaction with cdk’s. At the indicated time intervals co-immunoprecipitation ofthe cyclins and their cdk’s (cdk4 and cdk2, respectively) is suggested in order to extract the activecomplexes. An in vitro kinase assay would thereafter be necessary to quantify the complexes and tocheck whether the cyclins are indeed more active. We would have liked to include these experiments inour research, however due to time limitations this is beyond the scope of our project.However, this hypothesis might show that cyclin D1 / E directly influence Mfn2 by phosphorylation.Whether this phosphorylation results in activation or deactivation has to be explored. In case ofdeactivation, this could mean that Mfn-2 is no longer able to bind to Ras and subsequently induce cellcycle arrest. In addition, the protein would no longer be able to mediate OXPHOS. Both would bedesirable in progression from G1 to S phase. Since experiment 5.2 will be conducted in vitro, the resultscannot be generalized to in vivo circumstances. Further research could explore the relation in vivo.SCI 332 Advanced Molecular Cell Biology Research Proposal 89