Mitochondrial form and function: an investigation of the mechanism and significance of mitochondrial remodelling in rat cortical astrocytes

Elucidations of the mechanisms that regulate mitochondrial morphology have contributed to a greater understanding of mitochondrial function in eukaryotic cells. To date, mitochondrial morphological changes have mostly been attributed to fission and fusion. Mitochondrial fission is a calcium (Ca2+)/calcineurin mediated process that activates the dynamin related mechanoenzyme DRP1 to cleave the mitochondrial membranes. This thesis explores new evidence that elevations in intracellular calcium produces some mitochondrial fission but the change in morphology is predominately caused by mitochondrial “remodelling”. Mitochondrial remodelling results from a structural change (rounding or elongating) in the membrane of a single mitochondrion without fission or fusion. Due to the nature of tools utilized to assess mitochondrial morphology, remodelling has been largely overlooked in the literature. Using real-time live cell fluorescence microscopy I show that remodelling can occur concomitantly with fission and have provided evidence that the mechanism of remodelling is distinct from fission. Throughout these studies, I used mitochondrially targeted yellow fluorescent protein (mt-eYFP), ratiometric ROS-sensitive GFPs, the mitochondrial membrane potential dye TMRM as well as Ca2+ and ATP FRET probes in rat cortical astrocytes to measure mitochondrial morphological and functional changes in real time. In the first objective chapter, I blocked mitochondrial fission using FK506 and Cyclosporine A and showed that Ca2+ induced mitochondrial remodelling was unaffected. In the second objective, I induced fission and remodelling by applying ROS generating agents such as rotenone and Ca2+ and demonstrated that remodelling was blocked using antioxidants but fission was not attenuated, indicating that remodelling is regulated by ROS. In the final objective chapter, I further investigated the mechanism of remodelling as well as the functional significance of remodelling. I provided evidence that inhibition of glycogen synthase kinase 3β (GSK3β), an enzyme previously associated with fission, induced only mitochondrial remodelling. Furthermore, I showed that remodelling protects cells against some staurosporine induced cell death and that the mechanism of protection may occur through reduced Ca2+ uptake into the mitochondrial matrix. Through exploration of the mechanism and function of mitochondrial remodelling this thesis provides a greater understanding of the role of mitochondria in cellular maintenance and survival.