# Modeling the Effects of Calcium Overload on Mitochondrial Ultrastructual Remodeling
## 3 Results and Discussion
That said, cristae are specialized compartments of dynamic inner membrane folds whose curvatures are dictated by the dimerization of F1F0-ATP synthases [37]. In addition to ATP synthase, these specialized microcompartments contain within their membrane the respiratory machinery, or the-so-called electron transfer chain, in addition to energy-linked transhydrogenase, uncoupling proteins, and metabolic carriers. Such a rich environment in ATP synthases not only dictates the cristae shape, but it also supports ATP synthesis by the chemiosmotic coupling resulting from the electrochemical gradient generated by the movement of electrons across the various complexes of the electron transport chain. The sac-like structure shape is stabilized by the mitochondrial contact site and cristae organizing system (MICOS) [81]. This complex is composed of a myriad of proteins, known as Mic proteins, with a variety of roles including membrane shaping, contact site formation, lipid trafficking, protein biogenesis, cristae stability, and scaffolding [81]. While dynamic, the cristae shape has been mostly divided into two main morphotypes: lamellar and tubular. The former is known for its elongated, plate-like three-dimensional appearance, whereas the latter has a blob- or tube-like three-dimensional appearance. Despite the vast knowledge gathered since the discovery of cristae, little is known about how such a dynamic system is tightly regulated or the impact of the multiple morphotypes. It has been hypothesized that the dynamic adaptation of the cristae shape is dependent on the energetic demand, stage of development of the cell, and the cell type [81]. The latter adds a layer of complexity, provided that cells can have predominantly one kind of cristae morphology over the other or a mixture of them [82]. A recent study by [PERSON] et al. showed that while these morphotypes are stabilized by dimers of F1F0 ATP-synthase and MICOS complexes [83], a lamellar--but not tubular--cristae morphology involves the optic atrophy factor 1 (OPA1), which is known for driving inner membrane fusion events and establishing the cristae junctions [84], whereas studies conducted by [PERSON] et al. using super-resolution structured illumination microscopy identified mitochondrial calcium uptake 1 (MICU1), a protein involved in controlling the activity of the mitochondrial calcium unipporter complex, as a calcium-dependent regulator of the cristae junction [84]. These studies revealed that knocking down MICU1 caused widening of the cristae junctions, membrane depolarization, and higher levels of matrix-resting calcium, similar to the results obtained upon OPA1 knockdown. Hence, while calcium exerts some control over cellular bioenergetics and metabolism [25], these studies revealed a role for calcium in cristae dynamics. While the modeling approach described herein faithfully reproduced the inner membrane morphology in the normal and calcium-overloaded states, it did have some limitations. For example, we did not include the possible effect that free calcium (calcium ions in the solution) may have had on the proteins that regulated the cristae structure. While we do not yet know of any direct binding of calcium to the putative cristae regulators (e.g., OPA1 and MICOS), there are published studies that indirectly link calcium ions to cristae remodeling [84; 85; 86; 87; 88]. We intend to explore this in future studies, identifying the relationship between the total calcium content, ultrastructural changes, and ATP production rates. In this study, the total mitochondrial calcium content was not varied in the cryo-EM studies. If done, we would expect to see the total number and size of the granules play a direct role in the maximal rate of ATP production, as described in Figure 2.5 In addition, it was not coupled to reaction-diffusion equations to simulate the effects of the ultrastructure on metabolic fluxes, and the location and ultrastructural shaping effects of F1F0 ATP synthase and other mitochondrial enzymes were not accounted for.