Hoppins, SuzanneMartinez Bocanegra, Jennifer2025-05-122025-05-122025-05-122025MartinezBocanegra_washington_0250E_27940.pdfhttps://hdl.handle.net/1773/52937Thesis (Ph.D.)--University of Washington, 2025Mitochondria are often depicted in textbooks as static organelles whose principal purpose is to power cells; however, they are constantly moving and morphing shape in response to cellular requirements which go beyond the energetic needs of the cell. The dynamic properties exhibited by mitochondria include transport and positioning via interactions with cytoskeleton, and fission and fusion of the mitochondrial membranes, all of which are critical for maintaining mitochondrial function. The implications of disrupted mitochondrial transport and division have been extensively described in comparison to disrupted mitochondrial fusion. However, both mitochondrial fission and fusion are required to maintain a balanced and healthy network of mitochondria able to effectively respond to cellular demands. Mitochondrial fission and fusion are mediated by large GTPases from the Dynamin Superfamily of Proteins. Mitochondrial outer membrane fusion is mediated in mammals by the paralogs Mfn1 and Mfn2, collectively known as the mitofusins. Mutations in MFN2 are the cause for the most common form of Charcot-Marie-Tooth Type 2A Disease (CMT2A), though the mechanistic relationship between dysfunction of mitochondrial outer membrane fusion and the presentation of symptoms in patients with CMT2A has not been previously investigated. The following work describes the use of two biochemically characterized Mfn1 variants, Mfn1 S329P and Mfn1 S228A, to further investigate how mitofusin function is regulated and how regulation of Mfn1 contributes to cellular function. First, I present the use of the nematode Caenorhabditis elegans as a powerful new model to understand the contribution of Mfn1 S329P to neuronal dysfunction. This variant, along with its homologous disease-associated variant Mfn2 S350P, are fusion incompetent and exhibit phenotypes when expressed in cells that include redistribution of mitochondria to the perinuclear region. Mfn1 S329P was expressed in C. elegans and neuronal function was assessed by measuring neuron-dependent behaviors. Transgenic worms expressing Mfn1 S329P exhibited uncoordinated locomotion and decreased progeny that was likely due to an egg-laying defect. To better understand how mitofusin dysfunction contributes to disease, I performed genetic screens for suppressors of the behavioral phenotypes. Given that a candidate-based approach did not yield insight into the cellular functions contributing to the behavioral defects, I performed an unbiased genetic screen to identify candidates. I isolated agns-1 (um0017) in C. elegans and describe the formation of Mfn1 S329P associated mitochondrial clusters in neurons, which is facilitated by agns-1. Finally, I propose leveraging C. elegans as a tractable model for investigating how CMT2A-associated mitofusin variants contribute to neuronal dysfunction in real-time. In the following chapter I investigate the functional impacts of phosphorylation at Mfn1 S228 with respect to cellular health. I find that blocking phosphorylation of Mfn1 S228 renders cells slightly more susceptible to cell death induced by the ATP synthase inhibitor Oligomycin. Taken together, I demonstrate that dysregulation of Mfn1 contributes to cellular dysfunction and provide a novel model that bridges the gap between our understanding of mitofusin function in vitro.application/pdfen-USnoneFzoMembrane fusionMitochondriaMitochondrial dynamicsMitofusinsBiochemistryBiological chemistryThesis