Thompson, Caleb Omar Dion; (2025) Exploring Miro as a Nexus for Cellular Homeostasis: Key Regulators of Mitochondrial Dynamics and ER-Mitochondrial Communication. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The mitochondria are essential organelles, not only do they provide the majority of a cells energy, but they also serve important roles in cellular calcium homeostasis, lipid homeostasis and cell fate signalling. Therefore, a range of dynamic and inter-regulated mechanisms manage mitochondrial morphology, turnover, and distribution to ensure their adaptable support is provided through-out the cell. The mitochondrial Rho-GTPases, Miro1 and Miro2, support several of these features including the regulation of mitochondrial trafficking and mor-phology. Their importance is emphasised by the observation of severe neuro-degeneration in transgenic mouse lines devoid of Miro1 GTPase function. This project aimed to explore the functional nuances of Miro1 and Miro2, exploring the impact of their dysfunction on cellular homeostasis and mitochondrial func-tion. During this project I collaborated in work outlining the seminal discovery of a conserved Miro hydrophobic binding pocket (ELF pocket), through which the Miro-GTPases bind several functional partners. Work presented in this thesis validates the functional relevance of this mechanism of interaction, outlining the interplay between the Miro ELF pocket and Mitochondrial Fission Regulator 1-Like (Mtfr1l) -regulation of mitochondrial morphology. Mtfr1l attenuates the num-ber of mitochondrial fusion events, a role dependent on its phosphorylation by AMP-activated protein kinase (AMPK). Consequently, Mtfr1l knockout (KO) re-sults in an aberrantly hyperfused mitochondrial network. It was found that the Miro1-Mtfr1l interaction, through the ELF, is essential for Mtfr1l regulation of mi-tochondrial morphology. Indeed, abolishing the Mtfr1l-Miro1 interaction, through site-directed mutagenesis of key ELF-pocket binding residues, prevented the correction of the hyperfused mitochondrial network phenotype observed in Mtfr1l-KO U2OS cells. Importantly, this is not simply a consequence of Mtfr1l lo-calisation away from the mitochondria as the function of Mtfr1l mutants, unable to bind Miro, was not rescued by artificially recruiting them to the mitochondrial surface. These findings exemplify the functional relevance of this interaction. Moreover, excessive Mtfr1l-Miro1 interaction impaired readouts of mitochondrial distribution in neuronal models, disrupting neuronal morphogenesis. Thereby, suggesting that binding into the ELF may act as competitive junction across mi-tochondrial dynamics. Therefore, this data outlines an important axis bridging AMPK-dependent regulation of mitochondrial network morphology to Miro-dependent regulation of mitochondrial trafficking. Although once thought to act alone, organelles coordinate with one and other at inter-organellar contacts to regulate cellular homeostasis. A key exam-ple of this inter-organellar collaboration occurs between the endoplasmic reticu-lum (ER) and the mitochondria at ER-mitochondrial contacts sites (ERMCs). In-deed, the Miro GTPases have been implicated in ER-mitochondrial communica-tion. However, the role of the Miro GTPases at these important homeostatic landmarks is still not completely understood. This work separately investigates the roles of Miro1 and Miro2, importantly revealing their varying importance and regulation of ERMC organisation and dynamics. Indeed, using proximity ligation assays and split-fluorescent approaches to quantify ERMCs, I reveal that Miro1 and Miro2 differentially disrupt ERMC number and organisation. It would seem that Miro1 is more universally important to ER-mitochondrial connectivity disrupt-ing ERMC number in every assay applied. Moreover, Miro1 overexpression alone, and not Miro2 overexpression, was sufficient to significantly enhance ERMC number. Finally, Miro1 loss of function alone attenuated the enrichment of ERMCs in response to cellular starvation. Therefore, this data outlines a key role for the Miro GTPases in ERMC organisation, outlining the unique contribution of each family member. Finally, unbiased proteomic investigations assessed the signatures of Mi-ro1 and Miro2 dysfunction in vitro and in vivo. Indeed, label-free mass spectrom-etry was used to describe the proteome in cell models and hippocampi from transgenic mouse models of Miro1 (R1KO), Miro2 (R2KO) and Miro1/2 double KO (DKO). Miro GTPase function appears to be essential to the stability of the mitoribosome as a significant proportion of the large mitochondrial ribosomal subunits are downregulated in DKO mouse embryonic fibroblasts (MEFs) and transgenic mouse brains. This integral role in establishing mitochondrial func-tional integrity is emphasised by the simultaneous destabilisation of key compo-nents of the electron transport chain (ETC) in DKO MEFs and brains. These findings aligned with metabolomic data outlining impaired mitochondrial metab-olism in DKO MEFs. Importantly, the destabilisation of ETC components is again prevalent in R1KO brains, potentially indicating that this Miro role is skewed to-wards Miro1 function. This is a particularly important point, as metabolomic data from R2KO MEFs shows evidence of impaired mitochondrial metabolism, de-spite an absence of the destabilised ETC phenotype in MEFs or brains. There-fore, R2KO may confer a disruption of mitochondrial metabolism through a unique mechanism which is independent of ETC component stability. This works also provides a wealth of data for target identification and pathway finding for future investigation into Miro1 and Miro2 function, some of which are dis-cussed here. For example, I identify ARMC1, a novel regulator of mitochondrial morphology and dynamics and an interactor of both Mtfr1l and Miro1, which has disrupted mitochondrial recruitment and stability in DKO MEFs. Altogether, this work provides a broader understanding of the Miro GTPases as central regula-tors of mitochondrial homeostasis and dynamics. Furthermore, this work provides important insights into the unique signatures of Miro1 and Miro2 dysfunction.

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