Covill-Cooke, Christian Michael; (2019) Regulation of organelle dynamics by the Miro-GTPases. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Eukaryotic cells are dependent on the ability to compartmentalise processes within membrane-bound organelles. For these organelles to function optimally within the cell they must be regulated in position, morphology and content, in response to alterations in subcellular microenvironments. The importance of maintaining organelles dynamics is emphasised by perturbations in these pathways leading to a wide range of diseases. The mitochondrial Rho-GTPases, Miro1 and Miro2, are critical regulators of many aspects of mitochondrial dynamics, including long-range, microtubule-dependent trafficking. To better understand how they carry out these functions a yeast-two hybrid with Miro1 as bait was carried out; identifying the previously undescribed protein, Fam54b, as a novel Miro interactor. Using a range of microscopy and biochemical techniques Fam54b is found to have a partial mitochondrial localisation dependent on the presence of functional GTPase activity and calcium binding of Miro1, but not Miro2. Furthermore, overexpression of Fam54b leads to defects in mitochondrial trafficking and mitochondrial morphology, providing additional mechanistic insight into how mitochondrial dynamics can be regulated through Miro1. Alongside mitochondria as key sites of metabolism, peroxisomes are single-membrane organelles required for a multitude of processes, including fatty acid β-oxidation. Despite the importance of functional peroxisomes to cellular health, the mechanisms which underlie peroxisomal dynamics are poorly understood. This work describes that alongside its well documented localisation to mitochondria, Miro is also found to localise to peroxisomes. Despite the role of Miro in microtubule-dependent trafficking of mitochondria, loss of Miro1 and Miro2 has only minor effects on long-range peroxisomal transport. Instead, loss of Miro leads to a reduction in shorter-range peroxisomal trafficking events and alters peroxisomal morphology. As a result, this study identifies a non-mitochondrial role for Miro, adding to the complexity of Miro function. Taken together, this study provides a broader appreciation of how Miro regulates the dynamics of metabolic organelles.

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