Nano- and chromobodies for the structural and functional analysis of the mitochondrial outer membrane associated components, Miro1 and DRP1

Abstract

Mitochondrial outer membrane (MOM) associated proteins are critical players in mitochondrial transport, dynamics, and quality control. The MOM-anchored GTPase, Miro1, is a key player in mitochondrial transport, homeostasis and mitophagy. Aberrant Miro1 function has been implicated in Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS), suggesting that Miro1 may be a potential biomarker or drug target in neuronal disorders. However, the molecular functionality of Miro1 under normal and diseased conditions is poorly known. Another MOM associated GTPase, DRP1 is required for mitochondrial and peroxisomal fission. Though abnormal mitochondrial dynamics caused by DRP1 dysregulation in neurodegenerative disorders has been implied, concise information on its involvement remain elusive. Therefore, considering the roles played by Miro1 and DRP1 in neurodegenerative diseases and the lack of precise knowledge on their molecular functionality in these conditions, there is a great need for novel tools to study Miro1 and Drp1 in relevant research contexts. In this thesis, nanobodies (Nbs) were selected and generated as potential tools to characterize the molecular interactions, intracellular localization, and dynamics of Miro1 and DRP1. High affinity Nbs were selected from immune libraries by stringent phage display-based techniques and validated by detailed biochemical and functional assays. Using state-of-the-art methods, selected monovalent and generated bivalent Nbs were functionalized as nanotraps which efficiently capture their endogenous and exogenous antigens for proteomic applications. Bivalent Miro1-Nbs conjugated to fluorophores by advanced site-specific labelling methods were engineered and applied for the detection of Miro1 in immunofluorescence studies. Additionally, intracellularly functional Nbs were formatted into chromobodies (Cbs) which could trace Miro1 in real time by live cell imaging. As a further step towards the in vivo modulation of Miro1, intracellularly functional Miro1-Nbs were combined with an F-box domain to yield Nb-degrons, which were applied for the targeted degradation of Miro1. In summary, this study introduces a collection of novel Nbs that are promising tools for the biochemical characterization, intracellular visualization, and modulation of Miro1 and DRP1. The generation and application of these Nbs demonstrate the potential of Nbs as tools for the functional characterization of mitochondrial proteins.