Optimization, Functionalization and Application of Nanobodies against Miro1 and Drp1 to study Mitochondrial Dynamics

Abstract

To fulfil their multiple cellular functions, mitochondria constantly adapt to changing requirements by fusion, fission and active transport within the cell. These processes are coordinated by two key proteins: Drp1, which is responsible for the membrane constriction during mitochondrial fission, and Miro1, which connects the mitochondria to motor proteins, thus enabling mitochondrial movement. Drp1 dysregulation was linked to multiple diseases including Parkinson’s and cardiovascular diseases thus making Drp1 an attractive biomarker and a potential therapeutic target. To leverage this potential, a deeper understanding of Drp1 and Miro1 is required, which is currently impeded by the available research tools. To facilitate biochemical and cell biological studies of endogenous Drp1, Drp1-specific nanobodies (Nbs) previously identified by a phage display screen were formatted into versatile research tools. Functionalized into capture matrices, the Drp1-Nbs allowed the enrichment of endogenous Drp1 and thereby the identification of Drp1 interaction partners by proteomic analysis. Furthermore, a fluorophore-labelled bivalent Drp1-Nb (bivD7AF647) was generated and successfully applied for super-resolution STORM microscopy of endogenous Drp1. For real-time imaging of Drp1, Nb-fluorescent protein fusions, so-called chromobodies (Cbs), were intracellularly expressed in mammalian cells and the signal-to-noise ratio further improved by generating “turnover-accelerated” Cbs. With these probes, the dynamic relocalization of endogenous Drp1 upon compound-mediated induction of fission was visualized in living cells. Additionally, the intracellularly functional Drp1-Nbs were engineered into inducible TRIM21-based degrons for targeted depletion of Drp1 in live cells. For proteomic studies of endogenous Miro1, a previously generated Miro1-specific Nb was formatted into a bivalent affinity matrix, which was successfully applied to analyse the Miro1 interactome. To establish an approach for bringing fluorescently labelled Nbs into live cells, a recently described cholesterol-coomassie based approach for transducing proteins in live cells was tested for different Nbs. Although successful transduction of the Nbs into the cytosol was observed in principle, the method suffered from high cellular toxicity and low efficiency. Due to their versatility, the Nb-based research tools developed and validated in this study can facilitate further investigations into Drp1 and Miro1 and thus help answer open questions regarding their role in mitochondrial dynamics and pathophysiology.