Nanobodies as novel tools to monitor the mitochondrial fission factor Drp1

Abstract

AbstractIn cells, mitochondria undergo constant fusion and fission. An essential factor for fission is the mammalian dynamin-related protein 1 (Drp1). Dysregulation of Drp1 has been linked to neurodegenerative diseases including Parkinson’s as well as cardiovascular diseases and cancer. Here, we developed nanobodies (Nbs) for proteomics, advanced microscopy and live cell imaging of Drp1. To specifically enrich endogenous Drp1 with interacting proteins for proteomics, we functionalized high-affinity Nbs as capture matrices. Furthermore, we detected Drp1 by bivalent Nbs combined with site-directed fluorophore labelling in super-resolution STORM microscopy. For real-time imaging of Drp1, we intracellularly expressed fluorescently labelled Nbs, so-called chromobodies (Cbs). To improve the signal-to-noise ratio, we further converted Cbs into a “turnover-accelerated” format. With these imaging probes, we visualized the dynamics of endogenous Drp1 upon compound-induced mitochondrial fission in living cells. Considering the wide range of research applications, the presented Nb toolset will open up new possibilities for advanced functional studies of Drp1 in disease-relevant models.