Nanobodies as novel tools to monitor the mitochondrial fission factor Drp1.

Autor:	Froehlich T; Pharmaceutical Biotechnology, Eberhard Karls University Tübingen, Tübingen, Germany., Jenner A; Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany., Cavarischia-Rega C; Quantitative Proteomics, Department of Biology, Institute of Cell Biology, Eberhard Karls University Tübingen, Tübingen, Germany., Fagbadebo FO; Pharmaceutical Biotechnology, Eberhard Karls University Tübingen, Tübingen, Germany., Lurz Y; Center for Plant Molecular Biology (ZMBP), Eberhard Karls University Tübingen, Tübingen, Germany., Frecot DI; Pharmaceutical Biotechnology, Eberhard Karls University Tübingen, Tübingen, Germany., Kaiser PD; https://ror.org/03a1kwz48 NMI Natural and Medical Sciences Institute at the University of Tübingen, Reutlingen, Germany., Nueske S; Livestock Center of the Faculty of Veterinary Medicine, Ludwig Maximilians University Munich, Munich, Germany., Scholz AM; Livestock Center of the Faculty of Veterinary Medicine, Ludwig Maximilians University Munich, Munich, Germany., Schäffer E; Center for Plant Molecular Biology (ZMBP), Eberhard Karls University Tübingen, Tübingen, Germany., Garcia-Saez AJ; Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany.; Max Planck Institute of Biophysics, Frankfurt, Germany., Macek B; Quantitative Proteomics, Department of Biology, Institute of Cell Biology, Eberhard Karls University Tübingen, Tübingen, Germany., Rothbauer U; Pharmaceutical Biotechnology, Eberhard Karls University Tübingen, Tübingen, Germany ulrich.rothbauer@uni-tuebingen.de.; https://ror.org/03a1kwz48 Cluster of Excellence iFIT (EXC2180) 'Image-Guided and Functionally Instructed Tumor Therapies,' University of Tübingen, Tübingen, Germany.
Jazyk:	angličtina
Zdroj:	Life science alliance [Life Sci Alliance] 2024 May 30; Vol. 7 (8). Date of Electronic Publication: 2024 May 30 (Print Publication: 2024).
DOI:	10.26508/lsa.202402608
Abstrakt:	In cells, mitochondria undergo constant fusion and fission. An essential factor for fission is the mammalian dynamin-related protein 1 (Drp1). Dysregulation of Drp1 is associated with neurodegenerative diseases including Parkinson's, cardiovascular diseases and cancer, making Drp1 a pivotal biomarker for monitoring mitochondrial status and potential pathophysiological conditions. Here, we developed nanobodies (Nbs) as versatile binding molecules 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 into advanced 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. (© 2024 Froehlich et al.)
Databáze:	MEDLINE
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