Investigating the nuclear actin cytoskeleton in DNA repair

• Applicant: Dr. Hans-Peter Wollscheid
• Subject Area: General Genetics and Functional Genome Biology ; Cell Biology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 555596903

The actin cytoskeleton exerts vital cellular functions ranging from intracellular trafficking and cell division to morphology and motility. Although actin has been studied for more than a century, only very recently functions of filamentous actin (F-actin) in the nucleus were discovered. This was mainly achieved by the development of nanobody-based tools and revealed important roles of nuclear F-actin during DNA repair, DNA replication and mitotic exit, leading to a fast-growing excitement over potential nuclear functions of the actin cytoskeleton. However, the contribution of myosins to these nuclear processes still remains poorly understood in the context of human cells. Myosins are actin-based molecular motor proteins among which myosin VI stands out by its unique directionality, moving from the plus to the minus end of actin filaments. Based on our initial observation of interactions with a multitude of nuclear proteins, we set out to explore the nuclear functions of myosin VI. Inspired by the recent advances in actin research, we selected myosin VI-specific affinity probes (DARPins) with nanobody-like properties, which – fused to functional domains – enable us to manipulate the localization and stability of endogenous myosin VI. Importantly, we discovered a functional contribution of myosin VI to two major nuclear processes impinging on genome stability: DNA double-strand break (DSB) repair and replication fork protection. We found that Myosin VI-deficient cells are impaired in DNA end resection and double-strand break (DSB) mobility, key steps of the homology directed repair (HDR). In addition, we discovered a reduced checkpoint activation upon induction of replicative stress, concomitant with de-protection of replication forks upon loss of myosin VI. Interestingly, ovarian cancer cells selectively express the only myosin VI isoform that is detectable in the nucleus, suggesting that the oncogenesis-associated functions of Myosin VI may be nuclear. The aim of this project is to explore the mechanism of myosin´s VI function in DSB-repair, explicitly during Ku70/80-removal to allow long-range DNA end resection. Based on the motor-domain dependency of myosin VI´s nuclear functions, we expect to unravel the mechanism of nuclear F-actin´s action in DNA repair and beyond. Furthermore, we aim to expand DNA-repair analyses of other (plus-end directed) myosins as well as actin-binding proteins to DSB repair pathway. The application and further development of the affinity probes will help us in addressing our research questions. We expect to obtain novel mechanistic insights into how the actin cytoskeleton contributes to genome stability. Results obtained from this project should provide a fundamental understanding of the pathological events during oncogenesis in prostate and ovarian carcinomas and hence help to identify molecular targets for cancer therapy.

• DFG Programme: Research Grants
• Co-Investigator: Professor Dr. Robert Grosse