Role of the Diaphanous Formin FHOD1 and its interaction with nesprin-2-giant in nuclear migration
Final Report Abstract
FHOD1 is an atypical member of the Diaphanous-related formin (DRF) protein family that bundles actin filaments, decorates cellular actin structures, and coordinates actin with microtubule cytoskeletons. Our previous work had identified FHOD1 as component of the machinery governing nuclear movement of migrating fibroblasts that interacts with nesprin-2-giant (N2G) and F-actin that is essential for actinmediated positioning of the nucleus as well as for centrosome reorientation. Having established this basic principle of how FHOD1 facilitates actin dependent nuclear positioning, the first goal was to gain insight into this unusual mechanism at the structural and functional level. To this end we sought to define the molecular determinants that govern the association between FHOD1 and N2G and actin and found that the actin binding site (ABS) of FHOD1 interacts with spectrin repeats 11 and 12 of N2G. To gain further insight in the mechanism of action of FHOD1, we next adressed the relevance of FHOD1 ligands in nuclear migration and centrosome positioning. We identified the kinesisn-2 protein KIF3C as novel interaction partner of FHOD1 by co-immunoprecipitation. Although KIF3C typically forms a heteromeric complex with KIF3A, silencing of KIF3A expression did not affect the ability of KIF3C to interact with FHOD1. Furthermore, we mapped the interaction site for KIF3C in FHOD1 to the cterminal DAD motif which required the integrity of the DAD core motif. Pull down assays using recombinant GST-FHOD1 confirmed that FHOD1 interacts with KIF3C via the core DAD motif and that this interaction is specific to KIF3C but not KIF3A, suggesting that KIF3C exerts its role in nuclear migration independently of KIF3A. Using the same approach, we also identified the neck region on KIF3C as the FHOD1 interaction site. To address the relevance of the FHOD1-KIF3C interaction, an siRNA-mediated silencing approach was used and the effects on nuclear migration studied. The results revealed that KIF3C is as important for centrosome reorientation and nuclear movement as FHOD1 and N2G and that a KIF3C binding-deficient FHOD1 mutant fails to support centrosome reorientation. Finally, we explored the relationship between FHOD1, KIF3C and N2G, which revealed that all three proteins may physically associate together in a tripartite complex with the N2G spectrin repeats 11 and 12 as well as the KIF3C neck region being instrumental for these interactions. We are currently investigating the organization and mechanism of action of this tripartite complex in preparation of a new grant proposal.
Publications
- (2015) Reinforcing the LINC complex connection to actin filaments: the role of FHOD1 in TAN line formation and nuclear movement. Cell Cycle, 14: 2200-2205
Antoku, S., Zhu, R.,Kutscheidt, S., Fackler, O.T. and Gundersen, G.G.
(See online at https://doi.org/10.1080/15384101.2015.1053665)
