Unravelling the Multifaceted Role of FNBP4 in Regulating Actin Cytoskeleton Dynamics through Non-Diaphanous Formin FMN1 and Direct Actin Interactions in the Nucleus

Das, Shubham (2025) Unravelling the Multifaceted Role of FNBP4 in Regulating Actin Cytoskeleton Dynamics through Non-Diaphanous Formin FMN1 and Direct Actin Interactions in the Nucleus. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Shubham Das (19RS081)) 19RS081.pdf - Submitted Version Restricted to Repository staff only Download (24MB)

Abstract

Nuclear actin cytoskeleton is crucial for transcription regulation, DNA damage repair, and chromatin organization. Actin-binding proteins, including formins, dynamically shuttle between the cytoplasm and nucleus to regulate nuclear actin dynamics. Mammalian formins are categorized into diaphanous-related formins and non-diaphanous formins, distinguished by their regulatory mechanisms. Notably, the non-diaphanous formin FMN1 has emerged as a nuclear regulator, influencing transcriptional activity. However, the regulatory mechanisms governing nuclear formins, particularly non-diaphanous ones like FMN1, remain largely unexplored. In contrast, FNBP4 has previously been annotated as a formin-binding protein, but its interaction with formins remains largely uncharacterized. Here, using surface plasmon resonance (SPR) and ELISA, we characterized the binding kinetics between the N-terminal WW1 domain of FNBP4 and the FH1 domain of FMN1, highlighting their specific interaction. Functional actin reconstitution assays, including TIRF microscopy and pyrene-actin polymerization assays, revealed that FNBP4 inhibits FMN1-mediated actin assembly, preventing processive capping activity at filament barbed ends and restricting FMN1's bundling activity by imposing spatial constraints on the FH2 domain. Moreover, our subcellular localization studies identified a monopartite nuclear localization signal (NLS) in FNBP4, demonstrating its nuclear colocalization with FMN1. This suggests that FNBP4 acts as a nuclear inhibitor of actin polymerization. Furthermore, we identified FNBP4 as an actin-binding protein, and mutational analyses identified the C-terminal region of FNBP4 as critical for the direct actin-binding site, with key residues KRRK (1009-1012 aa) mediating the interaction. Additionally, immunostaining and overexpression of 3x-NLS-actin demonstrate the colocalization of FNBP4 with nuclear actin, supporting their association. In summary, FNBP4 emerges as a multifunctional regulator of nuclear actin dynamics, modulating FMN1 activity and directly interacting with nuclear actin. Thus, it seems tempting to speculate that FNBP4 is a potential key player in nuclear functions.

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