Beside its prominent role in cytoplasmic F-actin formation, actin nucleation also affects processes within the cell nucleus, such as gene expression through SRF and its coactivator MAL/MRTF. However, the precise connection between the nucleation promoting factors (NPFs), nuclear actin and MAL/SRF is unclear. The aim of this proposal is to elucidate the role of WH2-domains and -NPFs in nuclear processes, i.e. MAL/SRF-mediated transcription and formation of nuclear actin polymers. Previous work demonstrated that MAL is activated by its release from a repressive G-actin complex. NPFs often contain G-actin binding WH2 domains. Preliminary results and structural data are consistent with our working hypothesis that WH2 domains displace MAL from G-actin and thereby activate SRF-mediated transcription; we will test this directly in cellular assays. Further, we will analyse the WH2-MAL competition by biochemical experiments and in living cells by imaging. Using both candidate approaches and unbiased screening, we will attempt to identify the critical WH2-containing NPFs. Moreover, we will determine whether they are nuclear, affect nuclear MAL:actin complexes, and nuclear actin polymerisation. We expect to characterise a new function of NPFs in transcription, resolve the mechanism underlying MAL:actin dissociation, and to shed light on nuclear actin assembly.
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