The role of actin nucleators and nucleation promoting factors in nuclear gene expression via MRTF

除了其在细胞质F-肌动蛋白形成中的突出作用之外，肌动蛋白成核还影响细胞核内的过程，例如通过SRF及其共激活因子MAL/MRTF的基因表达。然而，成核促进因子（NPFs），核肌动蛋白和MAL/SRF之间的确切联系尚不清楚。该建议的目的是阐明WH 2-结构域和-NPFs在核过程中的作用，即MAL/SRF介导的转录和核肌动蛋白聚合物的形成。以前的工作表明，MAL是激活其从一个压抑的G-肌动蛋白复合物的释放。NPF通常含有G-肌动蛋白结合WH 2结构域。初步结果和结构数据与我们的工作假设，WH 2结构域取代MAL从G-肌动蛋白，从而激活SRF介导的转录是一致的，我们将测试这直接在细胞测定。此外，我们将通过生物化学实验和活细胞成像分析WH 2-MAL竞争。使用两种候选方法和无偏筛选，我们将试图确定关键的WH 2含NPF。此外，我们将确定它们是否是核的，影响核MAL：肌动蛋白复合物和核肌动蛋白聚合。我们希望能揭示NPFs在转录中的新功能，解决MAL的潜在机制：肌动蛋白解离，并阐明核肌动蛋白组装。

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.