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

肌动蛋白核化除了在细胞质F-肌动蛋白的形成中发挥重要作用外，还通过SRF及其辅活化子MAL/MRTF影响细胞核内的基因表达。然而，核成核促进因子(NPF)、核肌动蛋白和MAL/SRF之间的确切联系尚不清楚。这一建议的目的是阐明WH2结构域和NPF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是通过从抑制性G-肌动蛋白复合体中释放出来而激活的。NPF通常含有G-肌动蛋白结合WH2结构域。初步结果和结构数据与我们的工作假设一致，即WH2结构域取代了G-肌动蛋白中的MAL，从而激活了SRF介导的转录；我们将直接在细胞检测中对此进行测试。进一步，我们将通过生化实验分析WH2-MAL的竞争，并通过成像分析活细胞中的竞争。使用两种候选方法和无偏见筛选，我们将尝试确定关键的含有WH2的NPF。此外，我们将确定它们是否是核的，是否影响核MAL：肌动蛋白复合体，以及核肌动蛋白聚合。我们期望描述NPF在转录中的一个新功能，解决MAL的机制：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.