Mechanisms of actin polymerization in T lymphocyte nuclei

虽然肌动蛋白骨架在哺乳动物细胞细胞质中的作用已经得到了很好的研究，但核肌动蛋白动力学的功能相关性直到最近才开始出现。越来越多的研究报道，核肌动蛋白丝在调节哺乳动物细胞与其环境的相互作用中起着重要作用，不同的肌动蛋白成核体在不同的途径中控制着核肌动蛋白丝的形成。在第一个资助期开始时，缺乏关于核肌动蛋白丝在免疫细胞中的一般作用和在T细胞受体（TCR）信号传导中的具体作用的信息。在第一个资助期，我们建立了核肌动蛋白聚合的短暂爆发作为CD4 T细胞激活的基本效应功能，这是一组选择的靶基因的表达所必需的，大量的细胞因子表达，因此有效的辅助功能。我们还将Arp2/3复合物定义为该过程中必不可少的肌动蛋白成核体，其活性受核Ca2+瞬态调节。虽然核Ca2+可能作为核肌动蛋白动力学的一般调节剂，但不同的肌动蛋白成核因子以细胞类型和刺激依赖的方式参与核肌动蛋白网络的形成。我们的初步结果表明，在CD4 T细胞中，Arp2/3异构体C5L和C5的特异性参与控制了TCR参与后肌动蛋白聚合的区隔化，并分别选择性地驱动核和细胞质肌动蛋白动力学。在第二个资助期，我们现在的目标是更详细地定义核肌动蛋白聚合的室特异性调节，并剖析核肌动蛋白动力学如何与选择性靶基因表达相耦合（特异性目标1）。在第二个工作包中，我们将利用我们生成的新小鼠模型来表达核f -肌动蛋白的荧光报告蛋白，通过活体显微镜在体内观察核肌动蛋白动力学，解剖核肌动蛋白动力学如何驱动体液免疫反应的增加，并评估核动力学在各种免疫细胞中的保守触发。总之，这些分析将为CD4 T细胞和其他免疫细胞中核肌动蛋白动力学的机制和相关性提供重要的见解，并建立核f -肌动蛋白一般功能影响的原则。

While the roles of the actin cytoskeleton in the cytoplasm of mammalian cells are well studied, the functional relevance of nuclear actin dynamics has only recently begun to emerge. An increasing number of studies report that nuclear actin filaments play important roles in regulating interactions of mammalian cells with their environment and that distinct actin nucleators govern the formation of nuclear actin filaments in individual pathways. At the beginning of the first funding period, information was lacking on the role of nuclear actin filaments in immune cells in general and in T Cell Receptor (TCR) signaling specifically. During the first funding period we established a transient burst of nuclear actin polymerization as essential effector function of CD4 T cell activation that is required for expression of a select set of target genes, ample cytokine expression, and thus efficient helper function. We also defined Arp2/3 complex as an essential actin nucleator for this process, whose activity is regulated by nuclear Ca2+ transients. While nuclear Ca2+ may act as general regulator of nuclear actin dynamics, different actin nucleators are involved in the formation of nuclear actin networks in a cell type and stimulus dependent manner. Our preliminary results suggest that in CD4 T cells, the specific involvement of the Arp2/3 isoforms C5L and C5 govern the compartmentalization of actin polymerization following TCR engagement and selectively drive nuclear and cytoplasmic actin dynamics, respectively. In the second funding period we will now aim at defining in more detail the compartment specific regulation of nuclear actin polymerization and dissect how nuclear actin dynamics is coupled to selective target gene expression (specific aim 1). In a second work package we will make use of a new mouse model we generated to express a fluorescent reporter for nuclear F-actin to visualize nuclear actin dynamics by intravital microscopy in vivo, dissect how nuclear actin dynamics drives mounting of humoral immune responses, and assess how conserved triggering of nuclear dynamics is in various immune cells. Together, these analyses will provide important insight in mechanism and relevance of nuclear actin dynamics in CD4 T cells and other immune cells and establish principles of the functional impact of nuclear F-actin in general.