Remodeling of the structure and function of the nuclear lamina by LINC complex-dependent tension

总结 核纤层是一种组成复杂的结构，在染色质组织中起作用， 转录调节、基因组保护和机械转导。在细胞和组织中， 纤层通过机械方式整合到肌动蛋白、微管和中间丝细胞骨架中， 核骨架和细胞骨架（LINC）复合物的跨核连接体。通过这些 细胞骨架连接，从细胞外基质或细胞外基质对质膜粘附施加的力， 从邻近细胞的信号可以传递到核内部。在LINC复合体功能已被破坏的细胞中， 改变后，研究人员观察到基因调控的相关变化。然而，迄今为止， 非常具有挑战性的是，要破译LINC复合体传递的机械力， 通过在核层的相互作用，直接影响特定的遗传程序。我们的出版物和 初步研究表明LINC复合物能够在细胞粘附之间产生临界串扰， 细胞骨架和核周边的组成部分。最重要的是，我们使用了鼠标 模型揭示了A型核纤层蛋白和LINC复合体对促纤维化信号传导的相反作用， 典型地由TGFβ下游的SMAD依赖性信号传导驱动。将这些见解与 我们的全球转录组研究表明，LINC复合物对核纤层施加的张力 影响SMAD基因靶点所必需的核事件被TGFβ输入适当调节（尽管 驱动该信号传导途径所必需的正常细胞质事件）。在此基础上，我们提出三个 互补的目的，将解决两个分子机制以及生理背景，其中 这些机制发挥着关键作用。首先，我们将采取无偏的方法来定义LINC复合体， 与来自细胞外基质的底物输入结合，影响核纤层相互作用组， 原位，最终采用交联质谱法。第二，我们将调查 LINC复合体消融影响细胞核中SMAD功能的机制，包括分析 SMAD靶基因的核定位，以及完整的内核的影响， 膜蛋白对SMAD依赖性基因输出的影响。最后，我们将测试是否（以及如何）LINC复杂功能 与A型纤层蛋白在TGFβ-SMAD纤维化轴中的交叉， 方法，包括心肌间质纤维化的小鼠模型和肺损伤模型， 肺纤维化总之，这项建议的目的将揭示两种分子机制， 通过LINC复合体的机械转导，同时也将这种详细的理解纳入其 生理和疾病背景。

SUMMARY The nuclear lamina is a compositionally complex structure that serves functions in chromatin organization, transcriptional regulation, genome protection and mechanotransduction. In cells and tissues, the nuclear lamina is mechanically integrated into the actin, microtubule, and intermediate filament cytoskeletons via nuclear envelope-spanning Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes. Through these cytoskeletal connections, forces exerted on plasma membrane adhesions from either the extracellular matrix or from adjacent cells can be transmitted to the nuclear interior. In cells in which LINC complex function has been altered, investigators have observed correlative changes in gene regulation. However, to date it has been extremely challenging to decipher whether mechanical forces transmitted by the LINC complex, potentially through interactions at the nuclear lamina, directly influence specific genetic programs. Our published work and preliminary studies demonstrate that the LINC complex enables a critical crosstalk between cellular adhesions, the cytoskeleton, and components of the nuclear periphery. Most important for this proposal, we used a mouse model to reveal that A-type lamins and the LINC complex drive opposite effects on pro-fibrotic signaling, which is classically driven by SMAD-dependent signaling downstream of TGFβ. Taking these insights together with our global transcriptome studies, we suggest that tension exerted on the nuclear lamina by the LINC complex influences nuclear events necessary for SMAD gene targets to be properly regulated by TGFβ inputs (despite normal cytoplasmic events necessary to drive this signaling pathway). Building on this, here we propose three complementary Aims that will address both molecular mechanisms as well as physiological contexts in which these mechanisms play critical roles. First, we will take an unbiased approach to define how the LINC complex, in combination with substrate inputs from the extracellular matrix, influences the nuclear lamina interactome in situ, ultimately employing a cross-linking mass spectrometry approach. Second, we will investigate the mechanisms by which LINC complex ablation influences SMAD function in the nucleus, including the analysis of SMAD target binding, nuclear position of SMAD target genes, and the influence of integral inner nuclear membrane proteins on SMAD-dependent gene output. Lastly, we will test if (and how) LINC complex function intersects with that of A-type lamins in this TGFβ–SMAD-fibrotic axis using both in vitro and in vivo approaches, including mouse models of interstitial fibrosis of the myocardium and lung injury models of pulmonary fibrosis. Taken together, the Aims of this proposal will reveal both molecular mechanisms of mechanotransduction through the LINC complex while also placing this detailed understanding into its physiological and disease contexts.