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

摘要 核层是一种成分复杂的结构，其在染色质组织中起作用， 转录调控、基因组保护和机械转导。在细胞和组织中，细胞核 膜被机械地整合到肌动蛋白、微管和中间丝细胞骨架中。 核骨架和细胞骨架(LINC)复合体的跨核膜连接物。通过这些 细胞骨架连接，来自细胞外基质或细胞外基质的作用于质膜粘连的力 可以从相邻的细胞传播到细胞核内部。在LINC复合体功能已经被 经过改变，研究人员观察到了基因调控的相关变化。然而，迄今为止，它一直是 要破译LINC复合体传递的机械力是否潜在地 通过核层的相互作用，直接影响特定的遗传程序。我们出版的作品和 初步研究表明，LINC复合体能够在细胞黏附之间产生关键的串扰， 细胞骨架，以及核外周的组成部分。对于这个提案，最重要的是，我们使用了一只鼠标 模型揭示了A型Lamins和LINC复合体在促纤维化信号转导中的相反作用，这 传统上是由转化生长因子β下游依赖SMAD的信号驱动的。将这些洞察力与 我们的全球转录组研究表明，LINC复合体对核层施加的张力 影响转化生长因子β输入适当调节SMAD基因靶点所需的核事件(尽管 驱动该信号通路所需的正常细胞质事件)。在此基础上，我们提出三点建议 互补的目标，将解决分子机制以及生理环境，在 这些机制起着至关重要的作用。首先，我们将采取公正的方法来定义LINC建筑群， 结合来自细胞外基质的底物输入，影响核层相互作用体 原位，最终采用了交联质谱方法。第二，我们将调查 LINC复合体消融影响核内SMAD功能的机制 SMAD靶结合、SMAD靶基因的核位置以及完整内核的影响 膜蛋白对SMAD依赖基因输出的影响。最后，我们将测试LINC复杂函数是否(以及如何) 与A型板层蛋白在转化生长因子β-SMAD-纤维化轴上的交叉点 方法包括小鼠心肌间质纤维化模型和肺损伤模型。 肺纤维化。综上所述，该提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置于其 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.