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

LINC 复合物依赖性张力重塑核层的结构和功能

基本信息

批准号：
10247783
负责人：
MEGAN C KING
金额：
$ 33.5万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2023-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10247783
关键词：
Ablation Actins Actomyosin Address Adhesions Architecture Area Binding Biochemical Biological Cell Adhesion Cell Nucleus Cell membrane Cells Chromatin Collaborations Complex Cytoplasm Cytoskeleton Data Disease Environment Etiology Event Extracellular Matrix Fibrosis Functional disorder Gene Expression Regulation Gene Targeting Genes Genetic Genetic Diseases Genetic Transcription Genome Homeostasis In Situ In Vitro Intermediate Filaments Investigation Lamin Type A Lamins Mass Spectrum Analysis Mechanics Membrane Proteins Methods Microtubules Modeling Molecular Mutation Myocardium Nature Nuclear Nuclear Accidents Nuclear Envelope Nuclear Inner Membrane Nuclear Lamina Nuclear Translocation Output Pathway interactions Physiological Physiology Play Positioning Attribute Process Proteins Publishing Pulmonary Fibrosis Regulation Research Personnel Role Signal Pathway Signal Transduction Structure System Techniques Testing Tissues Transcriptional Regulation Transforming Growth Factor beta Translating Work base crosslink in vivo insight interstitial keratinocyte lung injury mechanical force mechanotransduction mouse model novel programs protein protein interaction response transcription factor transcriptome transmission process

项目摘要

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.

概括核层是一种合成的复杂结构，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，核通过机械整合薄片，通过核骨骼和细胞骨架（LINC）复合物的核包膜跨膜接头。通过这些细胞骨架连接，从细胞外基质或质膜上导出的力可以从相邻的细胞传播到核内部。在林格复合功能的单元格中变化，研究人员观察到基因调节的相关变化。但是，到目前为止非常挑战地破译林克复合物是否传输的机械力，潜在的通过核层次的相互作用，直接影响特定的遗传程序。我们已发表的工作和初步研究表明，LINC复合物可以在细胞粘合剂之间进行临界串扰，细胞骨架和核周围的成分。对于此建议最重要的是，我们使用了鼠标模型揭示A型层粘连和LINC复合驱动器对促纤维化信号的影响相反，这在TGFβ下游的SMAD依赖信号传导上，经典驱动。将这些见解与我们的全球转录组研究，我们建议LINC Complex上施加紧张影响SMAD基因靶标所必需的核事件，以适当地受到TGFβ输入的调节（尽管驱动此信号通路所需的正常细胞质事件）。在此基础上，我们在这里提出三个完全旨在解决分子机制以及物理环境的目标这些机制起着关键作用。首先，我们将采取一种公正的方法来定义林克综合体如何结合细胞外基质的底物输入，影响核薄片相互作用组原位，最终采用交联质谱法。第二，我们将调查 LINC复合消融影响核中SMAD功能的机制，包括分析 SMAD靶标结合，SMAD靶基因的核位置以及积分内核的影响依赖SMAD的基因输出上的膜蛋白。最后，我们将测试（以及如何）LINC复合功能使用体外和体内的TGFβ-SMAD-纤维化轴与A型层粘蛋白的相交方法，包括心肌间质纤维化的小鼠模型和肺损伤模型肺纤维化。综上所述，该提案的目的将揭示两种分子机制通过LINC复合物的机械转导，同时还将这种详细的理解置于其生理和疾病环境。