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.

概括核层是一种组成复杂的结构，在染色质组织中发挥作用，细胞和组织、细胞核中的转录调控、基因组保护和机械转导。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）复合物的跨核膜连接体。细胞骨架连接，从细胞外基质或细胞外基质施加在质膜粘附上的力来自邻近细胞的信号可以传输到细胞核内部，其中 LINC 复合功能已经发挥作用。然而，迄今为止，研究人员已经观察到基因调控的相关变化。破译 LINC 复合体传输的机械力是否可能具有极大的挑战性通过核层的相互作用，直接影响我们发表的工作和初步研究表明，LINC 复合物能够在细胞粘附之间实现关键的串扰，对于这个提议最重要的是，我们使用了小鼠。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产生相反的作用，这结合这些见解，传统上是由 TGFβ 下游的 SMAD 依赖性信号驱动的。我们的全球转录组研究表明，LINC 复合体对核层施加了张力影响 SMAD 基因靶标所需的核事件，以通过 TGFβ 输入正确调节（尽管在此基础上，我们提出了三个必要的正常细胞质事件。互补的目标将解决分子机制以及生理背景首先，我们将采用公正的方法来定义 LINC 如何复杂、与细胞外基质的底物输入相结合，影响核层相互作用组原位，最终采用交联质谱方法其次，我们将研究。 LINC 复合物消融影响细胞核内 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完整内核的影响最后，我们将测试 LINC 复合体是否（以及如何）发挥作用。在体外和体内实验中，与 TGFβ-SMAD 纤维化轴中的 A 型核纤层蛋白相交方法，包括小鼠心肌间质纤维化模型和肺损伤模型综上所述，该提案的目的将揭示肺纤维化的两种分子机制。通过 LINC 复合体进行机械转导，同时也将这种详细的理解融入到其生理和疾病背景。