Impact of a Disease-Associated Dynactin Variant on Motile Phenomena in Lung Epithelial Cells

疾病相关的 Dynactin 变体对肺上皮细胞运动现象的影响

基本信息

批准号：
10704306
负责人：
TRINA A SCHROER
金额：
$ 36.69万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-22 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10704306
关键词：
Actins Acute Respiratory Distress Syndrome Affect Behavior Binding Biochemical Biochemistry Biological Assay Biosensor Biotinylation Cell Adhesion Cell membrane Cell physiology Cell surface Cells Cellular biology Characteristics Complement Coupled Cues Cystic Fibrosis Cytoskeleton Data Defect Detection Disease Disease Progression Dynein ATPase Epithelial Epithelial Cells Event Fibrosis Focal Adhesion Kinase 1 Focal Adhesions Genes Genetic Genetic Transcription Goals Guanine Guanine Nucleotide Exchange Factors Human Human Cell Line Image Integrins Lead Link Lung Lung Adenocarcinoma Lung diseases Maintenance Membrane Mesenchyme Methods Microscopy Microtubules Mitosis Motor Movement Pathology Phase Phenotype Plus End of the Microtubule Positioning Attribute Process Proteins Proteome Proteomics Pulmonary Pathology Receptor Protein-Tyrosine Kinases Risk Role Secretory Vesicles Sepsis Signal Pathway Signal Transduction Sorting - Cell Movement Surface Total Internal Reflection Fluorescent Transitional Epithelium Variant Work assault base bronchial epithelium cell cortex cell motility cofactor cohort contrast imaging crosslink cytokine dynactin ephexin exome sequencing experimental study imaging study in vivo insight interest lung repair macromolecular assembly migration monolayer novel protein transport repaired response rho GTP-Binding Proteins stem tool trafficking tumor wound wound healing

项目摘要

Project Summary The microtubule-based motor, cytoplasmic dynein-1, is best known for providing motile functions in membrane trafficking and mitosis. Complete abrogation of dynein function yields gross cellular and organismal defects that preclude detection of its contributions to other important cellular phenomena. The work described in this proposal focuses on a human dynein variant, DCTN4Y270C, that is associated with increased risk of extreme lung pathology. DCTN4Y270C (rs35772018) was first identified as a genetic modifier of cystic fibrosis but has also been associated with increased risk of lung adenocarcinoma and acute respiratory distress syndrome following sepsis. Homozygous DCTN4Y270C has no obvious impact on its own, indicating that any defects associated with DCTN4Y270C exacerbate pathology but do not cause it directly. Lung epithelial cells must be able to undergo a reversible epithelial-mesenchyme transition (EMT) to provide the ongoing wound-healing that is required for repair and maintenance of the airway. Imbalance in reversible EMT is believed to underlie the fibrosis seen in severe lung disease. Phenotypically, DCTN4Y270C expression yields reduced cell movement into experimental wounds and facilitates acquisition of epithelial polarity, both hallmarks of the mesenchyme-epithelial transition (MET). Transcriptionally, cultured bronchial epithelial cells expressing DCTN4Y270C show with hallmarks characteristic of EMT. These incongruent findings indicate that DCTN4Y270C positions cells in an aberrant, yet tolerated, state that lies between epithelia and mesenchyme, similar to the partial EMT seen in tumors. The overarching goal of the proposed work is to understand how DCTN4Y270C affects lung cell biology, specifically the cytoskeleton- dependent phenomena that underlie directed cell migration. To obtain mechanistic insight into how DCTN4Y270C alters cell function we determined the interactomes of DCTN4WT and DCTN4Y270C using BioID proximity biotinylation. This revealed a small cohort of differential interactors that others have linked to the phenomena we know to be altered in cells expressing DCTN4Y270C. The aims of this project are to establish mechanistic connections between dynein/dynactin and these novel interactors, taking advantage of DCTN4Y270C as a unique tool for modulating and perturbing cell function.

项目概要基于微管的马达，细胞质动力蛋白-1，以提供运动功能而闻名。膜运输和有丝分裂。动力蛋白功能的完全废除产生总细胞和妨碍检测其对其他重要细胞现象的贡献的有机体缺陷。这该提案中描述的工作重点关注人类动力蛋白变体 DCTN4Y270C，它与极端肺部病理的风险增加。 DCTN4Y270C (rs35772018) 首次被鉴定为遗传性囊性纤维化的修饰剂，但也与肺腺癌和肺腺癌风险增加有关脓毒症后的急性呼吸窘迫综合征。纯合子DCTN4Y270C对无明显影响其本身，表明与 DCTN4Y270C 相关的任何缺陷都会加剧病理，但不会导致病理直接地。肺上皮细胞必须能够经历可逆的上皮间质转化（EMT）提供修复和维护气道所需的持续伤口愈合。不平衡据信，可逆的 EMT 是严重肺部疾病中纤维化的基础。表型上，DCTN4Y270C 表达减少了细胞向实验伤口的移动，并促进上皮细胞的获得极性，这是间质-上皮转化（MET）的两个标志。转录、培养表达 DCTN4Y270C 的支气管上皮细胞显示出 EMT 特征的特征。这些不一致的发现表明 DCTN4Y270C 将细胞置于异常但可耐受的状态，即上皮细胞和间充质之间，类似于肿瘤中看到的部分 EMT。该组织的总体目标是拟议的工作是了解 DCTN4Y270C 如何影响肺细胞生物学，特别是细胞骨架 - 定向细胞迁移的依赖性现象。为了深入了解 DCTN4Y270C 如何改变细胞功能，我们确定了以下相互作用组 DCTN4WT 和 DCTN4Y270C 使用 BioID 邻近生物素化。这揭示了一小群差异其他人已经将相互作用因子与我们已知的在细胞表达中发生改变的现象联系起来 DCTN4Y270C。该项目的目的是建立动力蛋白/动力蛋白之间的机械连接以及这些新颖的相互作用器，利用 DCTN4Y270C 作为调制和干扰细胞功能。