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

项目摘要 基于微管的马达，细胞质动力蛋白-1，最为人所知的是提供运动功能， 膜运输和有丝分裂。动力蛋白功能的完全消除产生了大量的细胞和 有机体缺陷，妨碍检测其对其他重要细胞现象的贡献。的 这项提案中描述的工作集中在人类动力蛋白变体DCTN 4 Y270 C上，该变体与以下疾病相关： 会增加肺部严重病变的风险DCTN 4 Y270 C（rs35772018）首次被鉴定为一种遗传性 但也与肺腺癌的风险增加有关， 脓毒症后急性呼吸窘迫综合征纯合子DCTN 4 Y270 C对 这表明与DCTN 4 Y270 C相关的任何缺陷都会加剧病理，但不会导致病理 直接. 肺上皮细胞必须能够经历可逆的上皮-间充质转化（EMT）， 提供修复和维护气道所需的持续伤口愈合。不平衡 可逆EMT被认为是严重肺病中所见纤维化的基础。表型，DCTN 4 Y270 C 表达产生减少的细胞运动到实验伤口中，并促进上皮细胞的获得。 极性，两者都是间充质-上皮转化（MET）的标志。转录，培养 表达DCTN 4 Y270 C的支气管上皮细胞表现出EMT的特征。这些 不一致的发现表明，DCTN 4 Y270 C将细胞定位在一种异常但可耐受的状态， 在上皮和间质之间，类似于在肿瘤中看到的部分EMT。的首要目标 拟议的工作是了解DCTN 4 Y270 C如何影响肺细胞生物学，特别是细胞骨架- 依赖性现象是定向细胞迁移的基础。 为了获得DCTN 4 Y270 C如何改变细胞功能的机制，我们确定了DCTN 4 Y270 C的相互作用组。 使用BioID邻近生物素化的DCTN 4 WT和DCTN 4 Y270 C。这揭示了一个小队列的差异 其他人已经将其与我们所知的细胞表达改变的现象联系起来， DCTN4Y270C。本项目的目的是建立动力蛋白/动力蛋白之间的机制联系 这些新型的相互作用，利用DCTN 4 Y270 C作为一种独特的工具， 扰乱细胞功能

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.