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

• 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
• 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

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。完全取消动力蛋白功能会产生大量的细胞和 器官缺陷，使人们无法检测到它对其他重要细胞现象的贡献。这个 该提案中描述的工作重点是人类动力蛋白变异体DCTN4Y270C，它与 增加了极端肺部病变的风险。DCTN4Y270C(Rs35772018)首次被鉴定为遗传性 囊性纤维化的修饰物，但也与肺腺癌和 脓毒症后的急性呼吸窘迫综合征。纯合子DCTN4Y270C对 它本身，表明任何与DCTN4Y270C相关的缺陷都会加剧病理，但不会导致病理 直接去吧。 肺上皮细胞必须能够经历可逆的上皮-间充质转化(EMT) 提供修复和维持呼吸道所需的持续伤口愈合。不平衡 可逆性EMT被认为是严重肺部疾病所见纤维化的基础。表型：DCTN4Y270C 表达减少了细胞在实验创面中的移动，并促进了上皮细胞的获得 极性，这两个标志间充质-上皮转化(MET)。转录上，培养的 表达DCTN4Y270C的支气管上皮细胞具有EMT的特征。这些 不一致的发现表明，DCTN4Y270C将细胞定位在一种异常的、但仍可耐受的状态 位于上皮和间质之间，类似于肿瘤中的部分EMT。的首要目标是 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- 细胞定向迁移背后的依赖现象。 为了深入了解DCTN4Y270C如何改变细胞功能，我们确定了 DCTN4WT和DCTN4Y270C使用BioID邻近生物素化。这揭示了一小群不同的 其他人已经将其与我们已知的在细胞中表达的改变的现象联系起来的相互作用 DCTN4Y270C。这个项目的目的是在动力蛋白/动力蛋白之间建立机械联系。 和这些新型的互感器，利用DCTN4Y270C作为一种独特的工具来调制和 扰乱细胞功能。