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

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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.