Uncovering the Roles of PIP5KDuring Cell Migration - Cardiovascular diseases and pulmonary fibrosis remain leading global health challenges, with cardiovascular diseases accounting for 17.9 million deaths annually and pulmonary fibrosis carrying a median survival time of only 3 to 5 years post-diagnosis. Despite current treatment options, patients with pulmonary fibrosis continue to experience high mortality rates. A major challenge to developing targeted treatments is the limited understanding of the molecular and biophysical mechanisms driving these diseases, specifically, how a cell initiates membrane asymmetry, establishes polarity, and enables directional migration—the fundamental biological processes involved in developing and maintaining cardiovascular and pulmonary systems. My recent findings reveal that phosphatidylinositol 4-phosphate 5-kinase (PIP5K), which synthesizes phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), undergoes dynamic partitioning and reciprocally inhibits Ras small GTPase activity during cell migration. Therefore, we target PIP5K and our overall hypothesis is that PIP5K and its effector Arf GTPase regulate cell migration through differential diffusion-driven dynamic partitioning, modulating Ras activity and downstream signaling pathways on specific membrane regions. The proposed hypothesis will be addressed in three specific aims. Aim 1 will determine how dynamic partitioning and binding affinity of PIP5K contribute to symmetry breaking and polarity establishment in migrating cells. I will measure PIP5K diffusion coefficients with photo-conversion techniques and single-molecule imaging under varying conditions. I will investigate how actin and actomyosin networks influence PIP5K partitioning using CRISPR-based gene disruption and pharmacological inhibitors. These experimental data will be integrated into stochastic simulations of an excitable network to validate models of symmetry breaking. Aim 2 seeks to identify and characterize the binding partners of PIP5K to understand its role in regulating Ras activity. I will use TurboID proximity labeling and Mass Spectrometry to map PIP5K’s interactome, with validation through co-immunoprecipitation and genetic perturbation. Functional studies will investigate how these partners influence PIP5K and Ras activities during cell migration. Aim 3 focuses on elucidating the roles of Arf GTPases in regulating cell migration and PIP5K functions. Using optogenetic tools and CRISPR, I will study how Arf GTPases and their regulators (ArfGEFs and ArfGAPs) modulate PIP5K localization, Ras signaling, and cytoskeletal dynamics. Biochemical assays will explore interactions between Arf proteins and PIP5K and their joint regulation of Ras activity. This study will provide valuable insights into how PIP5K, Arf, and Ras form a signaling cascade to regulate cell migration and polarity through the Ras/PI3K/Akt pathway and potentially uncover novel therapeutic targets for cardiovascular and pulmonary diseases. This project aligns with NHLBI’s mission to advance research on heart, lung, and blood diseases, improving public health through innovative science.
