Investigating the role of signaling feedback in directed cell migration

Weill Medical Coll Of Cornell Univ, New York NY

Investigators

Abstract

Project Summary Cell migration is critical for animal development, homeostasis, and is altered in many diseases. During migration, a cell’s ability to change direction is essential for processes such as wound healing, immune surveillance, and tissue morphogenesis. The polarization of cells and the key feedback loops maintaining this polarization allow for faithful execution of cell migration, however, little is known about how cells remain able to integrate new information to change its direction. The experiments outlined in this proposal are designed to dissect the mechanisms by which cells regulate key signaling pathways to control navigation. I will focus on PI3K, Ras, and Rac signaling, which are active at the front of migrating cells, and membrane-proximal F-actin (MPA) – which is localized to the rear. Many of these components have been shown to maintain their polarized activity through positive feedback but it is unclear how exactly negative feedback and crosstalk of these signaling pathways promote directional change. A better understanding of signaling feedback for these pathways will not only increase our understanding of how cells maintain faithful cell migration but are likely to shed light on several other biological processes such as cell division, polarity, and trafficking where these pathways also play critical roles. In Aim 1, the canonical signaling pathways involved in cell migration signaling at the leading edge, such as phosphatidylinositol-3 kinase (PI3K) and Ras signaling will be reinforced to determine the role of signaling feedback in migration speed, turning, and chemotaxis. Negative feedback signaling onto these pathways has been proposed as a mechanism to promote cell responsiveness to stimuli change-of-direction, but the consequences of preventing such negative signaling feedback remain unknown. This will be tested using unbiased front-localizing PIP3-binding domain to recruit protein fragments designed to augment endogenous cell signaling. I will also use fluorescence live-cell biosensors to determine if reinforcement of PI3K and Ras alter the ability of other signaling pathways to regulate cell turning. In Aim 2, I will determine how membrane-proximal F-actin (MPA) regulates cell migration speed, turning, and chemotaxis. Our laboratory has recently shown that areas high in MPA are restrictive to protrusion generation but the mechanism by which this occurs is still unclear. The experiments in this aim will determine how MPA recruitment to the front of the cell alters migration speed, as well as key signaling pathways, such as RhoA and Rac1, that regulate turning and chemotaxis. I will combine the use of high throughput quantitative microscopy, fluorescent biosensors, micropatterned migration substrates, 3D matrices, and cell tracking analysis to address these aims.