title: Synthetic spatially graded Rac activation drives cell polarization and movement.
abstract: Migrating cells possess intracellular gradients of active Rho GTPases, which serve as central hubs in transducing signals from extracellular receptors to cytoskeletal and adhesive machinery. However, it is unknown whether shallow exogenously induced intracellular gradients of Rho GTPases are sufficient to drive cell polarity and motility. Here, we use microfluidic control to generate gradients of a small molecule and thereby directly induce linear gradients of active, endogenous Rac without activation of chemotactic receptors. Gradients as low as 15% were sufficient not only to trigger cell migration up the chemical gradient but to induce both cell polarization and repolarization. Cellular response times were inversely proportional to the steepness of Rac inducer gradient in agreement with a mathematical model, suggesting a function for chemoattractant gradient amplification upstream of Rac. Increases in activated Rac levels beyond a well-defined threshold augmented polarization and decreased sensitivity to the imposed gradient. The threshold was governed by initial cell polarity and PI3K activity, supporting a role for both in defining responsiveness to Rac activation. Our results reveal that Rac can serve as a starting point in defining cell polarity. Furthermore, our methodology may serve as a template to investigate processes regulated by intracellular signaling gradients.
authors.affiliation: Department of Biomedical Engineering, Institute for Cell Engineering, Johns Hopkins University, Baltimore, MD 21205, USA.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Lin
authors.forename: William R
authors.initials: WR
authors.lastname: Holmes
authors.forename: C Joanne
authors.initials: CJ
authors.lastname: Wang
authors.forename: Tasuku
authors.initials: T
authors.lastname: Ueno
authors.forename: Andrew
authors.initials: A
authors.lastname: Harwell
authors.forename: Leah
authors.initials: L
authors.lastname: Edelstein-Keshet
authors.forename: Takanari
authors.initials: T
authors.lastname: Inoue
authors.forename: Andre
authors.initials: A
authors.lastname: Levchenko
mesh_terms: D002465:Cell Movement / Q000187:drug effects; D016764:Cell Polarity / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D008954:Models, Biological; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D055503:Protein Multimerization / Q000187:drug effects; D020123:Sirolimus / Q000494:pharmacology; D013997:Time Factors; D020744:rac GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D019869:Phosphatidylinositol 3-Kinases; D020744:rac GTP-Binding Proteins; D020123:Sirolimus