Spatial and Temporal Analysis of Rac Activation during Live Neutrophil Chemotaxis

Abstract

The ability of cells to recognize and respond with directed motility to chemoattractant agents is critical to normal physiological function. Neutrophils represent the prototypic chemotactic cell in that they respond to signals initiated through the binding of bacterial peptides and other chemokines to G protein-coupled receptors with speeds of up to 30 microm/min. It has been hypothesized that localized regulation of cytoskeletal dynamics by Rho GTPases is critical to orchestrating cell movement. Using a FRET-based biosensor approach, we investigated the dynamics of Rac GTPase activation during chemotaxis of live primary human neutrophils. Rac has been implicated in establishing and maintaining the leading edge of motile cells, and we show that Rac is dynamically activated at specific locations in the extending leading edge. However, we also demonstrate activated Rac in the retracting tail of motile neutrophils. Rac activation is both stimulus and adhesion dependent. Expression of a dominant-negative Rac mutant confirms that Rac is functionally required both for tail retraction and for formation of the leading edge during chemotaxis. These data establish that Rac GTPase is spatially and temporally regulated to coordinate leading-edge extension and tail retraction during a complex motile response, the chemotaxis of human neutrophils.

Synthetic spatially graded Rac activation drives cell polarization and movement

“…For example, state I cells exhibit initially low, mostly homogeneous Rac activity, followed by the induction of high Rac activity at a newly formed front when a gradient of rapamycin is applied. This pattern of Rac activity is similar to that seen in neutrophils polarizing to a gradient of f-MetLeu-Phe (47). Additionally, state III cells repolarize when given sufficiently high gradients of rapamycin by forming a new front at the rear and retracting the previous front.…”

Abr and Bcr, Two Homologous Rac GTPase-Activating Proteins, Control Multiple Cellular Functions of Murine Macrophages

“…It is believed that localized activation/deactivation of Rac is critical for CSF-1-induced motility, because the microinjection of both dominant negative Rac and constitutively active Rac prevented cells from developing the proper polarization required for migration, thereby inhibiting motility (3,26). This view is further supported by the finding of Gardiner et al (16) showing that Rac is periodically activated at the leading edge of neutrophils migrating toward chemokines. Many studies have focused on the positive regulators of Rac, such as phosphatidylinositol 3-kinase and Vav that link CSF-1 stimulation to the localized Rac activation at the cell membrane and to motility (45,54,60).…”

Antagonistic Cross-talk between Rac and Cdc42 GTPases Regulates Generation of Reactive Oxygen Species

“…To specifically inhibit Cdc42 during neutrophil activation, the CRIB domain of WASP, which binds Cdc42 but not Rac (or Rho) (41), was introduced into human neutrophils using Bioporter. We have previously shown that this method successfully delivers fluorescently labeled p21-activated kinase CRIB domain into human neutrophils (31). As shown in Fig.…”

“…The proteins were expressed and purified from E. coli and quantified by BCA protein assay (Pierce). The proteins were delivered into neutrophils as previously described (31). Briefly, Bioporter reagent (Gene Therapy Systems, La Jolla, CA) was reconstituted with chloroform according to the manufacturer's instructions, and 2-3 l was aliquoted into Eppendorf tubes and evaporated overnight.…”