Coordination of Rho GTPase activity in simulated protrusions of macrophages

Abstract

CX3CL1- and Colony Stimulating Factor-1 (CSF-1)-mediated monocyte/macrophage infiltration is a hallmark event in an array of inflammatory diseases, such as atherosclerosis, rheumatoid arthritis, and the metastatic progression of cancer, yet the chemotactic signaling events downstream of these potent pro-inflammatory chemokines are not well-defined. Rho GTPases are master regulators of actin cytoskeletal remodeling, a fundamental requirement for efficient cell migration. Rac1, Rac2 and Cdc42, members of the Rho GTPase family, have been implicated in the formation of actin-rich protrusions and chemotaxis by macrophages. However the individual roles of these Rho GTPases downstream of the receptors for CSF-1 and CX3CL1, the receptor tyrosine kinase (RTK) CSF-1R and the G protein-coupled receptor (GPCR) CX3CR1 respectively, remains unclear. To clearly define the individual as well as the coordinated spatiotemporal dynamics of these Rho GTPases, we used Forster Resonance Energy Transfer (FRET)-based biosensors. Our laboratory recently developed a single-chain Rac1 biosensor. We successfully extended this approach to Rac2 and Cdc42, and demonstrated proper performance through extensive in vitro characterizations. Furthermore, we optimized the biosensors for facile and stable expression in hematopoietic cells, and performed functional validations in a subline of murine monocyte/macrophage RAW264.7 cells. We performed morphodynamic analysis of Rac1, Rac2 and Cdc42 activities in random protrusions in the presence of serum. Rac2 appeared to play a leading role in the generation of random protrusions, where Rac2 was strongly activated first in distal regions from the leading edge, followed by the activation of Rac1, a second distinct pool of Rac2, and lastly Cdc42 immediately behind the leading edge. Protrusions induced by global stimulation with CSF-1, CX3CL1 or fMLP (mediated by another GPCR) revealed differential Rac1 and Rac2 spatiotemporal activities, suggesting differential regulation of Rho GTPases downstream of RTK versus VI GPCR. Previous reports indicated that genetic deletion of Rac1 had no significant effect on CSF-1-induced macrophage chemotaxis. However, a range of abnormalities were observed both in cell morphology and behavior, suggestive of compensation by other family members, such as Rac2 or Cdc42. To investigate the compensatory potential of Rac2 and Cdc42 in the absence of Rac1, we performed ratiometric imaging of actin-rich protrusions in response to a global stimulation with CSF-1 using control and shRNAmediated Rac1-depleted macrophages. Macrophages with reduced levels of Rac1 still produce actin-rich protrusions, but to a much lesser extent compared to control cells. Rac1 activity is highly localized at CSF-1-stimulated actin-rich protrusions in wild-type macrophages, and we speculated that Rac2 or Cdc42 may compensate in the absence of Rac1 either by increased and/or re-localized activity. In control cells we found no measurable increase in Rac2 activity at the whole cell levels or in actin-rich protrusions, whereas whole cell level of Cdc42 activity was significantly elevated during CSF-1 response with modest localization to protrusions. Rac2 activity and localization in Rac1- depleted macrophages was indistinguishable from control cells, indicating no compensatory potential, however we found an unexpected effect on Cdc42 activity; it was significantly diminished, remaining at basal levels, in the absence of Rac1 during CSF-1 response. Surprisingly, a significant elevation in Cdc42 activity in Rac1-depleted macrophages was detectable, although at lower levels compared to control cells, during GPCR-mediated CX3CL1 stimulation, suggesting that Rac1-dependent activation of Cdc42 is CSF-1-specific. Our preliminary results indicate a guanine nucleotide exchange factor-mediated regulatory mechanism as the basis for Cdc42 potentiation by Rac1. This had functional relevance, as Rac1-depleted macrophages failed to migrate towards CSF-1, while they were able to respond to CX3CL1 in chemotactic assays. Overall, our studies provide insight into the differential regulation of Rho GTPases during RTK versus GPCR signaling, providing a basis for the identification of potential therapeutic targets.