Rac1 is the small GTPase responsible for regulating the neutrophil chemotaxis compass

Abstract

Although both of the small Rho guanosine triphosphatases (GTPases) Rac1 and Rac2 have been demonstrated to play a role in chemotaxis, the precise and possible unique roles performed by each of these 2 Rac isoforms in neutrophil chemotaxis have not been defined. To elucidate the specific roles of Rac1 and Rac2 in neutrophils during the process of chemotaxis, we generated mice deficient in Rac1, Rac2, or in both Rac1 and Rac2 in cells of myeloid lineage including neutrophils by mating Rac2 null mice with mice carrying a conditional allele for Rac1 and expressing the Cre recombinase downstream of a specific myeloid promoter, lysozyme M. We demonstrate here that although Rac1 null neutrophils display normal chemokinesis, they are unable to migrate toward the source of the chemoattractant. By contrast, Rac2 null neutrophils can orient toward the chemoattractant source but are unable to migrate efficiently. We show that Rac1 is essential for gradient detection and orientation toward the chemoattractant source through spatially constrained regulation of phosphoinositol-3,4,5-trisphosphate (PIP 3 ) and Akt in the leading edge and confirm that Rac2 is the primary regulator of actin assembly providing the molecular motor for neutrophil translocation during chemotaxis. (Blood.

Protein O-GlcNAc Modulates Motility-associated Signaling Intermediates in Neutrophils

“…Rac1 is ubiquitously expressed, whereas Rac2 expression is restricted to hematopoietic cells (75). Although the precise roles of each of these isoforms remain largely undefined, recent evidence indicates that Rac2 may be the primary regulator of actin assembly and the molecular machinery associated with movement, whereas Rac1 may be important for chemoattractant-directed orientation and gradient detection (76). In addition, Rac1 and Rac2 appear to exhibit cross-talk with one another, and their activities appear to be closely associated (77).…”

Neutrophil transcriptional profile changes during transit from bone marrow to sites of inflammation

“…15,[42][43][44] The neutrophil literature contains numerous methodologies for neutrophil purification; ranging from density gradient centrifugation to immunomagnetic selection, cell sorting and culture models. 14,41,[45][46][47] In this study, obtaining high-quality mRNA from neutrophils for all three compartments was the greatest obstacle and required the most amount of refining. While the current optimal method for obtaining pure neutrophils is immunomagnetic negative selection, 15,43,48 this approach resulted in very low cell yields (loss of up to 90% of neutrophils-see the section on 'Materials and methods') and low mRNA quality.…”

“…14,41 In analyzing the transcriptome of murine neutrophils from BM, blood and PE, we Table 5 and DAVID analysis of each cluster can be found in Supplementary Table 7). Green represents downregulation and red upregulation.…”

“…With that in mind, we tested different methods for each of the three compartments: For isolation of PMN-BM and PMN-B we used Percoll gradient separation 14 or immunomagnetic separation. 15 Additionally, for PMN-B we used the FACS-sorted samples, while peritoneal exudate (PE) PMN were collected with or without Percoll purification as detailed below.…”

“…In brief, BM cells were resuspended 14 in 4 ml of a-MEM and overlaid on Percoll gradients (82%/65%/55%). After centrifugation (2500 r.p.m., 4 uC, 30 min) the lower band was collected and washed with PBS.…”