Rho GTPase CDC42 Maintains Neutrophil Polarity Axis during Directed Migration by Regulating CD11b Integrin Signals through Microtubules

Neutrophil (PMN) migration to sites of infection is the first line of cellular defense. Among others, a key event of cell migration is the maintenance of a polarized morphology characterized by a single protrusive leading edge of F-actin and a contractile uropod devoid of F-actin protrusions. We have previously reported a fundamental role for the small Rho GTPase Cdc42 in neutrophil polarity by suppressing membrane protrusions at the uropod of the cells during directed migration (Szczur et al, Blood 2006). Using mice with a conditional Cdc42 (flox) allele, we previously showed that Cdc42^−/− PMNs extended multiple membrane extensions in various directions and failed to maintain cell polarity and directionality towards fMLP gradient compared to WT cells, as assessed by video microscopy. This was due to a loss of clustering of the integrin CD11b at the uropod. We have further investigated mechanisms of these events and first explored the migration behavior of CD11b-null PMN by video microscopy. CD11b^−/− cells extended multiple protrusions back and front but most failed to migrate toward fMLP. Interestingly, the cells that successfully traveled at least for a short distance extended inappropriate lateral protrusions accompanied by frequent changes in direction, indicating defective polarity. Furthermore, F-actin organization analysis revealed that while WT cells developed polarity with single lamellipodia in the leading front, CD11b^−/− cells exhibited increased F-actin around the cells, which indicated that CD11b is required to suppress lateral protrusion similarly to Cdc42. Enforcing CD11b clustering by CD11b cross-linking in Cdc42^−/− PMNs partially rescued cell polarity such that F-actin distribution concentrated only at the leading edge of the cells to WT levels. These data suggest that Cdc42 controls neutrophil polarity via CD11b signalling. To establish a link between Cdc42, which is active at the front, and CD11b, which localizes at the back, we examined the microtubule (MT) network. MTs were oriented toward the rear of WT cells and exhibited curling at the cell cortex consistent with cortical capture in migrating neutrophils. In contrast, MTs of Cdc42^−/− neutrophils extended both toward the front and the back but rarely made contact with the cell cortex at the sides. Furthermore, MTs made contact with CD11b clusters in WT but not in Cdc42^−/− neutrophils. Cells treated with the disrupting MT agent nocodazole cells failed to redistribute CD11b at the uropod. Correspondingly, these cells showed defective polarity, which suggests that Cdc42 regulates CD11b clustering and polarity via MTs. Finally, additional mechanistic studies revealed that CD11b functions as a signalling molecule by recruiting a pathway that involves ROCK and myosin light chain (MLC), and regulates contraction to antagonize membrane protrusions. Altogether, this study suggests that Cdc42 regulates the formation of a complex between MT and CD11b at the uropod that functions as an amplifier of internal signals to mediate cortical tension and thus consolidate polarity in migrating neutrophils. This study uncovers a new mechanism that is critical in maintaining neutrophil polarity between the leading front and the trailing back during migration. As Cdc42^−/− neutrophils exhibited defect in migration into peritoneal cavities in vivo, this pathway may have physiological role during the neutrophil extravasation cascade.