The RHO GTPASE CDC42 Regulator CDC42GAP Plays a Critical Role in Neutrophil Migration Via Podosome-Like Formation.

Neutrophils (PMN) are a critical cell in inflammatory processes. In response to environmental stimuli, they activate various signal transduction pathways allowing them to move rapidly to a site of microbial invasion and to perform phagocytosis, cytokine and oxygen substrate release. Rho GTPase proteins, Rac1, Rac2, CDC42 and Rho, are central regulators of cell movement via actin rearrangement. We have demonstrated the specific role of Rac1 and Rac2 in PMN functions (Gu and Filippi et al, Science 2003; Filippi et al. Nat Immuol., 2004) which raises the question of the specificity of the other Rho GTPases. CDC42 primarily regulates the formation of filopodia. CDC42 controls cell polarity and migration in hematopoietic cell lines. Most of previous studies have utilized dominant active or negative mutants which lack specificity and cannot be easily used to define in vivo cell biology. Here, we used mice genetically deficient in the CDC42 negative regulator CDC42 GTPase Activating Protein (GAP) to study the role of CDC42 in neutrophil functions in vitro and in vivo. Heterozygote (CDC42GAP^+/−) or homozygote (CDC42GAP^−/−) mutant mice displayed normal neutrophil differentiation in vitro and in vivo. PMN deficient in CDC42GAP displayed 2-fold increased in CDC42 activity. In vivo recruitment of PMN in peritoneal cavities after thioglycollate exposure was significantly impaired in CDC42GAP^+/− mice compared with wild type (WT) mice (25.5±0.76 x 10⁵ vs 35.7±0.38 x 10⁵, p<0.05). Both CDC42GAP^+/− and CDC42GAP^−/− PMN demonstrated defective directed migration in vitro in response to fMLP in a Boyden chamber assay compared with WT (248±31 and 199±20 versus 314±29 migrated cells, p<0.05), suggesting that CDC42 plays a critical role in neutrophil migration in vitro and in vivo. To further understand the role of CDC42GAP in neutrophil migration, single-cell analysis by time-lapse videomicroscopy was performed. Surprisingly, CDC42GAP^+/− PMN demonstrated higher migration velocity compared with WT cells in response to fMLP, but this increased speed was associated with an abnormal shape. Upon beta-2 integrin ligation, CDC42GAP^+/− PMN demonstrated abnormal elongated trailing tail associated with increased tail filopodia. Importantly, the podosome-like structures identified by a vinculin ring surrounding F-actin at the ventral plasma membrane that are present in the leading edge of WT PMN was absent in the mutant cells. CDC42GAP^−/− PMN demonstrated more dramatic F-actin impairment upon integrin ligation compared with CDC42GAP^+/− and WT cells and remarkably showed complete loss of cell polarity, consistent with the known role of CDC42 in cell polarity. We hypothesize that the lack of podosome formation in mutant cells could account for the increased speed observed in CDC42GAP^+/− cells and therefore result in ineffective directed migration in vivo. Altogether, this suggests that regulation of CDC42 activity plays a pivotal role in neutrophil migration likely via integrin-dependent podosome-like formation. This reinforces the importance of turnover of attachment structures during cell movement and suggests a new role for CDC42 in actin-based attachment structure in neutrophils.