Mouse macrophages lacking both Pak1 and Pak2 exhibit preserved chemotaxis despite a strikingly altered migration mode

Macrophages migrate directionally along chemoattractant gradients through repeated cycles of membrane protrusion and trailing-end retraction. Chemoattractant receptor signaling activates the Rho GTPases Rac1, Rac2, and Cdc42 at the leading edge, which stimulate actin polymerization via actin-nucleating factors, such as the Arp2/3 complex. Simultaneously, these Rho GTPases stabilize membrane protrusions by activating p21-activated kinases (PAKs), which inhibit the actin-depolymerizing ADF/cofilin family through activation of LIM motif-containing kinases. Here, we used myeloid-restricted Pak2 conditional knockout (cKO), Pak1/Pak2 double knockout (dKO), and Pak1 knockout (Pak1^-/-) mouse models to determine whether PAKs are required for chemotactic navigation. In real-time chemotaxis assays, macrophages lacking Pak1 are indistinguishable from control cells, whereas Pak2 cKO macrophages frequently adopt a dendritic morphology and exhibit modestly impaired chemotaxis and cell velocity. In contrast, macrophages lacking both Pak1 and Pak2 exhibit robust chemotaxis and markedly increased cell velocity. Strikingly, Pak1/Pak2 dKO cells adopt a distinct, chemoattractant-induced rounded morphology, characterized by extensive membrane ruffling at the leading edge, which is rich in transverse actin fibers, and a compact trailing end, although these cells intermittently transition to slower, partially spread out or elongated morphologies. Phospho-cofilin (Ser3) levels are reduced in complement C5a-induced polarized dKO cells compared with control cells, whereas total cofilin levels are unchanged. In summary, whereas loss of Pak2 alone modestly impairs chemotaxis, combined deletion of Pak1 and Pak2 preserves directional navigation while enhancing cell motility, likely through reduced inhibition of ADF/cofilin and increased actin turnover at the leading edge.