Phosphoinositide 3-kinase enables phagocytosis of large particles by terminating actin assembly through Rac/Cdc42 GTPase-activating proteins

Abstract

Phagocytosis is responsible for the elimination of particles of widely disparate sizes, from large fungi or effete cells to small bacteria. Though superficially similar, the molecular mechanisms involved differ: engulfment of large targets requires phosphoinositide 3-kinase (PI3K), while that of small ones does not. Here, we report that inactivation of Rac and Cdc42 at phagocytic cups is essential to complete internalization of large particles. Through a screen of 62 RhoGAP-family members, we demonstrate that ARHGAP12, ARHGAP25 and SH3BP1 are responsible for GTPase inactivation. Silencing these RhoGAPs impairs phagocytosis of large targets. The GAPs are recruited to large—but not small—phagocytic cups by products of PI3K, where they synergistically inactivate Rac and Cdc42. Remarkably, the prominent accumulation of phosphatidylinositol 3,4,5-trisphosphate characteristic of large-phagosome formation is less evident during phagocytosis of small targets, accounting for the contrasting RhoGAP distribution and the differential requirement for PI3K during phagocytosis of dissimilarly sized particles.

The neglected terminators: Rho family GAPs in neutrophils

“…In an acellular system, Rho family GAPs associated with the cell membrane of human neutrophils have been specifically depleted by antibodies, and changes in the NADPH oxidase activity were followed. 6 Extraction of p50RhoGAP (Cdc42GAP) or ARHGAP25 Human (cell free) NADPH oxidase [6] Human/cell culture Phagocytosis Actin polymerization [7,8] ARHGAP1/Cdc42GAP/ p50RhoGAP/RHOGAP1…”

“…[14] ARHGAP15 Rac Mouse Migration, NADPH oxidase, phagocytosis, abdominal sepsis, retention in bone marrow [15,16] Human/cell culture Migration, actin polymerization [17] ARHGAP13/srGAP1 Cdc42 Mouse/human Neutrophil chemotaxis [18] BCR/ABR Rac/Cdc42 Mouse (double knockout) Acute inflammatory responses [19] BCR Rac/Cdc42 Human (cell free) NADPH oxidase [9] Mouse Septic shock NADPH oxidase [20] ARHGAP12 Rac Human/cell culture Actin polymerization [8] ARHGAP43/SH3BP1 Rac Human/cell culture Actin polymerization [8] ARHGAP21/ ARHGAP10…”

“…8 In a systematic screen, they eventually identified three Rho family GAPs-ARHGAP12, ARHGAP25 and SH3BP1-that showed PIP3-dependent translocation to the forming phagosome, and their downregulation significantly impaired FcγR-mediated phagocytosis of large particles. The fact that none of these GAPs was essential for phagocytosis of small particles indicates different molecular organization of the phagocytic process depending on the size of the particle to be engulfed.…”

Involvement of a Rac1-Dependent Macropinocytosis Pathway in Plasmid DNA Delivery by Electrotransfection

“…61 Additionally, Rho family GTPases are well recognized for their complex interaction networks and multiple roles played in regulation of actin cytoskeleton assembly. 62 It has also been reported that two of the Rho family GTPases, cell division control protein 42 (Cdc42) and Rac1, can regulate cell motility, [63][64][65] phagocytosis, 66 and macropinocytosis. 67 Therefore, we hypothesized that electrotransfection could lead to actin remodeling and enhance macropinocytosis through activation of Cdc42 and Rac1.…”

Rac GTPase Activating Protein ARHGAP25 Regulates Leukocyte Transendothelial Migration in Mice

“…Our findings strongly suggest that the leukocytespecific RacGAP ARHGAP25 is a critical player in TNF-a-induced, Rac-mediated actin reorganization in neutrophils. Two recent reports provide important information on its physiological role: ARHGAP25 was shown to be required for actin depolymerization in the course of phagocytosis (21,44), and it was demonstrated to undergo significant changes in its phosphorylation pattern and GAP activity upon biological stimulation (45). TNF-a-initiated modulation of the phosphorylation pattern of ARHGAP25 with subsequent alterations of its GAP function may provide the link between the cytokine effect and the actin cytoskeleton rearrangement.…”