Plasma membrane charge distribution during neutrophil chemotaxis

The subcellular targeting of proteins with cationic domains, including the Rac small GTPases is mainly determined by the electrostatic interactions with negatively charged membranes. Recent studies have shown that Rac1 and Rac2 differentially regulate important cell processes and this may be explained by the differential targeting of these molecules based on their c-terminal tail charge. OBJECTIVE: To determine whether neutrophil polarity/ chemotaxis depends on a highly regulated charge gradient at the plasma membrane that correlates with differential protein recruitment. METHODS: In this investigation, primary murine neutrophils were transfected with K-ras4B derived charged probes (+2,+3,+6,+8), Rac1 and Rac2 (Rac1CA-GFP, Rac2CA-CFP) constructs, Lact-C2 and subjected to Fc-mediated IgG-coated latex bead phagocytosis and micropipette-delivered fMLP chemotaxis. Control neutrophils were compared to T. denticola Msp treated neutrophils. Images were recovered in real time using a spinning disc confocal microscope. RESULTS: The results show that the more cationic Rac1 colocalizes with highly charged membrane compartments (probes +6, +8), and is selectively recruited to the leading edge membrane and the pseudopods (P<0.01). Rac2 is mostly associated with moderately charged areas, including internal membranes. Highly charged probes (+6, +8) accumulate at the leading edge of migrating neutrophils and colocalize with negatively charged lipids including phosphatidylserine and phosphoinositides. Msp increased the near plasma membrane charge and induced the release of active Rac1 (P<0.001) CONCLUSIONS: Using this novel approach, we show in real-time that neutrophils establish a charge gradient during chemotaxis with highly negative charges at the leading edge. Additionally, we show that a bacterial outer membrane protein (Msp) inhibits the normal neutrophil charge distribution, impacting migration.