Pivotal Advance: Phospholipids determine  net membrane surface charge resulting in  differential localization of active Rac1  and Rac2.
In this investigation, we used primary murine neutrophils to demonstrate that local changes in membrane phospholipid composition alter the net cytoplasmic membrane surface charge, which results in selective recruitment of Rac1 or Rac2 based on the net charge of their respective C-terminal domains. Murine neutrophils undergoing chemotaxis or carrying out phagocytosis were transfected with K-ras4B-derived membrane charge biosensors and lipid markers, which allowed us to simultaneously monitor the levels of PIP(2), PIP(3), and PS and net membrane charge of the newly developing phagosome membrane and plasma membrane. Our results indicate that the combination of PIP(2), PIP(3), and PS generates a high negative charge (-8) at the plasma membrane of actin-rich pseudopods, where active Rac1 preferentially localizes during phagosome formation. The lipid metabolism that occurs during phagosome maturation results in the localized depletion of PIP(2), PIP(3), and partial decrease in PS. This creates a moderately negative net charge that correlates with the localization of active Rac2. Conversely, the accumulation of PIP(3) at the leading-edge membrane during chemotaxis generates a polarized accumulation of negative charges that recruits Rac1. These results provide evidence that alterations in membrane lipid composition and inner-membrane surface charge are important elements for the recruitment of differentially charged proteins and localization of signaling pathways during phagocytosis and chemotaxis in neutrophils.
Magalhaes
Marco A O
MA
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York, USA.
Glogauer
Michael
M
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology / Q000235:genetics / Q000276:immunology*; D002633:Chemotaxis / Q000235:genetics / Q000276:immunology; D050356:Lipid Metabolism / Q000235:genetics / Q000276:immunology; D051379:Mice; D009479:Neuropeptides / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D009504:Neutrophils / Q000201:enzymology / Q000276:immunology*; D010587:Phagocytosis / Q000276:immunology; D010743:Phospholipids / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000235:genetics / Q000276:immunology; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011554:Pseudopodia / Q000201:enzymology / Q000235:genetics / Q000276:immunology; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology*; D014162:Transfection; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D020830:rac1 GTP-Binding Protein; D000097766:RAC2 GTP-Binding Protein
D009479:Neuropeptides; D010743:Phospholipids; D016283:Proto-Oncogene Proteins p21(ras); D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein; D000097766:RAC2 GTP-Binding Protein; C484602:Rac1 protein, mouse; C495971:Hras protein, mouse