Cdc42, Rac1, and Rac2 display distinct patterns of activation during phagocytosis.
The small G proteins Cdc42, Rac1, and Rac2 regulate the rearrangements of actin and membrane necessary for Fcgamma receptor-mediated phagocytosis by macrophages. Activated, GTP-bound Cdc42, Rac1, and Rac2 bind to the p21-binding domain (PBD) of PAK1, and this interaction provided a basis for microscopic methods to localize activation of these G proteins inside cells. Fluorescence resonance energy transfer-based stoichiometry of fluorescent chimeras of actin, PBD, Cdc42, Rac1, and Rac2 was used to quantify G protein activation relative to actin movements during phagocytosis of IgG-opsonized erythrocytes. The activation dynamics of endogenous G proteins, localized using yellow fluorescent protein-labeled PBD, was restricted to phagocytic cups, with a prominent spike of activation over an actin-poor region at the base of the cup. Refinements of fluorescence resonance energy transfer stoichiometry allowed calculation of the fractions of activated GTPases in forming phagosomes. Cdc42 activation was restricted to the leading margin of the cell, whereas Rac1 was active throughout the phagocytic cup. During phagosome closure, activation of Rac1 and Rac2 increased uniformly and transiently in the actin-poor region of phagosomal membrane. These distinct roles for Cdc42, Rac1, and Rac2 in the component activities of phagocytosis indicate mechanisms by which their differential regulation coordinates rearrangements of actin and membranes.
Hoppe
Adam D
AD
Department of Microbiology and Immunology and the Biophysics Research Division, University of Michigan Medical School, Ann Arbor, Michigan 48109-0620, USA.
Swanson
Joel A
JA
D000199:Actins / Q000032:analysis; D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis; D002460:Cell Line; D049452:Green Fluorescent Proteins / Q000032:analysis; D008164:Luminescent Proteins / Q000032:analysis; D008264:Macrophages / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D010587:Phagocytosis / Q000502:physiology*; D010588:Phagosomes / Q000502:physiology; D019869:Phosphatidylinositol 3-Kinases / Q000032:analysis / Q000378:metabolism; D025941:Protein Interaction Mapping; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D020764:cdc42 GTP-Binding Protein / Q000032:analysis / Q000378:metabolism*; D054462:p21-Activated Kinases; D020744:rac GTP-Binding Proteins / Q000032:analysis / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000032:analysis / Q000378:metabolism*; D000097766:RAC2 GTP-Binding Protein
D000199:Actins; D001426:Bacterial Proteins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D020764:cdc42 GTP-Binding Protein; D054462:p21-Activated Kinases; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein; D000097766:RAC2 GTP-Binding Protein; C485182:Cyan Fluorescent Protein; C054562:yellow fluorescent protein, Bacteria; C515905:Pak1 protein, mouse