Dynamics of cytoskeletal proteins during Fcgamma receptor-mediated phagocytosis in macrophages.
Particle ingestion by phagocytosis results from sequential rearrangements of the actin cytoskeleton and overlying membrane. To assemble a chronology of molecular events during phagosome formation and to examine the contributions of phosphoinositide 3-kinase (PI 3-kinase) to these dynamics, a method was developed for synchronizing Fcgamma receptor-mediated phagocytosis by murine macrophages. Erythrocytes opsonized with complement component C3bi were bound to macrophages at 37degrees C, a condition that does not favor particle phagocytosis. Addition of soluble anti-erythrocyte IgG resulted in rapid opsonization of the bound erythrocytes, followed by their immediate internalization via phagocytosis. Cellular content of F-actin, as measured by binding of rhodamine-phalloidin, increased transiently during phagocytosis, and this increase was not diminished by inhibitors of PI 3-kinase. Immunofluorescence localization of myosins in macrophages fixed at various times during phagocytosis indicated that myosins II and IXb were concentrated in early phagosomes, myosin IC increased later, and myosin V appeared after phagosome closure. Other cytoskeletal proteins showed similar variations in the timing of their appearance in phagosomes. The PI 3-kinase inhibitor wortmannin did not change the dynamics of PI 3-kinase or ezrin localization but prevented the loss of PAK1 from phagosomes. These results suggest that PI 3-kinase deactivates PAK1, and that this may be needed for phagosome closure.
Diakonova
Maria
M
Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0620, USA.
Bokoch
Gary
G
Swanson
Joel A
JA
D000199:Actins / Q000378:metabolism*; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008264:Macrophages / Q000502:physiology* / Q000648:ultrastructure; D051379:Mice; D008855:Microscopy, Electron, Scanning; D009218:Myosins / Q000378:metabolism*; D010587:Phagocytosis / Q000502:physiology*; D010588:Phagosomes / Q000502:physiology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017452:Receptors, IgG / Q000502:physiology*; D012756:Sheep; D015398:Signal Transduction / Q000502:physiology; D000077191:Wortmannin; D054462:p21-Activated Kinases
D000199:Actins; D000730:Androstadienes; D004791:Enzyme Inhibitors; D009218:Myosins; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D017452:Receptors, IgG; D000077191:Wortmannin; D054462:p21-Activated Kinases; C515905:Pak1 protein, mouse