Dynamics of cytoskeletal proteins during Fcgamma receptor-mediated phagocytosis in macrophages

Mol Biol Cell. 2002 Feb;13(2):402-11. doi: 10.1091/mbc.01-05-0273.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Actins / metabolism*
  • Androstadienes / pharmacology
  • Animals
  • Enzyme Inhibitors / pharmacology
  • Macrophages / physiology*
  • Macrophages / ultrastructure
  • Mice
  • Microscopy, Electron, Scanning
  • Myosins / metabolism*
  • Phagocytosis / physiology*
  • Phagosomes / physiology
  • Phosphatidylinositol 3-Kinases / metabolism
  • Phosphoinositide-3 Kinase Inhibitors
  • Protein Serine-Threonine Kinases / metabolism
  • Receptors, IgG / physiology*
  • Sheep
  • Signal Transduction / physiology
  • Wortmannin
  • p21-Activated Kinases

Substances

  • Actins
  • Androstadienes
  • Enzyme Inhibitors
  • Phosphoinositide-3 Kinase Inhibitors
  • Receptors, IgG
  • Pak1 protein, mouse
  • Protein Serine-Threonine Kinases
  • p21-Activated Kinases
  • Myosins
  • Wortmannin