RhoC regulates the actin remodeling required for phagosome formation during FcγR-mediated phagocytosis

J Cell Sci. 2017 Dec 15;130(24):4168-4179. doi: 10.1242/jcs.202739. Epub 2017 Nov 7.

Abstract

Phagosome formation is a complicated process that requires spatiotemporally regulated actin reorganization. We found that RhoC GTPase is a critical regulator of FcγR-mediated phagocytosis in macrophages. Our live-cell imaging revealed that RhoC, but not RhoA, is recruited to phagocytic cups engulfing IgG-opsonized erythrocytes (IgG-Es). RhoC silencing through RNAi, CRISPR/Cas-mediated RhoC knockout, and the expression of dominant-negative or constitutively active RhoC mutants suppressed the phagocytosis of IgG-Es. Moreover, RhoC-GTP pulldown experiments showed that endogenous RhoC is transiently activated during phagosome formation. Notably, actin-driven pseudopod extension, which is required for the formation of phagocytic cups, was severely impaired in cells expressing the constitutively active mutant RhoC-G14V, which induced abnormal F-actin accumulation underneath the plasma membrane. mDia1 (encoded by DIAPH1), a Rho-dependent actin nucleation factor, and RhoC were colocalized at the phagocytic cups. Similar to what was seen for RhoC, mDia1 silencing through RNAi inhibited phagosome formation. Additionally, the coexpression of mDia1 with constitutively active mutant RhoC-G14V or expression of active mutant mDia1-ΔN3 drastically inhibited the uptake of IgG-Es. These data suggest that RhoC modulates phagosome formation be modifying actin cytoskeletal remodeling via mDia1.

Keywords: F-actin; FcγR; Macrophages; Phagocytosis; RhoC; mDia1.

MeSH terms

  • Actins / genetics
  • Animals
  • CRISPR-Cas Systems / genetics
  • Carrier Proteins / genetics*
  • Carrier Proteins / metabolism
  • Cell Line
  • Cell Tracking / methods
  • Erythrocytes / metabolism
  • Formins
  • Humans
  • Macrophages / metabolism
  • Mice
  • Phagocytosis / genetics*
  • Phagosomes / genetics*
  • Phagosomes / metabolism
  • Receptors, IgG / genetics
  • Receptors, IgG / metabolism
  • rhoC GTP-Binding Protein / genetics*
  • rhoC GTP-Binding Protein / metabolism

Substances

  • Actins
  • Carrier Proteins
  • Diap1 protein, mouse
  • Fcgr1 protein, mouse
  • Formins
  • Receptors, IgG
  • rhoC GTP-Binding Protein