Efficient uptake of Yersinia pseudotuberculosis via integrin receptors involves a Rac1–Arp 2/3 pathway that bypasses N-WASP function

Authors

  • Michael A. Alrutz,

    1. Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Avenue M and V 409, Boston, MA 02111, USA.
    Search for more papers by this author
  • Amit Srivastava,

    1. Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Avenue M and V 409, Boston, MA 02111, USA.
    Search for more papers by this author
  • Ka-Wing Wong,

    1. Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Avenue M and V 409, Boston, MA 02111, USA.
    Search for more papers by this author
  • Crislyn D'Souza-Schorey,

    1. Department of Biological Sciences, University of Notre Dame, IN, USA.
    Search for more papers by this author
  • May Tang,

    1. Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Avenue M and V 409, Boston, MA 02111, USA.
    2. Howard Hughes Medical Institute, 136 Harrison Avenue M and V 409, Boston, MA 02111, USA.
    Search for more papers by this author
  • Lian-Ee Ch'Ng,

    1. Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Avenue M and V 409, Boston, MA 02111, USA.
    Search for more papers by this author
  • Scott B. Snapper,

    1. Gastrointestinal Unit, Massachusetts General Hospital, Boston, MA 02115, USA.
    2. Department of Medicine, Harvard Medical School, Boston, MA 02115, USA.
    Search for more papers by this author
  • Ralph R. Isberg

    Corresponding author
    1. Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Avenue M and V 409, Boston, MA 02111, USA.
    2. Howard Hughes Medical Institute, 136 Harrison Avenue M and V 409, Boston, MA 02111, USA.
    Search for more papers by this author

Abstract

Efficient uptake of Yersinia pseudotuberculosis into cultured mammalian cells is the result of high-affinity binding of invasin to β1 chain integrins. We demonstrate here that uptake requires Rac1 and Arp 2/3 function. Bacterial uptake was stimulated by GTPγS, but was inhibited in mammalian cells transfected with the interfering Rac1-N17 derivative. Rac1 was found to be activated in response to integrin engagement by invasin, whereas Rac1 and Arp 2/3 were found to be intensely localized around phagosomes bearing bacteria, indicating a specific role for Rac1 signalling from the nascent phagosome to downstream effectors. To determine whether the Arp 2/3 complex was a component of this proposed pathway, cells overproducing various derivatives of Scar1/WAVE1, an Arp 2/3-binding protein, were analysed. Sequestration of Arp 2/3 away from the phagocytic cup as a result of Scar1/WAVE1 overproduction dramatically inhibited uptake. To determine whether signalling from Rac1 to Arp 2/3 occurred via N-WASP, uptake was analysed in a cell line lacking expression of WASP and N-WASP. Uptake was unaffected by the absence of these proteins, indicating that β1 integrin signalling from Rac1 to Arp 2/3 can occur in the absence of N-WASP function.

Ancillary