Mammalian target of rapamycin and Rictor control neutrophil chemotaxis by regulating Rac/Cdc42 activity and the actin cytoskeleton

Mol Biol Cell. 2013 Nov;24(21):3369-80. doi: 10.1091/mbc.E13-07-0405. Epub 2013 Sep 4.

Abstract

Chemotaxis allows neutrophils to seek out sites of infection and inflammation. The asymmetric accumulation of filamentous actin (F-actin) at the leading edge provides the driving force for protrusion and is essential for the development and maintenance of neutrophil polarity. The mechanism that governs actin cytoskeleton dynamics and assembly in neutrophils has been extensively explored and is still not fully understood. By using neutrophil-like HL-60 cells, we describe a pivotal role for Rictor, a component of mammalian target of rapamycin complex 2 (mTORC2), in regulating assembly of the actin cytoskeleton during neutrophil chemotaxis. Depletion of mTOR and Rictor, but not Raptor, impairs actin polymerization, leading-edge establishment, and directional migration in neutrophils stimulated with chemoattractants. Of interest, depletion of mSin1, an integral component of mTORC2, causes no detectable defects in neutrophil polarity and chemotaxis. In addition, experiments with chemical inhibition and kinase-dead mutants indicate that mTOR kinase activity and AKT phosphorylation are dispensable for chemotaxis. Instead, our results suggest that the small Rho GTPases Rac and Cdc42 serve as downstream effectors of Rictor to regulate actin assembly and organization in neutrophils. Together our findings reveal an mTORC2- and mTOR kinase-independent function and mechanism of Rictor in the regulation of neutrophil chemotaxis.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Actin Cytoskeleton / metabolism*
  • Blotting, Western
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism*
  • Cell Line, Tumor
  • Cells, Cultured
  • Chemotaxis, Leukocyte*
  • Fluorescent Antibody Technique
  • HL-60 Cells
  • Humans
  • Mechanistic Target of Rapamycin Complex 2
  • Multiprotein Complexes / genetics
  • Multiprotein Complexes / metabolism
  • N-Formylmethionine Leucyl-Phenylalanine / pharmacology
  • Neutrophils / cytology
  • Neutrophils / drug effects
  • Neutrophils / metabolism
  • Phosphorylation / drug effects
  • Proto-Oncogene Proteins c-akt / metabolism
  • RNA Interference
  • Rapamycin-Insensitive Companion of mTOR Protein
  • Sirolimus / pharmacology
  • TOR Serine-Threonine Kinases / genetics
  • TOR Serine-Threonine Kinases / metabolism*
  • cdc42 GTP-Binding Protein / genetics
  • cdc42 GTP-Binding Protein / metabolism*
  • rac GTP-Binding Proteins / genetics
  • rac GTP-Binding Proteins / metabolism*

Substances

  • Carrier Proteins
  • Multiprotein Complexes
  • RICTOR protein, human
  • Rapamycin-Insensitive Companion of mTOR Protein
  • N-Formylmethionine Leucyl-Phenylalanine
  • MTOR protein, human
  • TOR Serine-Threonine Kinases
  • Mechanistic Target of Rapamycin Complex 2
  • Proto-Oncogene Proteins c-akt
  • cdc42 GTP-Binding Protein
  • rac GTP-Binding Proteins
  • Sirolimus