Two pathways through Cdc42 couple the N-formyl receptor to actin nucleation in permeabilized human neutrophils.
We developed a permeabilization method that retains coupling between N-formyl-methionyl-leucyl-phenylalanine tripeptide (FMLP) receptor stimulation, shape changes, and barbed-end actin nucleation in human neutrophils. Using GTP analogues, phosphoinositides, a phosphoinositide-binding peptide, constitutively active or inactive Rho GTPase mutants, and activating or inhibitory peptides derived from neural Wiskott-Aldrich syndrome family proteins (N-WASP), we identified signaling pathways leading from the FMLP receptor to actin nucleation that require Cdc42, but then diverge. One branch traverses the actin nucleation pathway involving N-WASP and the Arp2/3 complex, whereas the other operates through active Rac to promote actin nucleation. Both pathways depend on phosphoinositide expression. Since maximal inhibition of the Arp2/3 pathway leaves an N17Rac inhibitable alternate pathway intact, we conclude that this alternate involves phosphoinositide-mediated uncapping of actin filament barbed ends.
Glogauer
M
M
Hematology Division, Brigham and Women's Hospital, Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA. m.glogauer@rics.bwh.harvard.edu
Hartwig
J
J
Stossel
T
T
D000199:Actins / Q000097:blood*; D000328:Adult; D002462:Cell Membrane / Q000187:drug effects / Q000648:ultrastructure; D002463:Cell Membrane Permeability; D048429:Cell Size / Q000187:drug effects; D005960:Glucosides / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000737:chemistry; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D010446:Peptide Fragments / Q000494:pharmacology; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000097:blood*; D018000:Receptors, Peptide / Q000097:blood*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014923:Wiskott-Aldrich Syndrome; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D020764:cdc42 GTP-Binding Protein / Q000097:blood*
D000199:Actins; D005960:Glucosides; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D018000:Receptors, Peptide; C495771:WASL protein, human; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; C018619:octyl-beta-D-glucoside; D009240:N-Formylmethionine Leucyl-Phenylalanine; D006160:Guanosine Triphosphate; D020764:cdc42 GTP-Binding Protein