Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Actins / blood*
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Adult
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Cell Membrane / drug effects
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Cell Membrane / ultrastructure
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Cell Membrane Permeability
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Cell Size / drug effects
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Glucosides / pharmacology
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Guanosine Triphosphate / analogs & derivatives
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Guanosine Triphosphate / pharmacology
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Humans
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In Vitro Techniques
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Kinetics
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N-Formylmethionine Leucyl-Phenylalanine / pharmacology
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Nerve Tissue Proteins / chemistry
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Neutrophils / cytology
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Neutrophils / drug effects
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Neutrophils / physiology*
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Peptide Fragments / pharmacology
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Receptors, Formyl Peptide
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Receptors, Immunologic / blood*
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Receptors, Peptide / blood*
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Signal Transduction / drug effects
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Signal Transduction / physiology
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Wiskott-Aldrich Syndrome
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Wiskott-Aldrich Syndrome Protein, Neuronal
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cdc42 GTP-Binding Protein / blood*
Substances
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Actins
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Glucosides
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Nerve Tissue Proteins
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Peptide Fragments
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Receptors, Formyl Peptide
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Receptors, Immunologic
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Receptors, Peptide
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WASL protein, human
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Wiskott-Aldrich Syndrome Protein, Neuronal
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octyl-beta-D-glucoside
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N-Formylmethionine Leucyl-Phenylalanine
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Guanosine Triphosphate
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cdc42 GTP-Binding Protein