Original Article

Two Pathways Through Cdc42 Couple the N-formyl Receptor to Actin Nucleation in Permeabilized Human Neutrophils

Correspondence to: M. Glogauer, Hematology Division, Brigham and Women's Hospital, Department of Medicine, Harvard Medical School, Room 301, 221 Longwood Avenue, Boston, MA 02115. Tel:(617) 278-0389 Fax:(617) 734-2248 E-mail:mglogauer{at}rics.bwh.harvard.edu.

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.

Key Words: Arp2/3, actin assembly, signal transduction pathways, Rac, FMLP

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