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Published online 4 September 2000. doi:10.1083/jcb.150.5.1001
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© The Rockefeller University Press, 0021-9525/2000//1001 $5.00
The Journal of Cell Biology, Volume 150, Number 5, , 2000 1001-1012

Original Article

Profilin Enhances Cdc42-Induced Nucleation of Actin Polymerization



Changsong Yanga, Minzhou Huanga, John DeBiasioa, Martin Pringb, Michael Joycea, Hiroaki Mikic, Tadaomi Takenawac, and Sally H. Zigmonda

a Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6018
b Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6018
c Department of Biochemistry, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan
Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018.(215) 898-8780(215) 898-4559

szigmond{at}sas.upenn.edu

We find that profilin contributes in several ways to Cdc42-induced nucleation of actin filaments in high speed supernatant of lysed neutrophils. Depletion of profilin inhibited Cdc42-induced nucleation; re-addition of profilin restored much of the activity. Mutant profilins with a decreased affinity for either actin or poly-L-proline were less effective at restoring activity. Whereas Cdc42 must activate Wiskott-Aldrich Syndrome protein (WASP) to stimulate nucleation by the Arp2/3 complex, VCA (verpolin homology, cofilin, and acidic domain contained in the COOH-terminal fragment of N-WASP) constitutively activates the Arp2/3 complex. Nucleation by VCA was not inhibited by profilin depletion. With purified N-WASP and Arp2/3 complex, Cdc42-induced nucleation did not require profilin but was enhanced by profilin, wild-type profilin being more effective than mutant profilin with reduced affinity for poly-L-proline.

Nucleation by the Arp2/3 complex is a function of the free G-actin concentration. Thus, when profilin addition decreased the free G-actin concentration, it inhibited Cdc42- and VCA-induced nucleation. However, when profilin was added with G-actin in a ratio that maintained the initial free G-actin concentration, it increased the rate of both Cdc42- and VCA-induced nucleation. This enhancement, also seen with purified proteins, was greatest when the free G-actin concentration was low. These data suggest that under conditions present in intact cells, profilin enhances nucleation by activated Arp2/3 complex.

Key Words: actin polymerization • nucleation • Cdc42 • leukocytes • profilin

© 2000 The Rockefeller University Press

Abbreviations used in this paper: CA, cofilin homology and acidic tail contained in the COOH-terminal fragment of N-WASP; GST, glutathione S-transferase; PLP, poly-L-proline; VCA, verpolin homology, cofilin, and acidic domain contained in the COOH-terminal fragment of N-WASP; VDBP, vitamin D binding protein; WASP, Wiskott-Aldrich Syndrome protein.


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