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Published online
doi:10.1083/jcb.200706167
The Journal of Cell Biology, Vol. 179, No. 6, 1141-1148
The Rockefeller University Press, 0021-9525 $30.00
© Wong et al.
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PDZRhoGEF and myosin II localize RhoA activity to the back of polarizing neutrophil-like cells



Kit Wong1, Alexandra Van Keymeulen2, and Henry R. Bourne1

1 Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158
2 Interdisciplinary Research in Human and Molecular Biology Institute (IRIBHM), Faculty of Medicine, Université Libre de Bruxelles (ULB), 1070 Brussels, Belgium

Correspondence to Henry R. Bourne: bourne{at}cmp.ucsf.edu

Chemoattractants such as formyl-Met-Leu-Phe (fMLP) induce neutrophils to polarize by triggering divergent pathways that promote formation of a protrusive front and contracting back and sides. RhoA, a Rho GTPase, stimulates assembly of actomyosin contractile complexes at the sides and back. We show here, in differentiated HL60 cells, that PDZRhoGEF (PRG), a guanine nucleotide exchange factor (GEF) for RhoA, mediates RhoA-dependent responses and determines their spatial distribution. As with RNAi knock-down of PRG, a GEF-deleted PRG mutant blocks fMLP-dependent RhoA activation and causes neutrophils to exhibit multiple fronts and long tails. Similarly, inhibition of RhoA, a Rho-dependent protein kinase (ROCK), or myosin II produces the same morphologies. PRG inhibition reduces or mislocalizes monophosphorylated myosin light chains in fMLP-stimulated cells, and myosin II ATPase inhibition reciprocally disrupts normal localization of PRG. We propose a cooperative reinforcing mechanism at the back of cells, in which PRG, RhoA, ROCK, myosin II, and actomyosin spatially cooperate to consolidate attractant-induced contractility and ensure robust cell polarity.

Abbreviations used in this paper: CA, constitutively active; dHL60, differentiated HL60; DN, dominant-negative; fMLP, formyl-Met-Leu-Phe; FRET, fluorescence resonance energy transfer; GEF, guanine nucleotide exchange factor; KD, knock-down; PRG, PDZRhoGEF; PIP3, phospatidylinositol-3,4,5-tris-phosphate; PI3K, phospatidylinositol-3'-kinase; ROCK, Rho-dependent kinase; shRNA, short hairpin RNA.


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